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https://openalex.org/W2397522618	https://europepmc.org/articles/pmc4878296?pdf=render	English	null	Emerging infectious disease and fast-track publication: when public health gets priority over the formality of scholarly publishing	Memórias do Instituto Oswaldo Cruz	2,016	cc-by	784	Emerging infectious disease and fast-track publication: when public health gets priority over the formality of scholarly publishing It has long been acknowledged within the scientific community that in the mid-term future the Earth will undergo important changes beyond those deduced from the past behaviour of known physical phenomena. For example, cli mate change due to rising temperatures is expected to influence several systems, like ocean levels, wind current and rain regimes. Global travel, urbanisation, biomedical manipulation and intensive agriculture are additional factors affecting natural systems which together in turn influence the diversity of plant, animals, and microorganisms in general. As a result, zoonoses represent 60,3% of emerging infectious diseases, more than 70% of which are caused by pathogens of wildlife origin (Jones et al. 2008). Whatever the action suffered by these subsystems (increase or reduction in species number), the fact is that currently we are unable to predict the exact outcomes of these changes. hi li i i f h f d k h i f b bl h h This limitation to forecast the future does not prevent us to sketch scenarios for probable events, such as the emer gence of unknown and lethal infectious agents. Advances in research of infectious disease and epidemiology have empowered society to respond quickly to this kind of threat, though not so effective as to the point of controlling or completely eliminating a disease. The Ebola epidemics is an example of such an event and its response. Now we have another threat: the Zika virus (ZIKV) and its vector, the mosquito Aedes aegypti. The Director-Gen eral of the World Health Organization, on 1 February 2016, declared this to be a Public Health Emergency of Interna tional Concern (PHEIC). We can only speculate that the spread of both this virus and vector might be seen as signs of the changes anticipated by climatologists and ecologists. However, one thing is beyond any question or doubt: we have to start new ways to accelerate scientific collaboration, including access to data and exchange of research findings. Recently, the Forum of Scientific Editors of FIOCRUZ decided collectively to give priority to the publication of papers regarding the Zika epidemic in their journals. Emerging infectious disease and fast-track publication: when public health gets priority over the formality of scholarly publishing In the context of this decision and the declaration of the Director-General of the World Health Organization, we, as the editors of the Memórias do Instituto Oswaldo Cruz (MIOC), have decided to create a section on the MIOC website called “Zika Fast Track”, where research manuscripts relevant to the Zika epidemic will be posted. We have also decided to follow the example of the Bulletin of WHO (Dye et al. 2016) and make the data in these papers freely available for unrestricted use, distribution and reproduction provided that the original work is properly cited as indicated by the Creative Commons Attribution license (CC BY). All research manuscripts concerning the Zika epidemic, after submission to the Memórias do Instituto Oswaldo Cruz, will be evaluated by an Editor and those considered relevant will be assigned a digital object identifier and posted online in the “Zika Fast Track” section of the Web-site within 24 hours. At the same time, manuscripts will b evaluated by peer review. Data in these manuscripts will thus be attributed to the authors while being freely availabl for reader scrutiny and unrestricted use, distribution and reproduction provided that the original work is properly cited as indicated by the Creative Commons Attribution license (CC BY 3.0). In papers accepted by the MIOC fol lowing peer review, this period of open data within the Zika Fast Track will be mentioned in the final publication. In case of rejection after peer review, authors are free to seek publication elsewhere. Claude Pirmez Editor in chief Adeilton Alves Brandão Hooman Momen Editors Adeilton Alves Brandão Hooman Momen Editors Manuscripts can be submitted via the MIOC website memorias.ioc.fiocruz.br. Dye C, Bartolomeos K, Moorthy V, Kieny MP. Data sharing in public health emergencies: a call to researchers [Internet]. Geneva: World Health Organization; 2016 [updated 2016 February 4]. Available from: http://dx.doi.org/10.2471/BLT.16.170860. Jones KE, Patel NG, Levy MA, Storeygard A, Bal D, Gittleman JL, et al. Global trends in emerging infectious diseases. Nature. 2008; 451: 990-3. 285 285 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 111(5): 285, May 2016 REFERENCES Dye C, Bartolomeos K, Moorthy V, Kieny MP. Data sharing in public health emergencies: a call to researchers [Internet]. Geneva: World Health Organization; 2016 [updated 2016 February 4]. Available from: http://dx.doi.org/10.2471/BLT.16.170860. Jones KE, Patel NG, Levy MA, Storeygard A, Bal D, Gittleman JL, et al. Global trends in emerging infectious diseases. Nature. 2008; 451: 990-3. doi: 10.1590/0074-02760160001 online \| memorias.ioc.fiocruz.br
https://openalex.org/W2076828262	https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.1000589&type=printable	English	null	Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases	PLoS biology	2,011	cc-by	15,025	Vijay Parashar1, Nicolas Mirouze2, David A. Dubnau1,2, Matthew B. Neiditch1* 1 Department of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School, Newark, New Jersey, United States Institute Center, UMDNJ–New Jersey Medical School, Newark, New Jersey, United States of America Abstract Funding: This work was supported by National Institutes of Health (NIH) grants R01AI081736 to MBN and R01GM57720-40 to DAD. 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. Abbreviations: HPt, histidine phosphotransfer; TPR, tetratricopeptide repeat * E-mail: matthew.neiditch@umdnj.edu phosphoryl groups to water in autohydrolysis reactions (Figure 1, right panel; [5,6]). The dephosphorylation of intermediate response regulators causes the direction of phosphoryl flow to reverse (Figure 1, right panel). That is, the intermediate response regulator pulls phosphoryl groups from the HPt, which in turn dephosphorylates the downstream response regulator. The rate of response regulator dephosphorylation is determined by autohydrolysis activity intrinsic to the REC domain and the activity of response regulator-specific phosphatases, such as Rap proteins (Figure 1, right panel; [7,8]). Abstract Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic ‘‘switch’’ residue to an internal position when the b4-a4 loop adopts an active-site proximal conformation. Citation: Parashar V, Mirouze N, Dubnau DA, Neiditch MB (2011) Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases. PLoS Biol 9(2): e1000589. doi:10.1371/journal.pbio.1000589 Academic Editor: Robert B. Bourret, University of North Carolina, United States of America Received August 16, 2010; Accepted December 22, 2010; Published February 8, 2011 Received August 16, 2010; Accepted December 22, 2010; Published February 8, 2011 Copyright: 2011 Parashar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Parashar et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 RapH Structure The RapH-Spo0F X-ray crystal structure shows that RapHA contains an N-terminal 3-helix bundle (a1-a3) (Figure 2A and 2C). RapHB also appears to form an N-terminal 3-helix bundle, but in contrast to Spo0FA the electron density corresponding to residues 27–42 of helix a2 and 43–45 of the a2-a3 loop were not clearly interpretable and these residues were not modeled (Figures S2, S1A, and S1B). Buried at the core of the RapH 3-helix bundle is tryptophan-17. Without exception, Rap proteins have tryptophan at this position, and an N-terminal 3-helix bundle is probably a universal feature of Rap proteins (Figure 4). The RapH-Spo0F structure suggests that the conserved tryptophan is important for the folding of the Rap protein N-terminal 3-helix bundle, and as discussed in detail below the Rap protein 3-helix bundle is centrally important for phosphatase function. which is bound to Mg2+, an important constituent of the Spo0F active site. The RapH-Spo0F crystallographic asymmetric unit contains RapH-Spo0F complexes in two different conformations, reflecting different steps along the Spo0F dephosphorylation pathway. To our knowledge, the RapH-Spo0F X-ray crystal structure represents the first structure of a Rap protein or a Rap protein in complex with its substrate. As expected from their amino acid sequences, Rap proteins have an overall structure that is radically different from the known structures of other bacterial phosphatases. The RapH-Spo0F crystal structure, along with in vitro biochemical and in vivo genetic data, provides mechanistic insight into Rap protein function, specifically the molecular basis of Rap phosphatase activity. Connected to the C-terminus of the RapH 3-helix bundle via a flexible linker and a short helix (a4) is a large right-handed superhelical domain composed of seven pairs of antiparallel a- helices (Figures 2C and S1B). Each pair of antiparallel a-helices is a helix-turn-helix consisting of an N-terminal helix (helix A), a connecting loop, and a C-terminal helix (helix B) (Figure 4). The antiparallel a-helices comprising pairs 1–5 and pair 7 contain tetratricopeptide repeat (TPR) sequences which are 34 amino acid motifs frequently found in tandem (Figure 4). A pair of antiparallel a-helices (a15–a16) separating TPR5 and TPR6 resembles a TPR helix-turn-helix fold in its overall structure but does not encode the TPR motif signature residues [14]. Additionally, the loop (residues 289–296) connecting TPR5 helix B to helix a15 is longer than the other TPR fold-connecting loops, and residues 292–294 are disordered in RapHB (Figures 4 and S1B). Introduction B. subtilis spore development is regulated by a phosphorelay signal transduction pathway that is the prototype for all phosphorelay signal transduction pathways in bacteria (Figure 1; [1]). The B. subtilis sporulation signal transduction pathway is initiated by five histidine kinases—KinA, KinB, KinC, KinD, and KinE—that autophosphorylate in response to unknown environ- mental and physiological cues coincident with nutritional starvation (Figure 1, left panel; [2]). Signaling converges on the centrally important intermediate response regulator Spo0F, which autophosphorylates using phosphohistidines in a sporulation histidine kinase as a phosphoryl donor. Spo0F then phosphorylates the histidine phosphotransfer (HPt) protein Spo0B, which in the final pathway step relays phosphoryl groups to Spo0A. Phosphor- ylated Spo0A then directly activates or represses 121 genes comprising the Spo0A regulon [3]. In total, however, at least 520 B. subtilis genes are significantly upregulated or downregulated as a result of Spo0A phosphorylation [4]. Rap proteins, named after the founding members of the family, which were shown to be response regulator aspartate phosphatases, have been most thoroughly studied in B. subtilis, where they are central to the regulation of diverse developmental processes including sporulation and genetic competence [8,9]. In B. subtilis alone there are 11 homologous Rap proteins encoded on the chromosome, and there are at least four additional Rap proteins encoded on B. subtilis plasmids [10,11]. A subset of Rap proteins, consisting of RapA, RapB, RapE, and RapH, dephosphorylates Spo0F, consequently reducing the level of phosphorylated Spo0A in the cell and inhibiting sporulation (Figure 1, right panel; [8,12,13]). Phosphorelay pathway intermediate response regulator proteins like Spo0F, which consist entirely of a single receiver (REC) domain identical in structure to the N-terminal REC domains of transcription factor response regulators, dephosphorylate by transferring their We have determined the X-ray crystal structure of a Rap phosphatase, RapH, in complex with its target protein, Spo0F, February 2011 \| Volume 9 \| Issue 2 \| e1000589 February 2011 \| Volume 9 \| Issue 2 \| e1000589 1 Rap Phosphatase Structure and Function (Figures 2A, 2B, and S1A). To discuss the RapH-Spo0F structure, we have adopted the following nomenclature. The different RapH-Spo0F complexes in the crystallographic asymmetric unit are named RapHA-Spo0FA and RapHB-Spo0FB (Figures 2A and S1A). RapHA dimerizes around a crystallographic 2-fold symme- try axis forming a (RapHA-Spo0FA)2 complex (Figure 2A). RapH Structure Because the RapH C- terminal domain consists of six bona fide TPR folds and a helix- turn-helix fold that largely resembles a TPR fold and contributes like a TPR fold to the C-terminal domain superhelix, we refer to the entire region (amino acids 77–376) as the TPR domain. Introduction This complex is a heterotetramer comprised of RapHA, RapHA9, Spo0FA, and Spo0FA9, and it buries 4,127 A˚ 2 of surface area at the large RapHA-RapHA9 interface. Similarly, RapHB dimerizes around a crystallographic 2-fold symmetry axis forming (RapHB- Spo0FB)2 (Figure S1A). This heterotetramer is composed of RapHB, RapHB9, Spo0FB, and Spo0FB9, and it buries 4,187 A˚ 2 surface area at the RapHB-RapHB9 interface. Where subscripts and superscripts are omitted from the protein names below, we refer to both of the non-identical copies of RapH or Spo0F in the crystallographic asymmetric unit. Author Summary A phosphorelay signal transduction pathway regulates sporulation in numerous Bacillus species including the genetic model organism, B. subtilis, and the causative agent of anthrax, B. anthracis. Histidine kinases initiate the flow of phosphoryl groups along the phosphorelay pathway, which then shuttles them to a downstream response-regulator transcription factor called Spo0A. Ultimately, sporulation is governed by the cellular con- centration of phosphorylated Spo0A. In numerous Bacillus species, Rap phosphatases function in opposition to the histidine kinases, inhibiting Spo0A activation by dephos- phorylating an intermediate pathway protein called Spo0F. Here we present the structure of a Rap protein, RapH, in complex with Spo0F, as determined by X-ray crystallogra- phy. The RapH–Spo0F structure, along with biochemical and genetic studies, reveals the mechanism of Rap- protein-mediated Spo0F dephosphorylation. We used information gleaned from our structure–function analysis, first, to assign Spo0F phosphatase activity to an unchar- acterized Rap protein, RapJ, on the basis of sequence alone, and, second, to engineer Spo0F phosphatase activity de novo into a non-phosphatase Rap protein, RapF. We found that in addition to dephosphorylating Spo0F, Rap proteins can inhibit the sporulation phosphor- elay by sterically blocking the transfer of phosphoryl groups to and from Spo0F. Ultimately, new classes of drugs might be developed that disrupt the flow of phosphoryl groups along phosphotransfer signaling path- ways by mimicking the antagonistic effects of Rap proteins on response regulators. PLoS Biology \| www.plosbiology.org Overall RapH-Spo0F Crystal Structure To determine the mechanistic basis of Rap phosphatase activity, we crystallized B. subtilis RapH in complex with its target response regulator Spo0F. The single wavelength anomalous diffraction method was used to obtain experimental phases and the RapH- Spo0F structure was ultimately refined to 2.20 A˚ resolution (Table S1). RapH forms dimers in the RapH-Spo0F crystals; this is consistent with gel filtration studies showing that RapH forms stable dimers in solution (Figures 2A, 2B, and S1A). Each RapH protomer in the RapH2 dimer is bound to a monomer of Spo0F, forming a (RapH-Spo0F)2 complex, and each Spo0F monomer coordinates a magnesium ion in its active site, consistent with the fact that magnesium was included in the protein purification, crystallization, and cryoprotection buffers (Figures 2A, 3A, and 3B). Overall, RapHA and RapHB have very similar tertiary structures and align with a root mean-square deviation of 0.9 A˚ for 332 Ca atoms modeled in both structures; however, there are very important conformational differences between them (see Figure S2). We attribute these differences largely to the distinct crystal packing environments of the RapHA and RapHB 3-helix bundles and a4 helices. For example, the RapHA 3-helix bundle and helix a4 interact with the RapHB TPR domain, burying 1,146.2 A˚ 2 surface area (unpublished data). In contrast, residues in the RapHB 3-helix bundle make far fewer crystallographic contacts, burying only 191.2 A˚ 2 surface area. These differences may also explain why residues 27–42 of helix a2, 43–45 of the a2-a3 loop, and 77–85 of the region containing the connecting helix a4 are ordered in RapHA As discussed in detail below, within single RapH-Spo0F crystals there are two RapH-Spo0F complexes in different conformations February 2011 \| Volume 9 \| Issue 2 \| e1000589 2 Rap Phosphatase Structure and Function Figure 1. Rap proteins and Phr peptides regulate B. subtilis sporulation signal transduction. The B. subtilis sporulation kinases (KinA-E) are depicted as membranous proteins for the sake of simplicity; however, KinA and KinE are actually soluble cytoplasmic receptors. Phosphoryl groups are transferred to Spo0F, Spo0B, and ultimately to Spo0A. When the level of Spo0A,P reaches a critical level, sporulation is triggered (left panel). Rap proteins dephosphorylate Spo0F,P causing phosphoryl groups to flow away from Spo0A, inhibiting sporulation (right panel). The Spo0K permease (not pictured) imports Phr peptides into the cytoplasm where they positively regulate sporulation by binding to Rap proteins and inhibiting Spo0F dephosphorylation. Overall RapH-Spo0F Crystal Structure This disorder, particularly in the b4-a4 loop, which, as described below, makes important regulatory contacts with RapH in the RapHA-Spo0FA structure, implies that RapHB and Spo0FB may be more loosely associated than RapHA and Spo0FA. Consistent with this hypothesis, the Spo0FB switch residues are in the non- RapHA causes the Spo0FA switch residue Thr82, located at the C-terminal end of strand b4, to translate approximately 1 A˚ and its sidechain to rotate more than 120 degrees towards the Spo0FA active site (Figure 3A, right panel). The rotation of a conserved threonine or serine at this position in response regulators enables its sidechain hydroxyl oxygen to hydrogen bond with a phosphoryl group oxygen in the active-site phosphoaspartate [18,19]. The RapHA-induced conformational change in Spo0FA Thr82 is accompanied by the rotation of the Spo0FA His101 sidechain to an internalized position where it enters space vacated by Thr82 (Figure 3A, right panel). It is important to note that while Spo0F is not phosphorylated in the RapH-Spo0F structure, RapHA locks non-phosphorylated Spo0FA in the conformation corresponding to a phosphorylation-stabilized receiver domain, which is character- ized by the rotation of both switch residues, as described above, and the movement of the b4-a4 loop towards the active site [20]. Fi ll l li f h R H b d f but disordered in RapHB (Figure S2). As described below, consistent with the conformational differences observed in RapHA and RapHB, Spo0FA and Spo0FB are in different conformations. Overall RapH-Spo0F Crystal Structure The Spo0E family member phosphatases, Spo0E, YisI, and YnzD, dephosphorylate B. subtilis Spo0A [48,49]. REC, Receiver Domain; H, histidine; D, aspartic acid; P, phosphoryl group; Pi, inorganic phosphate. doi:10.1371/journal.pbio.1000589.g001 Figure 1. Rap proteins and Phr peptides regulate B. subtilis sporulation signal transduction. The B. subtilis sporulation kinases (KinA-E) are depicted as membranous proteins for the sake of simplicity; however, KinA and KinE are actually soluble cytoplasmic receptors. Phosphoryl groups are transferred to Spo0F, Spo0B, and ultimately to Spo0A. When the level of Spo0A,P reaches a critical level, sporulation is triggered (left panel). Rap proteins dephosphorylate Spo0F,P causing phosphoryl groups to flow away from Spo0A, inhibiting sporulation (right panel). The Spo0K permease (not pictured) imports Phr peptides into the cytoplasm where they positively regulate sporulation by binding to Rap proteins and inhibiting Spo0F dephosphorylation. The Spo0E family member phosphatases, Spo0E, YisI, and YnzD, dephosphorylate B. subtilis Spo0A [48,49]. REC, Receiver Domain; H, histidine; D, aspartic acid; P, phosphoryl group; Pi, inorganic phosphate. doi:10.1371/journal.pbio.1000589.g001 RapHA causes the Spo0FA switch residue Thr82, located at the C-terminal end of strand b4, to translate approximately 1 A˚ and its sidechain to rotate more than 120 degrees towards the Spo0FA active site (Figure 3A, right panel). The rotation of a conserved threonine or serine at this position in response regulators enables its sidechain hydroxyl oxygen to hydrogen bond with a phosphoryl group oxygen in the active-site phosphoaspartate [18,19]. The RapHA-induced conformational change in Spo0FA Thr82 is accompanied by the rotation of the Spo0FA His101 sidechain to an internalized position where it enters space vacated by Thr82 (Figure 3A, right panel). It is important to note that while Spo0F is not phosphorylated in the RapH-Spo0F structure, RapHA locks non-phosphorylated Spo0FA in the conformation corresponding to a phosphorylation-stabilized receiver domain, which is character- ized by the rotation of both switch residues, as described above, and the movement of the b4-a4 loop towards the active site [20]. Finally, structural alignment of the RapH bound structures of Spo0FA and Spo0FB revealed that they are in different conforma- tions (Figure 3B). The entire b4-a4 loop, all of helix a4, and a portion of the a4-b5 loop are disordered in Spo0FB as evidenced by a lack of interpretable electron density corresponding to these residues. Spo0F Conformational Changes Caused by RapH Divalent metal ions were previously shown to be required for both Rap-mediated Spo0F dephosphorylation as well as Spo0F autodephosphorylation [15,16]. Consistent with these observa- tions, both Spo0FA and Spo0FB are bound to Mg2+ (Figure 3B). Alignment of Spo0FA with the previously determined crystal structure of Spo0F bound to Mn2+ (PDB ID 1PEY) revealed that RapHA induces conformational changes in Spo0FA (Figure 3A, right panel; [17]). More specifically, this alignment showed that RapHA binding causes the entire Spo0FA b4-a4 loop to dramatically shift and flip toward the active site (Figure 3A, right panel). This conformational change is exemplified by Tyr84, positioned in the middle of the Spo0FA b4-a4 loop, which experiences 2.1–5.0 A˚ displacements in its main-chain atoms and 5.4–10.3 A˚ shifts in its side-chain atoms (see Figure 3A, right panel). As described in detail below, the interaction of Spo0FA Tyr84 with a conserved binding cleft in RapHA, formed by RapHA residues Glu45, Asp46, and Leu50, appears to be critically important for RapH-mediated dephos- phorylation of Spo0F, and alanine substituted for Tyr84 was previously shown to render Spo0F resistant to the phosphatase activity of the RapH homolog RapB [16]. Finally, structural alignment of the RapH bound structures of Spo0FA and Spo0FB revealed that they are in different conforma- tions (Figure 3B). The entire b4-a4 loop, all of helix a4, and a portion of the a4-b5 loop are disordered in Spo0FB as evidenced by a lack of interpretable electron density corresponding to these residues. This disorder, particularly in the b4-a4 loop, which, as described below, makes important regulatory contacts with RapH in the RapHA-Spo0FA structure, implies that RapHB and Spo0FB may be more loosely associated than RapHA and Spo0FA. Consistent with this hypothesis, the Spo0FB switch residues are in the non- PLoS Biology \| www.plosbiology.org PLoS Biology \| www.plosbiology.o PLo February 2011 \| Volume 9 \| Issue 2 \| e1000589 3 PLoS Biology \| www.plosbiology.org Figure 2. RapH-Spo0F structure. (A) The (RapHA-Spo0FA)2 heterotetramer comprised of RapHA (blue), Spo0FA (brown), RapHA’ (green), and Spo0FA’ (magenta). (B) Size exclusion chromatography shows that RapH (MWtheor 44.1 kDa) forms RapH2 dimers (MWexper 85.1 kDa) in solution. The peak positions of gel filtration standards are indicted by vertical lines above the absorbance trace. (C) To obtain this view of RapHA, the structure illustrated in panel A was rotated 90u in the direction indicated by the arrow. Spo0F Conformational Changes Caused by RapH The RapHA N-terminal 3-helix bundle (light blue) is connected to the C- terminal TPR domain by a flexible linker (dashed lines) and a short helix (black cylinder). HTH, helix-turn-helix. doi:10.1371/journal.pbio.1000589.g002 Rap Phosphatase Structure and Function Rap Phosphatase Structure and Function Figure 2. RapH-Spo0F structure. (A) The (RapHA-Spo0FA)2 heterotetramer comprised of RapHA (blue), Spo0FA (brown), RapHA’ (green), and Spo0FA’ (magenta). (B) Size exclusion chromatography shows that RapH (MWtheor 44.1 kDa) forms RapH2 dimers (MWexper 85.1 kDa) in solution. The peak positions of gel filtration standards are indicted by vertical lines above the absorbance trace. (C) To obtain this view of RapHA, the structure illustrated in panel A was rotated 90u in the direction indicated by the arrow. The RapHA N-terminal 3-helix bundle (light blue) is connected to the C- terminal TPR domain by a flexible linker (dashed lines) and a short helix (black cylinder). HTH, helix-turn-helix. doi:10.1371/journal.pbio.1000589.g002 The RapH N-terminal 3-helix bundle and C-terminal TPR domain interact with Spo0F; therefore, interfacial residues in both of these domains were targeted for mutagenesis (Figure 5A and 5C). The interaction of the RapHA 3-helix bundle domain and Spo0FA buries a total of 968 A˚ 2 surface area, and the vast majority of RapHA 3-helix bundle residues buried in the RapHA-Spo0FA interface are located in RapHA helix a3 and the a2-a3 loop (Figure 5A). The RapH 3-helix bundle contacts residues in Spo0FA helices a1, a2, and a5, and in the b1-a1, b2-a2, b3-a3, b4-a4, and b5-a5 loops, which surround the Spo0FA active-site pocket (Figures 5A and S3). Initially, we generated RapH proteins containing individual alanine substitutions targeting the following 3-helix bundle residues that we identified as being buried in the Spo0F interface: Glu45, Gln47, Leu50, Leu55, and Phe58 (Figure 5A). Purified RapH-L50A, RapH-L55A, and RapH-F58A were as soluble as wild-type RapH; however, RapH-E45A and RapH-Q47A were largely insoluble. RapH-E45A was not studied phosphorylated conformation. That is, the sidechain of Thr82 is pointing away from the active site, and the His101 side chain is in an external conformation (Figure 3B). Because the Spo0FB b4-a4 loop is disordered and could not be modeled, its conformation could not be directly compared to that of the Spo0FA b4-a4 loop. Functional Analysis of the RapH-Spo0F Interface In Vitro Functional Analysis of the RapH-Spo0F Interface In Vitro To determine the mechanism of RapH phosphatase activity, we systematically explored the functional significance of the RapH- Spo0F interactions observed in the RapH-Spo0F X-ray crystal structure. The phosphatase activity of RapH mutants containing single mutations distributed throughout the RapH-Spo0F interface was first tested in vitro using a biochemical phosphatase assay. Using the in vitro phosphatase assay, we compared the rate of Spo0F dephosphorylation catalyzed by mutant RapH proteins to that of wild-type RapH and no RapH (Spo0F,P autodepho- sphorylation) controls (Figure 5). PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 PLoS Biology \| www.plosbiology.org 4 Figure 3. Spo0F structural alignments. (A, left panel) RapHA (blue surface) in complex with Spo0FA-Mg2+ (yellow cartoon). (A, right panel) Expanded view of the area enclosed by the black rectangle in the left panel showing the structural alignment of Spo0FA-Mg2+ (grey ribbon with yellow sticks) and Spo0F-Mn2+ (grey ribbon with magenta sticks, PDB ID 1PEY). Mg2+ and Mn2+ are depicted as green and orange spheres, respectively. This alignment reveals that RapHA causes Spo0F Thr82 to rotate towards the active site, Spo0F His101 to adopt an internalized conformation, and the Spo0F b4-a4 loop to shift towards the active site as indicated by the white curved arrows. Spo0F Lys104 could hydrogen bond with a phosphoryl group oxygen in the active-site phosphoaspartate (see Figure 9C). The pseudodihedral angle created by the Ca atoms equivalent to the Spo0F Ca atoms in residues 82, 83, 84, and 85 was previously used to quantify conformational changes in the FixJ and CheY receiver domain b4-a4 loops [34,50,51]. A rotation of 51u around this pseudodihedral angle is observed in the RapHA-Spo0FA structure. The side chain of Spo0FA Lys56 Rap Phosphatase Structure and Function Rap Phosphatase Structure and Function Figure 3. Spo0F structural alignments. (A, left panel) RapHA (blue surface) in complex with Spo0FA-Mg2+ (yellow cartoon). (A, right panel) Expanded view of the area enclosed by the black rectangle in the left panel showing the structural alignment of Spo0FA-Mg2+ (grey ribbon with yellow sticks) and Spo0F-Mn2+ (grey ribbon with magenta sticks, PDB ID 1PEY). Mg2+ and Mn2+ are depicted as green and orange spheres, respectively. Functional Analysis of the RapH-Spo0F Interface In Vitro The Mg2+ ion coordinated by Spo0FB Asp11, Asp54, and Lys56 is depicted by an orange sphere. Structural alignments were performed with DaliLite [45]. doi:10.1371/journal.pbio.1000589.g003 mutant RapH protein activity in B. subtilis as a function of Spo0A activation using a luciferase reporter gene under the control of the Spo0A-driven promoter PspoIIG (Figure 6). Since RapH dephos- phorylates Spo0F, resulting in the repression of Spo0A transcrip- tional activity, overexpression of wild-type RapH inhibits the expression of the PspoIIG-driven luciferase reporter (Figure 6A and 6B). Conversely, less active RapH mutants will display elevated levels of luciferase expression from the PspoIIG reporter. further in vitro but was further analyzed in vivo as described below. However, additional effort to obtain highly soluble RapH protein containing an amino acid substitution at Gln47 was warranted because RapH Gln47 inserts into the Spo0F active-site pocket, and we hypothesized that Gln47 plays a particularly important role in RapH function. Therefore, we generated RapH-Q47N and RapH- Q47E which were as soluble as wild-type RapH. Our biochemical analysis showed that RapH-L50A and RapH- F58A displayed reduced phosphatase activity compared to wild- type RapH (Figure 5B). For example, after 30 min 26% of Spo0F,P remained in the RapH-L50A reaction, while only 13% remained in the wild-type control (Figure 5B). Similarly, after 30 min 28% of Spo0F,P remained in the RapH-F58A reaction, while only 3% remained in its wild-type RapH control (Figure 5B). While RapH-L55A displayed a wild-type phenotype, one mutant, RapH-Q47N, had an even greater phosphatase defect than the RapH-L50A and RapH-F58A mutants (Figure 5B). In fact, RapH- Q47N appears to be catalytically dead. That is, the RapH-Q47N reaction is nearly indistinguishable from the no RapH control. We first tested the effects of the RapH mutants described above on Spo0F dephosphorylation in B. subtilis. RapH-L55A, which displays wild-type activity in vitro, showed reduced activity in vivo; however, this appears to be the result of its significantly reduced expression compared to wild-type RapH (Figures 6A and S4). In close agreement with our biochemical studies, RapH-Q47N, RapH-L50A, and RapH-F58A displayed reduced activity in vivo (Figure 6A). Western blotting confirmed that all of these RapH mutants were produced at levels comparable to wild-type RapH in B. subtilis (Figure S4). Functional Analysis of the RapH-Spo0F Interface In Vitro To further extend our analysis of the RapH-Spo0F interaction surface and to identify additional regulatory contacts, we examined a number of additional RapH proteins containing 3- helix bundle mutations lying in the Spo0F interface (Figures 5A, 6A, and S4). While RapH-I51A displayed wild-type RapH function, RapH-E45A and RapH-D46A displayed drastically reduced activity in vivo (Figure 6A). Thus, the RapHA-Spo0FA complex shows that RapH residues Glu45, Asp46, and Leu50 surround the side chain of Spo0F Tyr84, and our functional analysis shows that mutating any of these RapH residues to alanine inhibits RapH activity (Figures 5A, 5B, and 6A). It is also important to note that the identity of the Spo0FA Tyr84 binding pocket residues is highly conserved in all Rap proteins known to dephosphorylate Spo0F and that both RapH Glu45 and the Spo0F Tyr84-containing b4-a4 loop are disordered in RapHB- Spo0FB (Figures 3B and 4). Thus, the observed interaction of RapH with Tyr84 appears to be important for locking Spo0F in the conformation observed in RapHA-Spo0FA and required for wild-type levels of RapH activity in vivo and in vitro. It is important to note that the mutation of RapH Gln47 to asparagine is rather conservative; glutamine and asparagine are uncharged and differ by the presence or absence of a single side- chain carbon atom, respectively. However, shortening the sidechain of RapH Gln47 by the length of one carbon-carbon bond (,1.5 A˚ ) was sufficient to completely eliminate RapH phosphatase activity in vitro (Figure 5B). Importantly, the RapH-Q47E mutant that replaces glutamine-47 with glutamate displays wild-type phosphatase activity (Figure 5B). As discussed in detail below, we hypothesize that the RapH Gln47 side chain orients a water molecule for nucleophilic attack on the Spo0F aspartylphosphate phosphorous atom. Since RapH-Q47E displays wild-type activity, it appears that an 2O2 group can substitute functionally for the 2NH2 group in the Gln47 side chain to coordinate the attacking water. In addition to interacting with the RapH 3-helix bundle, Spo0F interacts extensively with the RapH TPR domain, burying 968 A˚ 2 surface area in RapHA-Spo0FA and 953 A˚ 2 surface area in RapHB-Spo0FB (Figure 5C). While it is possible that Spo0F interactions with the RapH 3-helix bundle are sufficient for RapH phosphatase activity, we hypothesized that the RapH TPR domain contributes important regulatory contacts to the RapH- Spo0F interface as well. Functional Analysis of the RapH-Spo0F Interface In Vitro This alignment reveals that RapHA causes Spo0F Thr82 to rotate towards the active site, Spo0F His101 to adopt an internalized conformation, and the Spo0F b4-a4 loop to shift towards the active site as indicated by the white curved arrows. Spo0F Lys104 could hydrogen bond with a phosphoryl group oxygen in the active-site phosphoaspartate (see Figure 9C). The pseudodihedral angle created by the Ca atoms equivalent to the Spo0F Ca atoms in residues 82, 83, 84, and 85 was previously used to quantify conformational changes in the FixJ and CheY receiver domain b4-a4 loops [34,50,51]. A rotation of 51u around this pseudodihedral angle is observed in the RapHA-Spo0FA structure. The side chain of Spo0FA Lys56 Figure 3. Spo0F structural alignments. (A, left panel) RapHA (blue surface) in complex with Spo0FA-Mg2+ (yellow cartoon). (A, right panel) Expanded view of the area enclosed by the black rectangle in the left panel showing the structural alignment of Spo0FA-Mg2+ (grey ribbon with yellow sticks) and Spo0F-Mn2+ (grey ribbon with magenta sticks, PDB ID 1PEY). Mg2+ and Mn2+ are depicted as green and orange spheres, respectively. This alignment reveals that RapHA causes Spo0F Thr82 to rotate towards the active site, Spo0F His101 to adopt an internalized conformation, and the Spo0F b4-a4 loop to shift towards the active site as indicated by the white curved arrows. Spo0F Lys104 could hydrogen bond with a phosphoryl group oxygen in the active-site phosphoaspartate (see Figure 9C). The pseudodihedral angle created by the Ca atoms equivalent to the Spo0F Ca atoms in residues 82, 83, 84, and 85 was previously used to quantify conformational changes in the FixJ and CheY receiver domain b4-a4 loops [34,50,51]. A rotation of 51u around this pseudodihedral angle is observed in the RapHA-Spo0FA structure. The side chain of Spo0FA Lys56 PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 5 Rap Phosphatase Structure and Function Rap Phosphatase Structure and Function is disordered, thus avoiding a clash with the repositioned b4-a4 loop. (B) A stereoview depicting the structural alignment of Spo0FA (yellow) and Spo0FB (magenta) shows that Spo0FA adopts a ‘‘phosphorylated’’ conformation and Spo0FB adopts a ‘‘non-phosphorylated’’ conformation. The Spo0FB b4-a4 loop, helix a4, and a portion of the a4-b5 loop are disordered. The Mg2+ ion coordinated by Spo0FA Asp11, Asp54, and Lys56 is depicted by a green sphere. Functional Analysis of the RapH-Spo0F Interface In Vitro To begin to test this hypothesis, we generated RapH-D134A, RapH-E137A, and RapH-Y175A and measured the phosphatase activity of these mutants in vitro (Figure 5D). RapH-D134A, RapH-E137A, and RapH-Y175A were significantly impaired for phosphatase function (Figure 5D). After 30 min 13% and 54% of Spo0F,P remained in the RapH- D134A and RapH-Y175A reactions, respectively, while only 3% remained in their wild-type control (Figure 5D). Similarly, after 40 min 15% of Spo0F,P remained in the RapH-E137A reaction while only 7% remained in its wild-type control. Therefore, we conclude that Spo0F interactions with both the RapH N-terminal 3-helix bundle and the C-terminal TPR domain are required for wild-type phosphatase activity in vitro. In agreement with our biochemical results, RapH proteins containing mutations in residues lining the surface of the RapH TPR domain-Spo0F interface—L96A, D134A, E137A, and Y175A—displayed reduced activity in B. subtilis (Figures 5C, 6B, and S4). These results were not surprising because RapH residue Leu96, like Asp134 and Glu137, contributes to the formation of the Spo0F Tyr13 binding pocket, and Spo0F Tyr13 was previously shown to be important for Rap protein-mediated dephosphorylation of Spo0F (Figure 5C; [16]). Furthermore, the RapH-Spo0F crystal structure shows that RapH residue Tyr175 hydrogen bonds to the Spo0F Mg2+-coordinating residue Asp11 and may help stabilize Mg2+ coordination, which is required for dephosphorylation (Figure 5C; [15]). To complete our mutagenic analysis of the RapH-Spo0F interface, we also tested RapH- Q90A, which displayed wild-type activity in vivo (Figure 6B). RapH Gln90 is located in the loop connecting helix a4 to TPR1 and lies on the periphery of the RapH-Spo0F interface (Figure 5C). Including this mutation in our in vivo analysis resulted in complete mutational coverage of the RapH-Spo0F interface. Ultimately, we tested the effects of at least one mutation located in every RapH Rap Phosphatase Structure and Function Figure 4. Rap phosphatase sequence alignment. The amino acid sequences of Bacillus Rap proteins previously demonstrated to dephosphorylate Spo0F in vitro were aligned using the Blosum62 matrix in Geneious Pro (Biomatters Ltd.). The residue numbers indicated above the sequences refer to RapH. The highly conserved tryptophan at the core of the N-terminal 3-helix bundle is colored green. The ConSurf server was used to determine surface residue conservation [46]. The highly conserved residues in the RapH-Spo0F interface are surrounded by black boxes. Alanine substitutions at the positions highlighted by red boxes resulted in the greatest loss of RapH function in vivo. Alanine substitutions at the positions highlighted by blue or green boxes resulted in intermediate or no loss of RapH function in vivo, respectively. Asparagine substitution at RapH residue Gln47, which is highlighted by a black box, resulted in an intermediate loss of RapH function in vivo. Alanine substitutions at the positions marked with red arrowheads resulted in the greatest loss of RapH function in vitro. Alanine substitutions at the positions marked with blue or green arrowheads resulted in intermediate or no loss of RapH function in vitro, respectively. Asparagine or glutamate substitution at RapH residue Gln47, which is marked with a black arrowhead and in bold type, resulted in complete loss-of-function or no loss-of-function in vitro, respectively. RapH secondary structure assignments were made using the PyMOL algorithm [41]. The secondary structure cylinder colors correspond to the coloring scheme used in Figures 2C and S1B. doi:10 1371/journal pbio 1000589 g004 To test this hypothesis, we analyzed the sequence of other Bacillus Rap proteins and determined that B. subtilis RapJ, among other Rap proteins, contains a high degree of sequence similarity in these interfacial residues (Figure 4). In fact, RapJ encodes a residue that is identical to a residue in one of the other known Spo0F Rap phosphatases in 15 of the 18 highly conserved interfacial positions. The least well-conserved RapJ residues in the 18 highly conserved interfacial positions are Leu40, Asn46, and Phe53, corresponding to RapH residues Ile40, Asp46, and Tyr53, respectively (Figure 4). Modeling leucine in place of Ile40 in RapH showed that leucine could substitute for its positional isomer isoleucine without introducing atomic clashes (unpublished data). Furthermore, the RapH-Spo0F structure showed that RapH Asp46 makes only main-chain contacts with Spo0F, so it seemed likely that a Rap phosphatase could tolerate asparagine in this position. RapH Obstructs Phosphotransfer to and from Spo0F RapH Obstructs Phosphotransfer to and from Spo0F Taken together, our X-ray crystallographic, biochemical, and genetic results suggest that the RapH-Spo0F interfaces we identified in the RapH-Spo0F crystal structure are physiologically important. However, we found it curious that RapH mutants displaying a severe loss-of-function phenotype in vitro, such as RapH-Q47N, which is catalytically dead, still inhibited, at least to some degree, the flow of phosphoryl groups along the sporulation phosphorelay in vivo (Figures 5B and 6A). Interestingly, the RapH-Spo0F structure revealed that RapH binds to a Spo0F surface containing numerous residues previously shown to be important for its interaction with KinA and Spo0B (Figure S2; [21]). Therefore, we hypothesized that catalytically dead RapH mutants, and potentially wild-type RapH as well, could bind to Spo0F and obstruct its phosphorylation by the sporulation histidine kinases and phosphotransfer to Spo0B. In contrast with RapJ, the non-phosphatase Rap protein RapD conserves only 6 of the 18 highly conserved interfacial positions. Modeling RapD residues in place of the conserved RapH interfacial residues showed that many of them would clash with Spo0F (unpublished data). For example, RapD contains trypto- phan at residue 50 where RapH contains leucine. RapD also encodes alanine at residue 58 where RapH contains phenylala- nine, and alanine substituted for phenylalanine at this position in RapH resulted in a loss of function in vitro and in vivo. Furthermore, RapD contains threonine at the position equivalent to the RapH catalytic residue Gln47. To test this hypothesis, we mixed Spo0F with the catalytically dead RapH-Q47N mutant and found that it inhibited Spo0F phosphor- ylation by KinA in a phosphotransfer assay when KinA, RapH- Q47N, and Spo0F were present in a molar ratio of 0.006:0.4:1 (Figure 7). When the KinA:RapH-Q47N:Spo0F molar ratio was 0.006:4:1, phosphotransfer from KinA to Spo0F was nearly undetectable even after 1 h (unpublished data). Not surprisingly, RapH-Q47N also blocked phosphotransfer from Spo0F to Spo0B in vitro (unpublished data). Furthermore, when we more drastically disrupted the RapH-Spo0F interaction by incorporating another RapH-Spo0F interface mutation, RapH-F58A, into the RapH- Q47N mutant, we found that the double mutation relaxed the RapH-mediated inhibition of phosphotransfer from KinA to Spo0F and from Spo0F to Spo0B (Figure 7 and unpublished data). Thus, RapH-Q47N can bind Spo0F and restrict KinA and Spo0B access to the Spo0F active site. Sequence-Based Assignment of Rap Phosphatase Activity Sequence-Based Assignment of Rap Phosphatase Activity In the absence of a Rap protein-Spo0F X-ray crystal structure, sequence-based prediction of Rap protein function was not previously possible. Mapping the sequence conservation of B. subtilis RapA, RapB, RapE, and RapH, as well as B. anthracis Rap proteins BXA0205 and BA3790, which have been shown to dephosphorylate B. subtilis Spo0F, onto the structure of RapH revealed 18 highly conserved positions in the RapH-Spo0F interface (Figure 4; [22]). We hypothesized that these residues occupy functionally important positions and that we could predict whether other Rap proteins are Spo0F phosphatases based on their amino acid sequence identity in these positions. Rap Phosphatase Structure and Function Finally, the hydroxyl moiety of RapH Tyr53 is not involved in a hydrogen bond with Spo0F and its aromatic ring appears to be more important to the Spo0F interaction; thus Phe53 in RapJ would be unlikely to disrupt phosphatase function. helix and loop in the Spo0F interface. The results of both the in vivo and in vitro functional analysis are summarized in Figure 4. RapH Obstructs Phosphotransfer to and from Spo0F Finally, while RapH residue Gln47 is absolutely required for RapH to dephosphorylate Spo0F, our in vivo and in vitro results suggest that this residue might not contribute significantly to the RapH-Spo0F binding energy. To test our prediction that RapJ is a Spo0F phosphatase, we overexpressed and purified RapJ and evaluated its ability to dephosphorylate Spo0F in vitro. Indeed, RapJ dephosphorylated Spo0F (Figure 8A). After 30 min only 4% of Spo0F,P remained in the RapJ reaction, while 60% remained in the no RapJ control (Figure 8A). This result is consistent with microarray data showing that RapJ overproduction in B. subtilis affects the expression of 20 Spo0A-regulated operons [23]. Finally, like every other Rap protein that we have tested to date, RapJ is dimeric as determined by gel filtration chromatography (Figure 8B). Thus, insight obtained from the RapH-Spo0F X-ray crystal structure, together with biochemical and genetic analysis of the observed RapH- Spo0F interactions, has enabled us to assign Rap phosphatase function based on sequence alone. PLoS Biology \| www.plosbiology.org Functional Analysis of the RapH-Spo0F Interface In Vivo Functional Analysis of the RapH-Spo0F Interface In Vivo To confirm the physiological relevance of our X-ray crystallo- graphic and biochemical results, we measured wild-type and PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 6 Rap Phosphatase Structure and Function February 2011 \| Volume 9 \| Issue 2 \| e1000589 7 Rap Phosphatase Structure and Function PLoS Biology \| www.plosbiology.org Rap Phosphatase Structure and Function Figure 5. In vitro phosphatase activity of RapH mutants targeting the RapH-Spo0F interface. (A) The RapHA 3-helix bundle (blue) interaction with Spo0FA (brown surface). The phosphatase activity of RapH mutants corresponding to the residues depicted as green sticks was tested. The main-chain nitrogen atom of RapHA residue Asp46 hydrogen bonds with the 2OH group of Spo0FA Tyr84. The surface of Spo0F Tyr84 is colored red. (B) The ability of wild-type and mutant RapH mutants to dephosphorylate Spo0F,P was compared. Above each gel depicting the phosphatase activity of a RapH mutant is a gel containing a wild-type RapH control reaction as well as a no RapH (Spo0F,P autohydrolysis) control reaction. For the purpose of normalization across gels, identical samples (not shown) from the wild-type RapH control reactions were electrophoresed alongside every RapH mutant reaction. The 5-min time point was ultimately used for across-gel normalization. (C) The RapHA TPR domain (rainbow-colored cylinders) interaction with Spo0FA (brown surface). The phosphatase activity of RapH mutants corresponding to the residues depicted as green sticks was determined. The surfaces of Spo0F Asp11 and Spo0F Tyr13 are colored red. (D) The ability of wild-type and mutant RapH mutants to dephosphorylate Spo0F,P was compared as in (B). In order to measure the RapH-E137A phosphatase defect, 20 and 40 min time points were evaluated for RapH-E137A in lieu of 30 and 60 time points. Gels are representative of experiments repeated at least three times for each RapH mutant. doi 10 1371/journal pbio 1000589 g005 doi:10.1371/journal.pbio.1000589.g005 RapH and a catalytically dead RapH mutant that cannot dephosphorylate Spo0F inhibited phosphotransfer from KinA to Spo0B (Figure 7 and unpublished data). Because receiver domains readily adopt the phosphorylated conformation even in the absence of phosphorylation [26,27,28], we hypothesize that RapH could be binding to either phosphorylated or non-phosphorylated Spo0F and inhibiting the sporulation phosphorelay by sterically limiting kinase and phosphotransferase access to the Spo0F active site. Consistent with this hypothesis, RapA was previously shown to interact with phosphorylated and non-phosphorylated Spo0F, albeit the interac- tion with phosphorylated Spo0F appeared to be more stable than the interaction with non-phosphorylated Spo0F [29]. We expect that steric inhibition of Spo0F phosphorylation would have the most significant biological effect when the concentration of Spo0F and KinA are limiting and Rap proteins are in excess. In B. subtilis alone, RapA, RapB, RapE, RapH, and RapJ all dephosphorylate Spo0F and presumably block access to the Spo0F active site. Rap Phosphatase Structure and Function Therefore, it seems possible that the steric effects that Rap proteins have on Spo0F phosphotransfer in addition to their phosphatase activity may be physiologically relevant. Consistent with this hypothesis, overexpressed catalytically dead RapH inhibited phosphotransfer in B. subtilis cells (Figure 6A). non-phosphatase protein RapF, which targets the DNA binding domain of ComA and does not dephosphorylate its REC domain [24,25], contains a glutamine at residue 47 which aligns with the RapH catalytic residue Gln47. Additionally, RapF contains a residue identical to at least one of the other Spo0F Rap phosphatases in all of the highly conserved interfacial residues with only two exceptions (Table S2). More specifically, RapF contains histidine and lysine where RapH contains Leu50 and Ser133, respectively. We predicted that lysine could be tolerated in RapF at the position corresponding to RapH Ser133 without disrupting potential Spo0F phosphatase activity because (1) the RapHA Ser133 side chain 2OH points away from the Spo0FA interface and only the Cb side-chain atom and main-chain atoms are buried in the Spo0FA interface, (2) in RapHB only main-chain atoms belonging to Ser133 are buried in the Spo0FB interface, and (3) modeling lysine at RapH residue 133 revealed that nearly every lysine side-chain rotamer could be tolerated without clashing with Spo0F. However, modeling histidine at RapH Leu50 in the RapH-Spo0F structure showed that every histidine side-chain rotamer clashed with Spo0F or RapH itself (unpublished data). Based on these findings, we hypothesized that this one residue, His50, disables Spo0F phosphatase function in RapF. The RapH-Spo0F structure revealed that the sidechain of RapH Gln47 inserts into the Spo0F active site (Figure 5A). Importantly, without exception, glutamine is conserved at this position in Rap proteins known to dephosphorylate Spo0F (Figure 4), and replacing RapH Gln47 with asparagine was sufficient to completely eliminate phosphatase activity (Figures 5B). We propose that RapH Gln47 orients water for direct in-line hydrolytic attack on the Spo0F phosphoaspartate-54 phosphorous atom (Figure 9D). To test the above hypothesis, we purified RapF-H50L and measured its phosphatase activity in vitro. Indeed, RapF-H50L dephosphorylates Spo0F, while wild-type RapF does not. After 30 min only 16% of Spo0F,P remained in the RapF-H50L reaction while 54% remained in the wild-type RapF control (Figure 8C). Rap Phosphatase Structure and Function These results, along with our structural analysis, reveal the mechanistic basis underlying the inability of wild-type RapF to dephosphorylate Spo0F and confirm that we have identified the biologically important Rap protein surface and many of the structural determinants required for Rap phosphatase activity. Furthermore, this represents an interesting and rare example of rational surface design that generates new target protein specificity and de novo enzymatic function. g Consistent with our proposed model of RapH function and the critical role that RapH Gln47 plays in dephosphorylating the response regulator Spo0F, the CheZ and CheC/CheX/FliY families of response regulator phosphatases insert a conserved glutamine and asparagine, respectively, into their target response regulator active sites where they orient water for nucleophilic attack on the phosphoaspartate (reviewed in [7]). Aligning the Spo0F molecule of RapHA-Spo0FA with the CheY molecules of the CheZ-BeF3-CheY and CheX-BeF3-CheY structures shows that the amide nitrogens in RapHA Gln47, CheZ Gln147, and CheX Asn99 are positioned at nearly identical distances—6.1 A˚ , 5.7 A˚ , and 6.0 A˚ , respectively—from their corresponding receiver domain active-site aspartate (Figure 9A and 9B). Consistent with the hypothesis that RapHB-Spo0FB represents a conformation adopted following RapH-mediated Spo0F dephosphorylation, the amide nitrogen in RapHB Gln47 is located 1.1 A˚ farther away from the Spo0FB active-site aspartate than the RapHA Gln47 amide nitrogen is from the Spo0FA active-site aspartate. PLoS Biology \| www.plosbiology.org Engineering De Novo Rap Phosphatase Activity To rigorously test our understanding of the structural requirements for Rap phosphatase activity and also to confirm that we have identified the biologically important Rap-Spo0F interface, we engineered a non-phosphatase Rap protein to dephosphorylate Spo0F. Sequence alignment revealed that the PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 8 Rap Phosphatase Structure and Function PLoS Biology \| www.plosbiology.org PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 February 2011 \| Volume 9 \| Issue 2 \| e1000589 PLoS Biology \| www.plosbiology.org PLoS Biology \| www.plosbiology.org Mechanism of Rap Activity Our X-ray crystallographic, biochemical, and genetic studies suggest that RapHA-Spo0FA represents a complex formed in B. subtilis when RapH binds to phosphorylated Spo0F. Conversely, RapHB-Spo0FB depicts a conformation adopted immediately following RapH-mediated Spo0F dephosphorylation. The disorder we observed in residues comprising the RapHB-Spo0FB interface suggests that the RapHB-Spo0FB structure represents a destabilized RapH-Spo0F complex. Destabilization of the RapH-Spo0F inter- action following Spo0F dephosphorylation could enable RapH to dissociate from non-phosphorylated Spo0F, freeing RapH to participate in another round of Spo0F dephosphorylation. Furthermore, aligning the Spo0F molecules of the RapHA- Spo0FA and Spo0B-BeF3-Spo0F structures illustrates approxi- PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 10 Rap Phosphatase Structure and Function Figure 6. In vivo activity of RapH mutants targeting the RapH- Spo0F interface. RapH activity was measured as a function of PspoIIG::luc. (A) In vivo analysis of RapH proteins containing mutations in the 3-helix bundle lying in the Spo0F interface as depicted in Figure 5A. (B) In vivo analysis of RapH proteins containing mutations in the TPR domain lying in the Spo0F interface as depicted in Figure 5C. Each curve is representative of at least three independent experiments performed in duplicate. (A and B) Wild-type and mutant rapH overexpression was controlled by the IPTG-inducible promoter Phyperspank. RLU, Relative Luminescence Units. doi:10.1371/journal.pbio.1000589.g006 mately where RapH Gln47 would position water for nucleophilic attack on the phosphoaspartate phosphorous atom (Figure 9C; [19]). More specifically, a water molecule hydrogen bonded to RapH Gln47 Ne2 and lying in a position near those occupied by Spo0B His30 Ce1 and Ne2 would be perfectly positioned for in- line attack on the Spo0F phosphoaspartate phosphorous atom (Figure 9C). We suspect that direct observation of the attacking water molecule in the crystal structure will require crystallizing the RapH-Spo0F-Mg2+ complex in the presence of a non-hydrolyz- able phosphoryl mimic such as beryllium fluoride or vanadate. Consistent with our proposed mechanism of RapH-mediated Spo0F dephosphorylation, there are no data suggesting that Spo0F transfers phosphoryl groups to RapH resulting in the formation of a phosphoenzyme intermediate. We detect no RapH phosphoen- zyme intermediate in our in vitro assays, and it was previously shown that RapB dephosphorylation of Spo0F was not inhibited by the protein tyrosine phosphatase transition-state inhibitor vanadate ([16]; and unpublished data). Conclusion The X-ray crystal structure of the response regulator phospha- tase RapH in complex with one of its cellular targets, Spo0F, combined with extensive biochemical and genetic analysis of RapH mutants, reveals the mechanism of Rap-mediated phos- phatase activity along with many of the structural requirements for Rap phosphatase function. The approach presented here that enabled us to assign Spo0F phosphatase function to RapJ based only on its amino acid sequence could be used to predict the target specificity of non-Spo0F phosphatase Rap proteins following the determination of structures of representative complexes of the Rap proteins bound to their targets. Furthermore, identifying the non- Spo0F phosphatase Rap protein residues important for target recognition will facilitate (1) the identification of Rap proteins whose sequences suggest that they may not recognize previously identified substrates, (2) the identification of new Rap protein targets, and (3) rational engineering of de novo Rap protein target specificity. The process of developing Rap proteins with engineered target specificity will not only provide valuable insight into the principles governing protein-protein interaction but will also provide valuable reagents for studying bacterial signal transduction. Finally, insight gleaned from our structure-function analysis of RapH-Spo0F, along with additional studies of other Rap proteins in complex with their response regulators, could be used to develop new classes of drugs that disrupt the flow of Spo0F was PCR-amplified from B. subtilis strain 168 chromo- somal DNA using Phusion High-Fidelity DNA Polymerase and the oligonucleotide pair Spo0F-BamHI and Spo0F-NotI (Table S3). The PCR product was cloned in the BamHI and NotI restriction sites of pETDuet-1 (GE Healthcare) to yield pETDuetF1. The Spo0F-D54E expression vector was obtained by site-directed mutagenesis of pETDuetF1 using primers D54E_Spo0F_T and D54E_Spo0F_B to give pETD54E (Table S3). Spo0F and Spo0F- D54E were expressed in E. coli strain BL21(DE3) by first growing the cells at 37uC in LB medium containing 100 mM ampicillin to OD600 = 0.5 and then inducing expression with 1 mM IPTG for 16 h at 25uC. All subsequent purification steps were carried out at 4uC. Cells were collected by centrifugation and lysed in buffer D (25 mM Tris-HCl (pH 8.0), 10 mM KCl, 40 mM MgCl2, and 5 mM b-mercaptoethanol (b-ME)) supplemented with 1 mM Pepstatin, 1 mM Leupeptin, 20 mg/ml DNase, and 1 mM PMSF. The supernatant was applied to Glutathione Uniflow Resin equilibrated in buffer D. Protein Production for X-Ray Crystallography Protein Production for X-Ray Crystallography y y g p y RapH was PCR-amplified from B. subtilis strain 168 chromo- somal DNA using Phusion High-Fidelity DNA Polymerase (NEB, USA) and the oligonucleotide pair RapH-BamHI and RapH-NotI (Table S3). The PCR product was cloned in the BamHI and NotI restriction sites of pGEX4T1 (GE Healthcare) to yield pGEXH1. GST-RapH was overexpressed in E. coli strain BL21 by first growing the cells at 37uC in LB medium containing 100 mM ampicillin to OD600 = 0.6 and then inducing expression with 0.1 mM isopropyl b-D thiogalactopyranoside (IPTG) for 16 h at 16uC. All subsequent purification steps were carried out at 4uC. The cells were collected by centrifugation and lysed in buffer A (20 mM Tris-HCl (pH 8.0), 250 mM NaCl, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, 10% glycerol) supplemented with 1 mM Pepstatin, 1 mM Leupeptin, 20 mg/ml DNase, and 1 mM phenylmethanesulfonyl fluoride (PMSF). The lysate supernatant was applied to Glutathione Uniflow Resin (Clontech) equilibrated in buffer A. The resin was then washed and resuspended in buffer A, and thrombin was added at 0.3 mg/ml glutathione bed volume. Overnight incubation at 4uC resulted in complete cleavage of RapH from the GST affinity tag as determined by SDS-PAGE. Following thrombin digestion, RapH (residues 1– 376) contained two heterologous N-terminal residues (Gly-Ser) derived from the thrombin cleavage signal. RapH was eluted with buffer A and diluted 3-fold with buffer B (20 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, and 10% glycerol), passed through a 0.45 mm filter, and loaded onto an anion exchange column (Source 15Q; GE Healthcare) equilibrated in buffer B containing 50 mM KCl. RapH was then eluted in a 50– 1,000 mM KCl gradient of buffer B. Fractions containing RapH were pooled, concentrated by ultrafiltration through a 30 kDa filter, and further purified by gel filtration using a Superdex 200 (GE Healthcare) 16/70 column equilibrated in buffer C (20 mM Tris-HCl (pH 8.0), 150 mM KCl, 5 mM MgCl2, and 5 mM DTT). RapH was concentrated to 14 mg/ml and stored at 280uC. doi:10.1371/journal.pbio.1000589.g007 of this aromatic residue regulates intra- and inter-molecular protein-protein interactions that occur at the a4-b5-a5 surface of receiver domains (reviewed in [35]). Interestingly, the existing data support a T-loop-Y coupling model that is directional [34]. Mechanism of Rap Activity For the purpose of normalization across gels, identical samples (not shown) from the no RapH control reaction were electrophoresed alongside every RapH-containing reaction. The 60-min time point was ultimately used for across-gel normalization. The gels are representative of the results from three independent phospho- transfer experiments. doi:10.1371/journal.pbio.1000589.g007 information along bacterial phosphotransfer signaling pathways by mimicking the antagonistic effects of Rap proteins on response regulators. Figure 7. Rap proteins inhibit phosphotransfer from KinA to Spo0F. RapH-Q47N does not catalyze Spo0F dephosphorylation (see Figure 5B), but it sterically inhibits phosphotransfer from KinA to Spo0F. RapH-Q47N,F58A contains the catalysis-eradicating Q47N active-site mutation as well as the RapH-Spo0F interfacial mutation, F58A. RapH- Q47N,F58A does not dephosphorylate Spo0F or inhibit phosphotransfer from KinA to Spo0F. For the purpose of normalization across gels, identical samples (not shown) from the no RapH control reaction were electrophoresed alongside every RapH-containing reaction. The 60-min time point was ultimately used for across-gel normalization. The gels are representative of the results from three independent phospho- transfer experiments. d l b Protein Production for X-Ray Crystallography That is, a b4-a4 loop oriented toward the active site dictates the internalization of the aromatic switch residue side chain; however, internalization of the aromatic switch residue does not restrict the conformation of the b4-a4 loop. p Aligning Spo0FA with the structure of Spo0F bound to BeF3 and Spo0B showed that the Thr82 switch residues are in similar positions pointing toward the active site in both models (Figure 9C). However, His101 is in an internal position in the RapHA-Spo0FA structure, while it is in an external position in the Spo0B-BeF3-Spo0F structure (Figure 9C). In support of the T- loop-Y coupling model, it appears that the internalization of His101 in RapHA-Spo0FA, likely resulting from RapH-mediated stabilization of the b4-a4 loop in the active-site proximal conformation, is not observed in the Spo0F-BeF3-Spo0B structure. Also consistent with this model, the Spo0FB His101 side chain is free to adopt an external conformation, probably as a result of the disordered Spo0FB b4-a4 loop. Mechanism of Rap Activity While the catalytic mechanism of RapH-mediated Spo0F dephosphorylation appears to be similar to the one employed by CheZ and CheC/CheX/FliY family members, its overall interaction with its target response regulator is different. Along with the catalytic amide-containing sidechain, the CheZ and CheC/CheX/FliY phosphatases insert an acidic amino acid sidechain into the receiver domain active site where it forms a salt bridge with the active site lysine equivalent to Spo0F Lys104 [30,31,32]. Insertion of the acidic residue into the receiver domain active site is required for wild-type CheZ and CheC/CheX/FliY family phosphatase activity. RapH inserts only an amide- containing side chain into the target response regulator active site, and the RapH-Spo0F structure shows that a salt bridge between RapH and Spo0F Lys104 is not required for RapH phosphatase activity. PLoS Biology \| www.plosbiology.org 1 Finally, while the explicit mechanistic basis of Spo0E family phosphatase function is unknown, genetic and biochemical data suggest that an acidic residue may be coordinating the water for hydrolysis rather than an amide-containing side chain [33]. A RapH mutant containing glutamate in place of the catalytic residue, Gln47, could dephosphorylate Spo0F (Figure 5B). To- gether, the structural and functional data presented here suggest that glutamine or glutamate at the position equivalent to RapH Gln47 is a requirement for Rap protein-mediated Spo0F dephosphorylation. The RapH-Spo0F Structure Supports T-loop-Y Coupling In the T-loop-Y model of receiver domain allostery, the conformation of the hydroxyl-containing switch residue is coupled to the conformation of the b4-a4 loop, and the b4-a4 loop gates conformational changes in the aromatic switch residue in b5 [34]. More specifically, when the b4-a4 loop is in an active site proximal conformation, favored when the hydroxyl switch residue rotates toward the active site, e.g. in response to phosphorylation, it causes the internalization of the aromatic switch residue. Internalization PLoS Biology \| www.plosbiology.org PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 11 Rap Phosphatase Structure and Function Figure 7. Rap proteins inhibit phosphotransfer from KinA to Spo0F. RapH-Q47N does not catalyze Spo0F dephosphorylation (see Figure 5B), but it sterically inhibits phosphotransfer from KinA to Spo0F. RapH-Q47N,F58A contains the catalysis-eradicating Q47N active-site mutation as well as the RapH-Spo0F interfacial mutation, F58A. RapH- Q47N,F58A does not dephosphorylate Spo0F or inhibit phosphotransfer from KinA to Spo0F. PLoS Biology \| www.plosbiology.org Conclusion Following a wash with three bed volumes of buffer D, the resin was incubated with thrombin at 0.3 mg/ml glutathione resin and incubated overnight at 4uC. SDS-PAGE analysis confirmed that the GST tag was removed, and Spo0F was eluted with buffer D. Following thrombin digestion, Spo0F PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 PLoS Biology \| www.plosbiology.org 12 Rap Phosphatase Structure and Function Figure 8. Predicting and engineering Rap phosphatase activity. (A) RapJ dephosphorylates Spo0F,P. phosphatase assays repeated four times. (B) Size exclusion chromatography shows that RapJ (MWtheor 44.4 kDa) 92.0 kDa) in solution. The peak positions of gel filtration standards are indicated by vertical lines above the abso dephosphorylates Spo0F,P while wild-type RapF does not. The gels are representative of phosphatase assays repe doi:10.1371/journal.pbio.1000589.g008 PLoS Biology \| www.plosbiology.org 13 February 2011 \| Vo Figure 8. Predicting and engineering Rap phosphatase activity. (A) RapJ dephosphorylates Spo0F,P. The gel is representative of phosphatase assays repeated four times. (B) Size exclusion chromatography shows that RapJ (MWtheor 44.4 kDa) forms RapJ2 dimers (MWexper 92.0 kDa) in solution. The peak positions of gel filtration standards are indicated by vertical lines above the absorbance traces. (C) RapF-H50L dephosphorylates Spo0F,P while wild-type RapF does not. The gels are representative of phosphatase assays repeated three times. doi:10.1371/journal.pbio.1000589.g008 Figure 8. Predicting and engineering Rap phosphatase activity. (A) RapJ dephosphorylates Spo0F,P. The gel is representative phosphatase assays repeated four times. (B) Size exclusion chromatography shows that RapJ (MWtheor 44.4 kDa) forms RapJ2 dimers (MWe 92.0 kDa) in solution. The peak positions of gel filtration standards are indicated by vertical lines above the absorbance traces. (C) RapF-H dephosphorylates Spo0F,P while wild-type RapF does not. The gels are representative of phosphatase assays repeated three times. doi:10.1371/journal.pbio.1000589.g008 Figure 8. Predicting and engineering Rap phosphatase activity. (A) RapJ dephosphorylates Spo0F,P. The gel is representative of phosphatase assays repeated four times. (B) Size exclusion chromatography shows that RapJ (MWtheor 44.4 kDa) forms RapJ2 dimers (MWexper 92.0 kDa) in solution. The peak positions of gel filtration standards are indicated by vertical lines above the absorbance traces. (C) RapF-H50L dephosphorylates Spo0F,P while wild-type RapF does not. The gels are representative of phosphatase assays repeated three times. Conclusion doi:10.1371/journal.pbio.1000589.g008 February 2011 \| Volume 9 \| Issue 2 \| e1000589 PLoS Biology \| www.plosbiology.org 13 Rap Phosphatase Structure and Function p p PLoS Biology \| www.plosbiology.org 14 February 2011 \| PLoS Biology \| www.plosbiology.org 14 Feb PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 14 Rap Phosphatase Structure and Function Figure 9. Structural comparison of response regulator phosphatase and phosphotransferase active sites, and a model of RapH- Spo0F dephosphorylation chemistry. The Spo0FA molecule of RapHA-Spo0FA was aligned with the CheY molecule of CheZ-BeF3-CheY (PDB ID 1KMI) (panel A), the CheY molecule of CheX-BeF3-CheY (PDB ID 3HZH) (panel B), and the Spo0F molecule of Spo0B-BeF3-Spo0F (PDB ID 2FTK) (panel C). Structural alignments were performed using DaliLite [45] and PyMol [41]. (Panel A) The amide nitrogens of RapHA Q47 and CheZ Q147 are similarly positioned relative to their target receiver domain active-site aspartates. The RapHA Q47 and CheZ Q147 amide nitrogens are 5.9 A˚ and 5.7 A˚, respectively, from the sites of phosphoryl group addition in their target receiver domains. (Panel B) The amide nitrogens of RapHA Q47 and CheX N99 are similarly positioned relative to their target receiver domain active-site aspartates. The RapHA Q47 and CheX N99 amide nitrogens are 5.9 A˚ and 6.0 A˚, respectively, from the sites of phosphoryl group addition in their target receiver domains. The water molecule that attacks the CheY phosphoaspartate phosphorous atom in the CheX-BeF3-CheY structure is shown and reveals approximately where the attacking water may be positioned during RapH mediated dephosphorylation of Spo0F. (C) The phosphoryl group mimic beryllium fluoride in the Spo0B-BeF3-Spo0F structure shows where the phosphoryl group would lie in a RapH-Spo0F,P complex. The distances between the BeF3 fluorine atoms in the Spo0B- BeF3-Spo0F structure and the atoms that are positioned to coordinate them in the structures of Spo0F-BeF3-Spo0B and RapHA-Spo0FA are depicted as light blue dashed lines. The distances between the fluorine atoms and the coordinating Spo0FA atoms are in parentheses. The Spo0FA His101 side chain is in an internal position in the RapHA complex, while the Spo0F His101 side chain in the Spo0B-BeF3 complex is not. This is consistent with the active-site proximal and active-site distal conformations of their Spo0F b4-a4 loops (not shown), respectively. Crystallization and Diffraction Data Collection RapH-Spo0F crystals were produced by the vapor diffusion method at 20uC using a 1:1 mixture of protein (7.5 mg/ml RapH and 3.5 mg/ml Spo0F in 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 2.5 mM DTT, 5 mM MgCl2) and well solution (20% [w/v] PEG 3350, 200 mM lithium sulphate, and 100 mM Bis-Tris (pH 6.0)). RapH-Spo0F crystals were cryoprotected in mother liquor solution containing 16.66 mM potassium phosphate buffer (pH 6.0), 17.2 mM MgCl2, and 12.5% glycerol for 20 min. Crystals containing RapH and selenomethionyl Spo0F(D54E) were grown under similar conditions to the native RapH-Spo0F crystals with the exception that the Bis-Tris was at pH 5.8. Crystals containing the selenomethionyl derivatized Spo0F protein were cryoprotected for 2–3 s in their mother liquor solutions containing 10% glycerol, 3.5 mM MgCl2, and 1 mM DTT. Single-wavelength anomolous dispersion (SAD) and native data on nitrogen-cooled crystals were collected at NSLS beamline X29 and processed using the HKL software package [36]. Conclusion (D) A trigonal bipyramidal phosphoryl group and attacking water were modeled into the RapH-Spo0F active site to generate a graphical depiction of the dephosphorylation transition state. Amino acid positions reflect their conformations in RapHA-Spo0FA. Bonds are depicted as black dashed lines. doi:10.1371/journal.pbio.1000589.g009 coordinate and individual B-factor refinement, as well as a TLS model whose initial parameters were guided by the TLS Motion Determination (TLSMD) server [39]. During the final rounds of refinement in PHENIX, the stereochemistry and ADP weights were optimized, i.e. the weights yielding the lowest Rfree value were used for refinement. Water molecules were added only after the RapH and Spo0F models were complete. The vast majority of the modeled water molecules are bound to RapHA or RapHB. The water molecule B-factors are comparable to those of the residues they are bound to. Insufficient electron density was observed for the following residues and they were omitted from the model: RapHA 1–3 and 69–76; RapHB 1–3, 27–45, 67–85, 292– 294; Spo0FA 1–3 and 120–124; and Spo0FB 1–3, 83–98, and 120– 124. Two molecules each of glycerol, sulfate, and Mg2+ were built into clear electron density during the final stages of refinement. Ramachandran statistics were calculated in Molprobity [40]. Molecular graphics were produced with PyMOL [41]. (residues 1–124) contained two heterologous N-terminal residues (Gly-Ser) derived from the thrombin cleavage signal. The protein was then further purified using anion exchange (Source 15Q) and HiTrap Heparin HP (GE Healthcare) columns arranged in series and equilibrated in buffer D. Spo0F eluted in the flow-through was concentrated by ultrafiltration using a 10 kDa cutoff filter, and subjected to gel filtration using a Superdex 200 16/70 column equilibrated with buffer E (20 mM Tris-HCl (pH 8.0), 50 mM KCl, and 5 mM MgCl2). Selenomethionyl Spo0F-D54E was purified in an identical manner with the exception that buffer D contained 120 mM MgCl2. Spo0F and selenomethionyl Spo0F- D54E eluted exclusively as monomers were concentrated to 7.5 mg/ml and stored at 280uC. Protein Production for Phosphatase and Phosphotransfer Assays RapH and RapF mutant expression vectors were generated by site-directed mutagenesis of pGEXH1 and pGEXRapF using the ChangeIT Mutagenesis Kit (USB) (Table S3). The expression vectors contained only the desired mutations, as determined by DNA sequencing. RapH, RapH mutants, RapF, and RapF-H50L were overexpressed and purified as described for the RapH protein used for crystallography with the exception that dialysis was performed rather than gel filtration to exchange the SourceQ column buffers for buffer C containing 10% glycerol. g g y rapJ was amplified from B. subtilis strain 168 genomic DNA using Phusion High-Fidelity DNA Polymerase and the primer pair RapJ-Fwd and RapJ-Rev (Table S3). The PCR product was cloned into the SapI and XhoI sites of pTB146 using the In-Fusion method (Clontech) to give pTB146J [42]. His-Sumo-RapJ was overexpressed and purified using the RapH purification protocol described above with the exceptions that buffer A had 10 mM b- ME instead of 5 mM DTT, and instead of being applied to glutathione resin the lysate supernatant was applied to Ni-NTA agarose (Qiagen) equilibrated in buffer A. The Ni-NTA resin was then washed in buffer A and resuspended in 65 mM Tris-HCl (pH 8.0), 325 mM NaCl, 35 mM KCl, 7 mM MgCl2, 3.5 mM DTT, 10% glycerol, and 0.2% NP-40. Sumo protease was then added at 4 mg/ml Ni-NTA resin and incubated at 25uC for 2 h. RapJ contained no heterologous residues following removal of the N-terminal His-Sumo fusion. RapJ was then eluted, purified by gel filtration (Superdex 200), and concentrated to 75 mg/ml. Aliquots were stored at –80uC. PLoS Biology \| www.plosbiology.org Structure Determination and Refinement The RapH-Spo0F crystal structure was determined by the SAD method using crystals of native RapH bound to selenomethionyl Spo0F(D54E) that are isomorphous to native RapH-Spo0F crystals. PHENIX (AutoSol) was used to locate heavy atom positions, calculate phases, and generate an initial model at 2.70 A˚ resolution [37]. This model was then refined against 2.20 A˚ native data in PHENIX. The final model was generated through iterative cycles of building in COOT [38] and refinement in PHENIX. The RapH and Spo0F models were built de novo into the SAD- phased map. The earliest rounds of refinement in PHENIX employed simulated annealing as well as rigid body, individual atomic coordinate, and individual B-factor refinement. The later rounds of refinement in PHENIX employed individual atomic PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 15 Rap Phosphatase Structure and Function Spo0F was amplified from pGEXF using primers Spo0F-NdeI and Spo0F-XhoI, digested with NdeI and XhoI, and cloned into the pET21b (Novagen) NdeI and XhoI sites to give pET21bF (Table S3). Spo0F containing a C-terminal fusion to hexahistidine was then purified as previously described [21]. Spo0F was amplified from pGEXF using primers Spo0F-NdeI and Spo0F-XhoI, digested with NdeI and XhoI, and cloned into the pET21b (Novagen) NdeI and XhoI sites to give pET21bF (Table S3). Spo0F containing a C-terminal fusion to hexahistidine was then purified as previously described [21]. Construction of B. subtilis PspoIIG::luc Reporter Strains The B. subtilis IS75 rapH-phrH null strain was constructed by first replacing chromosomal rapH-phrH with a tetracycline resistance cassette. The primers RapH1 and RapH2 were used to amplify a 1 kb DNA fragment upstream of rapH in the IS75 chromosome, while primers RapH3 and RapH4 were used to amplify a 1 kb DNA fragment downstream of phrH (Table S3). These PCR products were then ligated to a tetracycline cassette to generate upstream(rapH)-Tet-downstream (phrH). This product was then PCR amplified using the primers RapH1 and RapH4, purified, and transformed into B. subtilis IS75. Double crossover between the chromosome regions homologous to the upstream(rapH) and downstream(phrH) portions of (rapH)-Tet-downstream(phrH) re- places the entire rapH-phrH operon with tetracyline. The DrapH- phrH::tet strain was named BD5031. The rapH-phrH deletion in BD5031 was confirmed by DNA sequencing using primers RapH1seq, RapH2seq, RapHseq5R, Tet_seq1, and Tet_seq2, as well as by Western blotting using anti-RapH rabbit antisera (Table S3 and Figure S4). Structure Determination and Refinement Chromosomal DNA from BD5031 was isolated and transformed into strain PP533, which contains PspoIIG::luc inserted by Campbell recombination at the native spoIIG promoter, to yield strain BD5035 DrapH-phrH::tet, PspoII- G::luc. To generate site-directed mutations in the BD5035 rapH gene, rapH was amplified from wild-type B. subtilis by PCR and inserted into the SalI and SphI sites in pDR111 (a kind gift from D. Rudner, Harvard Medical School). The resulting plasmid, pDRH1, was then mutagenized using a common forward primer (FWD AMP) in combination with the appropriate mutagenic primer using the ChangeIT Mutagenesis Kit (USB) (Table S3). DNA sequencing confirmed that the rapH plasmids were free of mutations other than those introduced by site-directed mutagen- esis. pDRH1-derived plasmids were then transformed into BD5035 DrapH-phrH::tet, PspoIIG::luc, which by double-crossover recombination at the amyE locus yields strains expressing the entire wild-type or mutant rapH locus under the control of the IPTG- inducible hyperspank promoter. KinA was amplified from B. subtilis str. 168 genomic DNA using the primer pair KinA-Fwd and KinA-Rev and cloned into the NdeI and XhoI sites of pET15b using the In-Fusion method to give pET15bK (Table S3). His-KinA was expressed in E. coli strain BL21(DE3)plysS by first growing the cells at 37uC in LB medium supplemented with 100 mg/ml ampicillin and chloramphenicol 17 mg/ml to OD600 = 0.4. His-KinA expression was then induced for 16 h at 16uC with 0.5 mM IPTG. All subsequent protein purification steps were carried out at 4uC. The cells were collected by centrifugation, lysed in buffer F (25 mM Tris-HCl (pH 8.0), 450 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 10% glycerol) supplemented with 1 mM Pepstatin, 1 mM Leupep- tin, 20 mg/ml DNase, and 1 mM PMSF. The supernatant was applied to Ni-NTA agarose equilibrated in buffer F. The column was then washed with buffer F containing 20 mM imidazole. To remove the hexahistidine affinity tag, thrombin was added to 0.3 mg/ml Ni-NTA agarose bed volume in buffer F and incubated overnight. KinA was eluted and passed over clean Ni-NTA resin equilibrated in buffer F to remove any uncleaved His-KinA. KinA was concentrated through a 30 kDa cutoff membrane and stored at –80uC. Luciferase Bioassays The Rap phosphatase assays were performed at 25uC and initiated by adding radiolabeled Spo0F,P prepared as described above to obtain a final reaction containing: 6.5 mM RapH, or 6.5 mM RapJ, or 26 mM RapF, and 6.0 mM radiolabeled Spo0F,P, 24 mM Spo0F, 2.85 mM KinA, 13.25 mM Tris pH (8.0), 50 mM EPPS (pH 8.5), 0.1 mM EDTA, 100 mM KCl, 23 mM MgCl2, 3 mM DTT, 11.6% glycerol 0.04 mM [c-32P] ATP, and 1 mM ATP. Aliquots were removed at the indicated times, mixed with an equal volume of 2X SDS loading buffer, and stored on ice until they were loaded onto 10%–20% polyacrylamide Tris-Tricine-SDS gels. Following PhosphorImager analysis of the gels, Spo0F,P levels were quantified using ImageQuant. Luciferase from Photinus pyralis was employed as a reporter because it is highly unstable in B. subtilis. We detected less than 50% luciferase activity after less than 8 min following the addition of the protein translation inhibitor puromycin to growing bioluminescence reporter strains (unpublished data). Thus, the response dynamics of the assay are such that we are measuring the rate of gene transcription in near real-time rather than the prolonged accumu- lation of a highly stable reporter such as b-galactosidase. The reporter strains were grown in LB medium to OD600<2, centrifuged, and resuspended in fresh Sporulation Medium (DSM) [44] to OD600 = 2. The cultures were then diluted 20-fold in fresh DSM supplemented with 0.25 mM IPTG, and 200 ml were dispensed per well in duplicate in a 96-well black plate (Corning). 10 ml of luciferin was added to each well at a final concentration of 4.7 mM. The cultures were then incubated at 37uC under agitation in a PerkinElmer Envision 2104 Multilabel Reader. The plate lids were heated to 38uC to avoid condensation. Relative Luminescence Unit (RLU) and OD600 were measured at 1.5 min intervals. Spo0F Labeling The Spo0F labeling reaction was performed at 25uC and contained 206 mM Spo0F-His, 20 mM KinA, 0.25 mM [c-32P] ATP, and phosphotransfer buffer (50 mM EPPS (pH 8.5), 20 mM MgCl2, 0.1 mM EDTA, and 5% glycerol). After 1 h the reaction was quenched with 7.5 mM cold ATP. Spo0F and Spo0F,P were obtained at a ratio of 4:1 as determined by native-PAGE electrophoresis followed by Coomassie staining and quantitation with ImageJ [15,43]. In Vitro Phosphotransfer Assay To measure RapH inhibition of KinA phosphotransfer to Spo0F, reactions were set up by preincubating 6.4 mM wild-type or mutant RapH with 0.1 mM KinA and 0.04 mM [c-32P] ATP in phosphotransfer buffer for 2 min at 25uC. Spo0F was then added to each reaction. The final concentration of the reaction components were: 6.5 mM wild-type or mutant RapH, 16.6 mM Spo0F, 0.1 mM KinA, 5.4 mM Tris (pH 8.0), 50 mM EPPS (pH 8.5), 0.4 mM Bis-Tris (pH 7.2), 0.1 mM EDTA, 41.7 mM KCl, 21.4 mM MgCl2, 1.4 mM DTT, 7.9% glycerol, and 0.04 mM [c-32P] ATP. Aliquots were removed at the indicated times. Spo0F,P levels were determined by Tris-Tricine-SDS PAGE analysis and quantified as described above. Supporting Information Figure S1 The (RapHB-Spo0FB)2 heterotetramer. (A) RapHB (blue), Spo0FB (brown), RapHB9 (green), and Spo0FB9 (magenta). RapHB residues 27–45 and 292–294 are disordered and represented by blue and green dashed lines in RapHB and RapHB9, respectively. Spo0FB residues 83–98 are disordered and are represented by brown and magenta dashed lines, respectively. (B) To obtain this view of RapHB, the structure illustrated in panel A was rotated 90u in the direction indicated by the arrow. The RapHB N-terminal 3-helix bundle (light blue) is connected to the C-terminal TPR domain by a flexible linker (black dashed lines) and a short helix (black cylinder). The RapHB disordered residues 27–45 and 292–294 are represented by light blue and grey dashed lines, respectively. Found at: doi:10.1371/journal.pbio.1000589.s001 (1.65 MB TIF) Found at: doi:10.1371/journal.pbio.1000589.s005 (0.07 MB DOC) Table S2 Rap protein amino acid identity in highly conserved positions lying in the RapH-Spo0F interface. RapA, RapB, RapE, RapF, RapH, RapJ, and Rap60 sequences refer to B. subtilis Rap proteins. BXA0205 and BA3790 sequences refer to B. anthracis Rap proteins. Sequences were aligned in Geneious Pro and analyzed using the ConSurf server [46]. Rap60 is included in this table because previous in vivo studies suggest that Rap60 is a phosphatase, and our alignments show that the Rap60 amino acid sequence conserves all of the residues found to be functionally important for Spo0F dephosphorylation [47]. RapF residue His50 disables its phosphatase activity (H shown in bold). Table S2 Rap protein amino acid identity in highly conserved positions lying in the RapH-Spo0F interface. RapA, RapB, RapE, RapF, RapH, RapJ, and Rap60 sequences refer to B. subtilis Rap proteins. BXA0205 and BA3790 sequences refer to B. anthracis Rap proteins. Sequences were aligned in Geneious Pro and analyzed using the ConSurf server [46]. Rap60 is included in this table because previous in vivo studies suggest that Rap60 is a phosphatase, and our alignments show that the Rap60 amino acid sequence conserves all of the residues found to be functionally important for Spo0F dephosphorylation [47]. RapF residue His50 disables its phosphatase activity (H shown in bold). Figure S2 RapHA and RapHB structural alignment. Structural alignment of RapHA (blue) and RapHB (dark pink) illustrates their conformational differences. A portion of the RapH 3-helix bundle (residues 27–45) is disordered in RapHB but ordered in RapHA, and residues in and adjacent to this region make regulatory contacts with Spo0F. Acknowledgments We gratefully acknowledge Robert Sweet and the staff of the National Synchrotron Light Source X29 beamline for assistance with data collection; Frederick Hughson and Melinda Baker for critical review of the manuscript and discussion; and Phil Jeffrey, Stephen Miller, and Arkady Mustaev for advice and discussion. This work was supported by National Institutes of Health (NIH) grants R01AI081736 to M.B.N and R01GM57720-40 to D.A.D. Figure S3 RapH-Spo0F interface. (Bottom panel) Expanded view highlighting the RapH-Spo0F interaction contained within the area enclosed by the black rectangle in the top panel. Spo0F residues 11–18, 21–22, 25, 34–38, 56–60, 83, 84, 104–106, 108, 109, and 112 (magenta) are buried in the RapH (blue) interface. The Spo0F secondary structure elements containing residues buried in the RapH interface are labeled with magenta type. Spo0F residues not buried in the RapH interface are colored yellow. To help illustrate the fact that the RapH-Spo0F interface surrounds the Spo0F active site, the side chain of the Spo0F active- site aspartate-54 is depicted (green sticks). Supporting Information The N-terminal region of RapHA, extending from the 3-helix bundle to TPR1 helix A, is rotated slightly away from the C-terminal TPR domain. The most prominent displacements occur near the C-terminal ends of helices a1 and a3, helix a4, the loop connecting a4 to the N-terminus of TPR1 helix A (a5), and in the residues near the N-terminus of TPR1 helix A. Structural alignments were performed with Dali-Lite [45]. Found at: doi:10.1371/journal.pbio.1000589.s002 (1.55 MB TIF) p p y ( ) Found at: doi:10.1371/journal.pbio.1000589.s006 (0.04 MB DOC) Found at: doi:10.1371/journal.pbio.1000589.s006 (0.04 MB DOC) Table S3 Oligonucleotides. Found at: doi:10.1371/journal.pbio.1000589.s007 (0.07 MB DOC) Author Contributions The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: VP NM DAD MBN. Performed the experiments: VP NM DAD MBN. Analyzed the data: VP NM DAD MBN. Contributed reagents/materials/analysis tools: VP NM DAD MBN. Wrote the paper: VP MBN. p p (g ) Found at: doi:10.1371/journal.pbio.1000589.s003 (2.48 MB TIF) p p g Found at: doi:10.1371/journal.pbio.1000589.s003 (2.48 MB TIF) Rap Phosphatase Structure and Function Rap Phosphatase Structure and Function conjugated antibody. Protein was detected by ECL chemilumi- nescence (Pharmacia). Figure S4 Western blot analysis of RapH expression. B. subtilis whole-cell extracts were analyzed by Western blotting to determine the expression level of wild-type and mutant RapH proteins. Western blotting also confirmed the absence of RapH in strain BD5035 (DrapH-phrH::tet, PspoIIG::luc). Found at: doi:10.1371/journal.pbio.1000589.s004 (0.47 MB TIF) Accession Numbers Atomic coordinates and structure factors for RapH-Spo0F have been deposited in the Protein Data Bank under accession code 3Q15. Table S1 Phasing and refinement statistics. Data for the highest resolution shell are given in parentheses. Rsym~ShSijIi h ð Þ{ SI h ð ÞTjShSiIi h ð Þ, where Ii(h) is the ith measurement of h and ,I(h). is the mean of all measurements of I(h) for reflection h. Rcryst~SjjFoj{jFcjj=SjFoj, calculated with a working set of reflections. Rfree is Rcryst calculated with only the test set (6.2%) of reflections. The protein molecule average B-factors were calculat- ed using values that included both B-residual and B-TLS. FOM, figure of merit. p y 10. Meijer WJ, Wisman GB, Terpstra P, Thorsted PB, Thomas CM, et al. (1998) Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and g p 9. Solomon JM, Lazazzera BA, Grossman AD (1996) Purification and character- ization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev 10: 2014–2024. 6. Lukat GS, Stock AM, Stock JB (1990) Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis. Biochemistry 29: 5436–5442. PLoS Biology \| www.plosbiology.org 8. Perego M, Hanstein C, Welsh KM, Djavakhishvili T, Glaser P, et al. (1994) Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis. Cell 79: 1047–1055. 6. Lukat GS, Stock AM, Stock JB (1990) Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis. Biochemistry 29: 5436–5442. 7. Silversmith RE (2010) Auxiliary phosphatases in two-component signal transduction. Curr Opin Microbiol 13: 177–183. 8. Perego M, Hanstein C, Welsh KM, Djavakhishvili T, Glaser P, et al. (1994) Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis. Cell 79: 1047–1055. 9. Solomon JM, Lazazzera BA, Grossman AD (1996) Purification and character- ization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev 10: 2014–2024. 10. Meijer WJ, Wisman GB, Terpstra P, Thorsted PB, Thomas CM, et al. (1998) Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and Antibody Production and Immunoblotting Anti-RapH antiserum was recovered from rabbits injected with purified RapH protein (Lampire Biological Laboratories). B. subtilis whole-cell lysates were electrophoresed on SDS-PAGE gels and blotted to PVDF membrane. Immunostaining was performed using anti-RapH rabbit antisera followed by anti-rabbit IgG HRP- PLoS Biology \| www.plosbiology.org February 2011 \| Volume 9 \| Issue 2 \| e1000589 16 7. Silversmith RE (2010) Auxiliary phosphatases in two-component signal transduction. Curr Opin Microbiol 13: 177–183. 3. 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Tzeng YL, Hoch JA (1997) Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis. J Mol Biol 272: 200–212. 42. Bendezu FO, Hale CA, Bernhardt TG, de Boer PA (2009) RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. EMBO J 28: 193–204. 22. Bongiorni C, Stoessel R, Shoemaker D, Perego M (2006) Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation. J Bacteriol 188: 487–498. J 43. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics International 11: 36–42. 44. Schaeffer P, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54: 704–711. 23. Rap Phosphatase Structure and Function comparisons with related plasmids from gram-positive bacteria. FEMS Microbiol Rev 21: 337–368. 31. Park SY, Chao X, Gonzalez-Bonet G, Beel BD, Bilwes AM, et al. (2004) Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX. Mol Cell 16: 563–574. 11. Gleave AP, Mountain A, Thomas CM (1990) Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance. J Gen Microbiol 136: 905–912. p 32. Pazy Y, Motaleb MA, Guarnieri MT, Charon NW, Zhao R, et al. (2010) Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate. Proc Natl Acad Sci U S A 107: 1924–1929. J 12. Jiang M, Grau R, Perego M (2000) Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis. J Bacteriol 182: 303–310. inhibitors of Rap phosphatases in Bacillus subtilis. J Bacteriol 18 33. Diaz AR, Stephenson S, Green JM, Levdikov VM, Wilkinson AJ, et al. (2008) Functional role for a conserved aspartate in the Spo0E signature motif involved in the dephosphorylation of the Bacillus subtilis sporulation regulator Spo0A. J Biol Chem 283: 2962–2972. 13. Smits WK, Bongiorni C, Veening JW, Hamoen LW, Kuipers OP, et al. (2007) Temporal separation of distinct differentiation pathways by a dual specificity Rap-Phr system in Bacillus subtilis. Mol Microbiol 65: 103–120. p y 14. Main ER, Xiong Y, Cocco MJ, D’Andrea L, Regan L (2003) Design of stable alpha-helical arrays from an idealized TPR motif. Structure 11: 497–508. 34. Dyer CM, Dahlquist FW (2006) Switched or not? the structure of unpho- sphorylated CheY bound to the N terminus of FliM. J Bacteriol 188: 7354–7363. 15. Zapf JW, Hoch JA, Whiteley JM (1996) A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates. Biochemistry 35: 2926–2933. 35. Gao R, Stock AM (2010) Molecular strategies for phosphorylation-mediated regulation of response regulator activity. Curr Opin Microbiol 13: 160–167. regulation of response regulator activity. Curr Opin Microbiol 13: 16 36. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. In: Charles W, Carter Jr., eds. Methods in enzymology: Academic Press. pp 307–326. 16. Tzeng YL, Feher VA, Cavanagh J, Perego M, Hoch JA (1998) Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB. Biochemistry 37: 16538–16545. 37. Rap Phosphatase Structure and Function Auchtung JM, Lee CA, Grossman AD (2006) Modulation of the ComA- dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides. J Bacteriol 188: 5273–5285. 45. Holm L, Park J (2000) DaliLite workbench for protein structure comparison. Bioinformatics 16: 566–567. p p 24. Core L, Perego M (2003) TPR-mediated interaction of RapC with ComA inhibits response regulator-DNA binding for competence development in Bacillus subtilis. Mol Microbiol 49: 1509–1522. 46. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N (2010) ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucl Acids Res 38: W529–W533. 25. Bongiorni C, Ishikawa S, Stephenson S, Ogasawara N, Perego M (2005) Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems. J Bacteriol 187: 4353–4361. 47. Koetje EJ, Hajdo-Milasinovic A, Kiewiet R, Bron S, Tjalsma H (2003) A plasmid-borne Rap-Phr system of Bacillus subtilis can mediate cell-density controlled production of extracellular proteases. Microbiology 149: 19–28. p y J 26. Simonovic M, Volz K (2001) A distinct meta-active conformation in the 1.1-A resolution structure of wild-type ApoCheY. J Biol Chem 276: 28637–28640. 48. Ohlsen KL, Grimsley JK, Hoch JA (1994) Deactivation of the sporulation transcription factor Spo0A by the Spo0E protein phosphatase. Proc Natl Acad Sci U S A 91: 1756–1760. 27. Volkman BF, Lipson D, Wemmer DE, Kern D (2001) Two-state allosteric behavior in a single-domain signaling protein. Science 291: 2429–2433. 49. Perego M (2001) A new family of aspartyl phosphate phosphatases targeting the sporulation transcription factor Spo0A of Bacillus subtilis. Mol Microbiol 42: 133–143. 28. Feher VA, Cavanagh J (1999) Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F. Nature 400: 289–293. 50. Roche P, Mouawad L, Perahia D, Samama JP, Kahn D (2002) Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101. Protein Sci 11: 2622–2630. 29. Ishikawa S, Core L, Perego M (2002) Biochemical characterization of aspartyl phosphate phosphatase interaction with a phosphorylated response regulator and its inhibition by a pentapeptide. J Biol Chem 277: 20483–20489. 51. Formaneck MS, Ma L, Cui Q (2006) Reconciling the ‘‘old’’ and ‘‘new’’ views of protein allostery: a molecular simulation study of chemotaxis Y protein (CheY). Proteins 63: 846–867. y p p p J 30. References 1. Burbulys D, Trach KA, Hoch JA (1991) Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64: 545–552. 2. Jiang M, Shao W, Perego M, Hoch JA (2000) Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis. Mol Microbiol 38: 535–542. 3. Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, et al. (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50: 1683–1701. 4. Fawcett P, Eichenberger P, Losick R, Youngman P (2000) The transcriptional profile of early to middle sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A 97: 8063–8068. 5. Zapf J, Madhusudan M, Grimshaw CE, Hoch JA, Varughese KI, et al. (1998) A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site. Biochemistry 37: 7725–7732. 5. Zapf J, Madhusudan M, Grimshaw CE, Hoch JA, Varughese KI, et al. (1998) A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site. Biochemistry 37: 7725–7732. PLoS Biology \| www.plosbiology.org 17 February 2011 \| Volume 9 \| Issue 2 \| e1000589 Rap Phosphatase Structure and Function Zhao R, Collins EJ, Bourret RB, Silversmith RE (2002) Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ. Nat Struct Biol 9: 570–575. 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https://openalex.org/W4236726742	https://zenodo.org/record/1535283/files/article.pdf	English	null	9. THE ROLE OF SALTS IN THE PRESERVATION OF LIFE	Harvard University Press eBooks	1,964	public-domain	757	THE R ~ L EOF SALTS IN TIIE PRESERVATION OF LIFE THE R ~ L EOF SALTS IN TIIE PRESERVATION OF LIFE INmy address on " The R6le of Salts in the Preservation of Life," published in No. 381 of SCIENCE,I' made the following statement " Sev- eral authors, Lillie, McClendon and Lyon, have suggested that the fertilized egg is more permeable to salts than the unfertilized egg." Mr. R. Lillie calls my attention to the fact that he never made this suggestion. I feel it my duty not only to express my regrets for my oversight but to add that if my paper had dealt fully with the literature of thc subject Mr. Lillie's ingenious experiments and orig- inal ideas should have occupied a prominent place in it, as those who are familiar with the subject will fully realize. 147A). In this case (Fig. 1)the joints of the rock are shown to be widened by solution in such a manner as to make a siphon spring (s) possible. On the left a normal siphon is shown in which the spring does not flow until the reservoir ABCD is filled to B, that is, until the water begins to flow through the long arm EF of the siphon. On the right of the valley is an inverted siphon. It is perhaps unnecessary to state that although intermit- tent springs are the commonest of all springs the intermittent character seldom depends upon the presence of a siphon. JACQUES LOEB 846 846 SCIENCE IN. S. VOL. XXXIV. NO. 885 of the disappearance of the water in a throat or funnel. HERDMAN CLELAND I?. WILLIAMSTOWN, Mass., November 3, 1911 nature, if it ever occurs, and physiographers have done well (and physicists would do well) to omit it from their text-books. But in a modified form the siphon is probably occasion- ally operative. A figure which, although im- perfect, is more in accord with the structure of limestone strata and the effect of solution upon them is given by de Martonne (" Traite de Geographie Physique," 1909, p. 347, fig. nature, if it ever occurs, and physiographers have done well (and physicists would do well) to omit it from their text-books. But in a modified form the siphon is probably occasion- ally operative. A figure which, although im- perfect, is more in accord with the structure of limestone strata and the effect of solution upon them is given by de Martonne (" Traite de Geographie Physique," 1909, p. 347, fig. of the disappearance of the water in a throat or funnel. HERDMAN CLELAND I?. WILLIAMSTOWN, Mass., November 3, 1911 SCIENTIFIC BOOKS Observations and I;nvestigations made at the Blue Hill Observatory, Massachusetts, U. 8. A., in the Years 1906, 1907 and 1908, under the Direction of A. LAWRENCE ROTCH Annals of the Astronomical Observatory of Harvard College. Vol. LXVIII., Part II., 4to. Cambridge, Mass. 1911. Pp. 99-229, Figs. 15. The work of the Blue Hill Observatory needs no introduction to the readers of SCIENCE. The progress of that unique insti- tution, so important for American meteorol- ogy, has been faithfully recorded in the col- umns of this journal ever since the founda- tion of the observatory in 1884. Meteorol- ogists have long since learned that the Blue Hill volumes of the Annals of the Harvard College Observatory are sure to contain re- sults worthy of careful note and study. Sink Holes.-Sinlr or swallow holes are formed in one of two ways: (1) by the falling in of the roof of a cavern and (2) by the solution and erosion of the rock along joint or fault planes, the latter being by far the com- moner origin. American writers of text-books of geology and physiography usually give but one explanation of the origin of these features and that the first and most unusual. Only two authors, as far as the writer is aware, give both. The popularity of the first explanation is probably due to the fact that the word "sink " implies a sinking in of the surface as well as the disappearance of the water by pour- ing into a funnel. The suggestion is offered that the older ( 2 ) term "swallow" hole be used, since it carries with it only the thought Volume LXVIII., Part II., of these Annals contains the observations made twice daily in 190648; the usual summaries; results from the kite meteorograph and simultaneous rec- ords at the ground 1906-08; data obtained by means of ballons-sondes at Pittsfield, Mass., in 1908; supplementary data for a manned
https://openalex.org/W4211250013	https://www.researchsquare.com/article/rs-146257/latest.pdf	English	null	Prevalence and Factors Predicting Preventable Trauma Induced Mortality	Research Square (Research Square)	2,021	cc-by	4,636	Prevalence and Factors Predicting Preventable Trauma Induced Mortality Abbas Hajian (  abbashajian@ymail.com ) Kashan University of Medical Sciences Faculty of Health https://orcid.org/0000-0002-0298-8302 Abdoulhossein Davoodabadi Kashan University of Medical Sciences Faculty of Health Esmail Abdourrahim Kashi Kashan University of Medical Sciences Faculty of Health Mojtaba Sehat Kashan University of Medical Sciences Faculty of Health Shahrzad Ale Mohammad Kashan University of Medical Sciences Faculty of Health Original research Keywords: death, ISS, preventable, probability of survival, trauma Posted Date: January 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-146257/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Prevalence and Factors Predicting Preventable Trauma Induced Mortality Abbas Hajian (  abbashajian@ymail.com ) Kashan University of Medical Sciences Faculty of Health https://orcid.org/0000-0002-0298-8302 Abdoulhossein Davoodabadi Kashan University of Medical Sciences Faculty of Health Esmail Abdourrahim Kashi Kashan University of Medical Sciences Faculty of Health Mojtaba Sehat Kashan University of Medical Sciences Faculty of Health Shahrzad Ale Mohammad Kashan University of Medical Sciences Faculty of Health Original research Keywords: death, ISS, preventable, probability of survival, trauma Posted Date: January 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-146257/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Prevalence and Factors Predicting Preventable Trauma Induced Mortality Abbas Hajian (  abbashajian@ymail.com ) Kashan University of Medical Sciences Faculty of Health https://orcid.org/0000-0002-0298-8302 Abdoulhossein Davoodabadi Kashan University of Medical Sciences Faculty of Health Esmail Abdourrahim Kashi Kashan University of Medical Sciences Faculty of Health Mojtaba Sehat Kashan University of Medical Sciences Faculty of Health Shahrzad Ale Mohammad Kashan University of Medical Sciences Faculty of Health Original research Keywords: death, ISS, preventable, probability of survival, trauma Posted Date: January 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-146257/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: January 18th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/13 Page 1/13 Abstract Introduction: Trauma is one of the most common causes of morbidity and mortality worldwide. Since the definition of preventable death has been described many studies, like current one, were conducted to evaluate this issue. Materials and Methods: This cohort retrospective study investigated archived medical files of trauma victims from 2017 to 2020 in a referral single-center trauma hospital. Registered demographic data, vital signs, Glasgow coma scale (GCS), timing of trauma and death, executed interventions, type and mechanism of trauma in addition to time errors, clinical mismanagements and missed injuries were extracted. Injury severity score (ISS), revised trauma score (RTS) and probability of survival (Ps) based on TRISS method for each case were calculated. Eventually preventable and non-preventable death groups were compared. Results: Finally from the all 413 trauma victims 246(54.9%) files were enrolled. Victims aged from 18 to 95 years. Of all 189(76.8%) were males. Analysis manifested 135(54.9%) of all deaths were potentially and 2(0.08%) were certainly preventable; while the other 49.1% were non-preventable for expiration(p=0.001). Data showed that from all variables systolic blood pressure (SBP) ≥80mmHg, respiratory rate (RR) ≥20 per minute, GCS ≥8, higher RTS, road traffic accidents and control of external bleeding were contribute to prediction of preventable trauma related mortality. Conclusion: This study implied on frequency of trauma related preventable death was regionally high and associated factors that could be lessen the number of these mortalities including of SBP, RR, GCS, RTS, mechanism of trauma and external bleeding of trauma patients should be paid more attention. Trial registration: Retrospectively registered. Introduction Trauma is globally the third common and the first cause of mortality during first four decades of life.1 It ranks fifth among reasons of disability worldwide.1 In Iran, trauma corresponds for 28,000 and 300,000 annual death and disability.2 Males especially during their second decade of life are highly susceptible for trauma induced death.1 Road-traffic accident is the most common mechanism followed by falling, assault and others.1,2 Data showed that more than 50% of trauma death occurred at the scene of the event or immediately at the trauma bay.4 Early death which is accompanied by first hours of injury forms 30% and late mortality that happens after days to weeks of trauma shares 20% of total deaths.4 Since 50 years ago that the term preventable trauma related death has been described, many studies were conducted to find effective factors potentially preventing and/or at least predicting cause of post traumatic expiration.5 Results were formation of different types of trauma scaling systems matured through the time and containing of variety of patient and/or trauma related variables. As instance injury severity score (ISS), abbreviated injury score (AIS), revised trauma score (RTS) and trauma related injury Page 2/13 Page 2/13 severity score (TRISS) are commonly used methods to score the severity of trauma and probability of survival. There are much data available regarding trauma, death and explained possible preventing factors. Results for the latter are also varied study by study. This diversity is originated from the fact that trauma is multifactorial. Difference in human, society, economy, income, education, civilization, knowledge, equipment and health service characteristics could affect the trauma and outcomes.1,6-8 Despite these differences investigations showed that one level develop of an average trauma hospital care system could lead to decrease in about 15,250 trauma related death annually.9 Considering above, regional evaluation of trauma outcomes and identifying possible factors that help to less trauma induced morbidity and mortality is certain. This study was aimed to evaluate trauma victims to find probable factors paly preventing role from death. Materials And Method This retrospective descriptive cohort study was conducted from the May 2017 to the October 2020. Data was extracted from registered data from trauma victims’ archived medical files. All trauma patients aged 18 years or over who have expired in Medical University of Kashan referral trauma hospital were enrolled. Victims with lacking, unreadable or unregistered data were excluded. Extracted data were included of patients medical file number, event date, gender, age, way of transferring to trauma bay (private or with emergency medical service(EMS) ambulance), timing of trauma (pre-hospital time including of event to EMS arrival plus the EMS to hospital interval, hospital to death time and total event to death interval), trauma type (blunt or penetrating) and mechanism (road-traffic accident, falling, assault, and other), initial vital sign(systolic blood pressure(SBP), heart rate(HR) and respiratory rate(RR)), Glasgow coma scale(GCS), AIS, ISS, radiologic evaluation(FAST and brain CT scan), intervention type (chest and tracheal tube insertion, surgical operation including craniotomy, thoracotomy, laparotomy, and orthopedic procedures), and death reason. Immediate, early, and late death were defined as post traumatic expiration at the scene or at the time of hospital arrival with unsuccessful resuscitation, during first 3 days, and after 3 days from event respectively. Then study errors including time errors, errors associated with clinical management of trauma victims, and also missed injuries were defined for every participant. Time errors were categorized in two major items including of pre-hospital transfer interval, and time from hospital arrival to operate if needed. These major items have divided totally to six parts. Regarding clinical mismanagements we evaluate eleven errors including resuscitation didn’t perform based on last version of ATLS guide, trachea was not intubated in GCS≤8, fluid therapy was inadequate in concurrent hemorrhage, insufficient external bleeding control, poor immobilization of patient, missing early pleural space decompression, chest tube was not inserted in hemothorax, FAST, diagnostic peritoneal lavage(DPL) or exploratory laparotomy was neglected when unstable hemoperitoneum was probable, avoiding emergent thoracotomy if needed, perform abdominal laparotomy in stable case of retroperitoneal hematoma or pure pelvic fracture, and lack of brain CT scanning during first 2 hours of hospitalization in GCS≤13. Missing injuries were considered for major injuries led to death because of loss of true clinical judge or misinterpreting paraclinic results. These included musculoskeletal (rib, hip, Page 3/13 Page 3/13 and femur fracture), abdominal (solid organ laceration), and major vessel injury missing diagnosis. Materials And Method According to TRISS method RTS and probability of survival (Ps) for every victim were calculated. For the latter calculated Ps<0.5 considered as non-preventable(NPD) and greater numbers defined as death which could be preventable(PD). Finally all trauma victims were appointed to PD and NPD group based on Ps score. Parametric variables were addressed by mean and standard deviation. Non parametric factors were presented by numbers and percent. To compare means the independent t-test and the ANOVA were used. Analysis of parametric variables was performed through the chi-squared exam. To examine correlation of variables in two groups of study the multivariable logistic regression test was applied and the ROC curve was customized. Significant level of analysis was considered as the p < 0.05. All statistical analysis was performed under the SPSS version 21 computer program. Results Overall 26,655 trauma medical files were archived. Of these there were 413(1.5%) victims. Through the all trauma victims finally 246 files were eligible to enroll. Males were dominant (189 cases, 76.8%) (p<0.001). The ages were from 18 to 95 years. Considering event to death time totally 27(11%), 138(56.1%), and 81(32.9%) victims died immediately, early, and late respectively (p<0.001). The calculated Ps showed 135 (54.9%) potentially PDs (p=0.001). Finding of registered trauma victims’ characteristics is shown in table 1 according to death probability status. Page 4/13 Table 1 Registered trauma victims’ characteristics Variable Unit Probability status of death P Preventable Non-preventable Gender Male 104(77) * 85(76.6) 0.9 Female 31(23) 26(23.4) Age(year) <55 82(60.7) 68(61.3) 0.9 ≥55 53(39.3) 43(38.7) Transfer type EMS 131(97) 109(98.2) 0.5 Private 4(3) 2(1.8) Injury type Blunt 132(97.8) 106(95.5) 0.04 penetrating 3(2.2) 5(4.5) Mechanism Road-traffic accident 103(76.3) 95(85) 0.001 Falling 26(19.3) 10(9) assault 3(2.2) 2(1.8) others 3(2.2) 4(36) Time interval(min) PHT1 28.2±13.1 31.6±13.2 0.4 HTD2 11502.3±14998 4644.2±9575.4 <0.001 pre-hospital transfer 2 hospital to death n(%) 1 pre-hospital transfer 2 hospital to death n(%) 1 pre-hospital transfer 2 hospital to death n(%) As table 1 shows no statistically significant difference between PD and NPD groups for gender, age, transfer type, and pre-hospital transfer interval was present; while other table content variables including of injury type, mechanism of trauma, and hospital to death interval time were obviously different. Registered clinical findings extracted from medical files prior to tracheal intubation –if performed- in addition to frequency of study errors are shown by table 2. Data analysis in table 2 shows that significantly different clinical features between PD and NPDs were SBP, RR, GCS, and breathing type. Findings consisted of no difference when HR, ISS, and external bleeding were regarded. Calculated RTS and TRISS also had significant difference between groups. Page 5/13 Table 2 Table 2 Comparison of clinical data and frequency of study errors among PD and NPDs. 1 focused assessment sonography for trauma 2 brain CT 3 operating room 4 central nervous system n(%) Results Variable Unit Probability status of death p Preventable Non-preventable SBP1(mmHg) <80 10(7.4)* 59(53.2) <0.001 ≥80 125(92.6) 52(46.8) HR2(per minute) <100 67(49.6) 65(58.6) 0.1 ≥100 68(50.4) 46(41.4) RR3(per minute) <20 66(48.9) 70(63.1) 0.02 ≥20 69(51.1) 41(36.9) GCS4 13-15 42(31.1) 2(1.8) <0.001 9-12 16(11.9) 2(1.8) 6-8 31(23) 5(4.5) 4-5 28(20.7) 15(13.5) 3 18(13.3) 87(78.4) ISS5 <16 20(14.8) 7(6.3) 0.3 ≥16 115(85.2) 104(93.7) Breathing type Normal 57(42.2) 18(16.2) 0.001 Assisted 78(57.8) 93(83.8) External bleeding severity None 68(50.4) 65(58.6) 0.3 Mild 47(34.8) 29(26.1) Moderate 12(8.9) 13(11.7) Severe 8(5.9) 4(3.6) RTS6 5.6±1.3 2.5±1.6 <0.001 TRISS7 0.7±0.3 0.2±0.1 <0.001 Time error 91(67.4) 83(73.9) 0.2 Mismanagement 97(71.9) 78(68.5) 0.5 Missing injury 33(45) 25(22.5) 0.06 clinical data and frequency of study errors among PD and NPDs. 1systolic blood pressure 2heart rate 3respiratory rate 4glasgow coma scale 5injury severity score 6 revised trauma score 7 trauma related ISS n(%) Although time, clinical management, and missing errors of the study generally had no difference in total numbers (p>0.05); there was obvious diversity when insufficient external bleeding control (14.8% vs. 6.3% respectively in PD and NPD) from clinical management errors was regarded(p=0.03). Performed interventions for patients prior to expiration whether invasive or not in addition to registered reason of death are introduced in table 3. As table 3 shows no significant difference was present between PD and NPD about positive FAST exam, time to transfer the patient to the operating room, and frequency of reasons made death. However, more emergent operation was performed among victims with potential PD (p=0.02). Frequent emergent operations for the latter subsequently included of craniotomy and exploratory abdominal laparotomy (both equal to 16.3%), thoracotomy (5.2%), and orthopedic (3.7%). Table 3 Comparison of performed intervention and cause of death among trauma victims Variable Probability status of death p Preventable Non-preventable FAST1 (if positive) 38(28.1) 24(21.6) 0.1 BCT2 102(75.6) 71(64) <0.001 Emergent operation 56(41.5) 33(29.7) 0.02 OR3 transfer time(min) 180.1±152 176.6±182.4 0.5 Cause of death Cardiac arrest 27(20) 20(18) 0.1 CNS4 66(48.9) 67(60.4) Thorcic 7(5.2) 5(4.5) Abdominopelvic 14(10.4) 11(9.9) Non specified 21(15.6) 8(7.2) 1 focused assessment sonography for trauma 2 brain CT 3 operating room 4 central nervous system 1 focused assessment sonography for trauma 2 brain CT 3 operating room 4 central nervous system n(%) *n(%) Independent significant predictive factors for preventable death including of SBP, RR, GCS, road-traffic accident, higher RTS, and adequate external bleeding control are shown in table 4. Results Independent significant predictive factors for preventable death including of SBP, RR, GCS, road-traffic accident, higher RTS, and adequate external bleeding control are shown in table 4. Page 7/13 Table 4 Assessment of predictive factors for preventable trauma induced mortality Factor Odds ratio 95% confident interval p Gender 1 0.5-1.8 0.9 Age<55 years 0.9 0.5-1.6 0.9 SBP≥80 mmHg 14.1 6.7-29.8 <0.001 HR<100 per minute 1.4 0.8-2.3 0.1 RR≥20 per minute 1.7 1-2.9 0.02 GCS≥8 3.3 1-10.3 0.03 Higher RTS 4.9 3.1-7.5 <0.001 Road-traffic accident 1.7 1-3.1 0.04 External bleeding 1.1 0.8-1.5 0.3 Adequate external bleeding control 3.4 1.2-9.7 0.02 The ROC curve statistic pointed to that RTS ≥4.46 was accompanied with 90% sensitivity to predict PD. Similarly RR ≥24 per minute, SBP ≥82 mmHg, and GCS ≥9 were associated with prediction sensitivity of 85, 68, and 60% respectively. The ROC curve statistic pointed to that RTS ≥4.46 was accompanied with 90% sensitivity to predict PD. Similarly RR ≥24 per minute, SBP ≥82 mmHg, and GCS ≥9 were associated with prediction sensitivity of 85, 68, and 60% respectively. Discussion Trauma is accused for 148 death and 2,000 disability per every hour worldwide.6-8 According to the WHO reports trauma would rank third for DALY and low income countries would affect more since 2030.1,10,11 Global distribution of trauma outcome is unclear and neither health systems nor reports optimally cover the latter.3,6-8 Additionally, trauma is a negative outcome of variety of life aspects of human whether at individual level or at society. Therefore, regional health system could discover its noxious points making susceptibility for trauma. Since 50 years ago that trauma induced death prevention has been described many scientific states declared considering contributive factors to lessen mortality from trauma. This study was conducted to extend the burden of the latter issue. Page 8/13 Overall regional rate of trauma induced mortalities in our referral trauma bay was 1.5%. Others claimed 5- 25% of mortality rate.12-14 Recent WHO report regarding trauma PDs noticed that about 20% of all annual trauma mortalities are preventable.10 Statistical analysis of this study after calculating the probability of survival via the TRISS method showed preventable death rate among victims over 18 years was about 55%. Although it seems a great number, identical studies regarded spectrum of this value from 1 to 81%.15,16 For example in Brazil, united states, New Zealand, Britain, and Iran PD frequency was 1,7, 24, 39, and 46% respectively.3 In case of certainly preventable death which in this study was 0.8%, other stated statistic was varied 0.5-4.2%.13,15,17-19 Notwithstanding the goal of trauma health care systems should be decrease PDs to as least as possible. and 46% respectively.3 In case of certainly preventable death which in this study was 0.8%, other stated statistic was varied 0.5-4.2%.13,15,17-19 Notwithstanding the goal of trauma health care systems should be decrease PDs to as least as possible. Although male victims like other studies were dominant (77 vs. 23%),3,6,11,15,17-21 younger ages and being male or female didn’t influence on survival probability in this study. However, opponents considered whether being female or being over 60-65 years could decrease survival rate.9,20,21 Post traumatic GCS ≥8 in this study was in association with better prognosis; namely patients with higher GCS than 7 had 3.3 times more chance to survive in comparison with some who had lower score(CI95%:1-10.3). Discussion Advocates declared that lower GCS was accompanied with either pre-hospital or in- hospital 10 to 13 times higher mortality rate respectively.20,21 The sensitivity for prediction of death in the latter study achieved to 68% for GCS≤5.5.20 Our survey manifested that GCS≥9 was accompanied with 60% sensitivity to predict preventability of trauma related mortality. Another independent predictive factor for PDs in this study was SBP ≥80 mmHg (OR:14.1; CI95%:6.7- 29.8). The systolic blood pressure over 82 mmHg was up to 68% sensitive to predict survival. Other authors have claimed that SBP lesser than 90 and 60 mmHg was contributed to 2.5 and 2.2 times more possibility for post traumatic death.20,21 This study showed that respiratory rate ≥20 per minute prior to every breathing assistance increased the probability of survival 1.7 times(CI95%:1-2.9). Based on our knowledge, no identical study was found to compare for the latter finding. Analysis didn’t clarify power of prediction in case of ISS ≥16. However, some other authors opposed by believe in that the lower the ISS the higher the possibility of survival.20 Implicitly for the latter sensitivity and specificity were introduced 94 and 60% respectively if ISS was less than 9.20 These difference could be due to sample size and study method diversity. They involved every over 13 years old sufferer with both death and survive outcome.20 Other opponents defined ISS ≥27 as a cut point predicting pre- hospital mortality.22 Beside these contrasts, many other authors presented their findings in lined with us.1,3,12,17,22-25 Current study calculated that RTS ≥4.46 could be predictable for survival in trauma patients with 90% sensitivity (OR:4.9; CI95%:3.1-7.5). an identical study revealed that RTS ≥7.69 was respectively 95 and 67% sensitive and specific for predicting survival among trauma subjects.20 Again in the latter study pre- hospital death event was contributed with RTS<7.6 (OR:6; CI95%:2-13.7).20 Hence, calculated RTS is a reliable value to predict prognosis of trauma. We found road-traffic accident as the trauma mechanism was a predicting factor for preventable death (Or:1.7; CI95%:1-3.1). Fortunately, it was also the most common among all type of trauma mechanisms Page 9/13 Page 9/13 (80%) followed by falling (15%). In almost all other studies road-traffic accident was the most prevalent mechanism.1,16,21,25 Regarding study errors, despite equivalence of external bleeding severity among all of the study subjects, insufficient external hemorrhage control was significantly more among PDs. Conclusion Preventable death prevalence was partially high in our region. Investigatory analysis identified a number of factors could independently predict potentially preventable death among post traumatic subjects. These included systolic blood pressure, respiratory rate, Glasgow coma scale, calculated revised trauma score, road-traffic accident, and external bleeding control. Paying attention to these factors could point to survival probability among trauma patients. Discussion Analysis revealed if bleeding control adequately it could promote survival rate 3.4 times more (CI9%:1.2-9.7). Similar studies manifested other type of errors including time errors mostly because of delay to initiate treatment (3- 53%), clinical management errors consisting of inappropriate clinical judge (5-90%), false diagnosis (4- 12%), and ineffective treatment (13%), and errors contributing to neglected injuries (6-40%).12,17,19,23 Undoubtedly regional aforementioned errors are exist irrespective to whether the type or the frequency. Therefore, continuous review of trauma health care system status recommends. In this report, no statistical results implied on that other rest variables including regarded times and type of trauma, heart rate, positive FAST exam, type of emergent operations, and cause of death had neither difference nor enough power to predict survival. Considering limitations of the study, it should be noted that this retrospective study was performed in a single-center referral trauma hospital. Data was extracted from archived medical files of trauma victims through a section of time. Because all subjects were not eligible to enroll, data was limited to medical files registered either complete or readable. In this report, no statistical results implied on that other rest variables including regarded times and type of trauma, heart rate, positive FAST exam, type of emergent operations, and cause of death had neither difference nor enough power to predict survival. Considering limitations of the study, it should be noted that this retrospective study was performed in a single-center referral trauma hospital. Data was extracted from archived medical files of trauma victims through a section of time. Because all subjects were not eligible to enroll, data was limited to medical files registered either complete or readable. List Of Abbreviations AIS: Abbreviated Injury Score; ANOVA: Analysis of Variance; ATLS: Advanced Trauma Life Support; BCT: Brain Computed Tomography; CNS: Central Nervous System; EMS: Emergency Medical Service; FAST: Focused Assessment Sonography for Trauma; GCS: Glasgow Coma Scale; HR: Heart Rate; HTD: Hospital to Death; ISS: Injury Severity Score; NPD: Non-Preventable Death; PD: Preventable Death; PHT: Pre- Hospital Transfer; Ps: Probability of Survival; RTS: Revised Trauma Score; RR: Respiratory Rate; SBP: Systolic Blood Pressure; TRISS: Trauma Related Injury Severity Score; WHO: World Health Organization. SS: Trauma Related Injury Severity Score; WHO: World Health Organization. Funding This study was conducted under order and supervision of Kashan University of Medical Sciences and all advantages referred back to this university. Availability of data and material The data used to support findings of this study is available in medical file archive unit of Beheshti Hospital, Kashan; Iran. Authors' contributions Abbas Hajian: study design, data collection, data Analysis, interpret results, drafting article Abdoulhodein Davoodabadi: study design, supervision, interpret results Esmail Abdourrahim Kashi: study design, supervision, interpret results Mojtaba Sehat: statistical advisement Shahrzad Ale Mohammad: data collection Shahrzad Ale Mohammad: data collection Consent for publication Not applicable Acknowledgements We present our great thanks to departments of emergency medicine, general surgery, neurosurgery, and orthopedy and also to Trauma Research Center of the University of Medical Sciences, Kashan, Iran. We also respect unlimitedly to medical files archive unit of Beheshti Hospital and also EMS institute of Kashan facilitating data availability. Ethics approval and consent to participate This study was performed under supervision of University of Medical Sciences and ethics committee has approved study design by registering code IR.KAUMS.MEDNT.REC.1399.088. Page 10/13 Competing interests The authors declare that they have no competing interests. References 1. Soreide K. Epidemiology of major trauma. The British journal of surgery. 2009;96(7):697-8. 2. Montazeri A. Road-traffic-related mortality in Iran: a descriptive study. Public Health. 1. Soreide K. Epidemiology of major trauma. The British journal of surgery. 2009;96(7):697-8. 1. Soreide K. Epidemiology of major trauma. The British journal of surgery. 2009;96(7):697-8. 2. Montazeri A. Road-traffic-related mortality in Iran: a descriptive study. Public Health. 2004;118(2):110-3. 3. Gholipour C, Samadi Rad B, Vahdati SS, et al. Assessment of Causes of Preventable Deaths in Pre- hospital SettingsAssessment of Causes of Preventable Deaths in Pre-hospital Settings. erciyes medical journal .2016;(38): 66-9. 3. Gholipour C, Samadi Rad B, Vahdati SS, et al. Assessment of Causes of Preventable Deaths in Pre- hospital SettingsAssessment of Causes of Preventable Deaths in Pre-hospital Settings. erciyes medical journal .2016;(38): 66-9. Page 11/13 4. Tien H, Chu PT, Brenneman F. Causes of death following multiple trauma. Current Orthopaedics. 2004;18(4):304-10. 5. Teixeira PG, Inaba K, Hadjizacharia P, et al. Preventable or potentially preventable mortality at a mature trauma center. J Trauma. 2007; 63(6): 1338-46. 6. Gad MA, Saber A, Farrag S, et al. Incidence, patterns, and factors predicting mortality of abdominal injuries in trauma patients. N Am J Med Sci. 2012;4(3):129-34. 7. Roudsari BS, Sharzei K, Zargar M. Sex and age distribution in transport-related injuries in Tehran. Accid Anal Prev. 2004; 36: 391-8. 8. 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https://openalex.org/W3159097135	https://repository.library.noaa.gov/view/noaa/45160/noaa_45160_DS1.pdf	English	null	Snowfall and snow accumulation processes during the MOSAiC winter and spring season	null	2,021	cc-by	28,566	Snowfall and snow accumulation during the MOSAiC winter and spring seasons David N. Wagner1,2, Matthew D. Shupe3,4, Christopher Cox3,4, Ola G. Persson3,4, Taneil Uttal3, Markus M. Frey5, Amélie Kirchgaessner5, Martin Schneebeli1, Matthias Jaggi1, Amy R. Macfarlane1, Polona Itkin6,7, Stefanie Arndt8, Stefan Hendricks8, Daniela Krampe8, Marcel Nicolaus8, Robert Ricker8, Julia Regnery8, Nikolai Kolabutin9, Egor Shimanshuck9, Marc Oggier10, Ian Raphael11, Julienne Stroeve12,13,14, and Michael Lehning1,2 3NOAA Physical Science Laboratory, Boulder, CO, USA 4Cooperative Institute for the Research in Environmental Sciences, University of Colorado Boulder, Boulder, CO, USA 5British Antarctic Survey – Natural Environment Research Council, Cambridge, UK 6UiT – The Arctic University of Norway, Tromsø, Norway 7Cooperative Institute for Research in the Atmosphere, Colorado State University, Fort Collins, CO, USA 8Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung, Bremerhaven, Germany 9Arctic and Antarctic Research Institute, St. Petersburg, Russia 10International Arctic Research Center, University of Alaska Fairbanks, Fairbanks, AK, USA 11Thayer School of Engineering, Dartmouth College, Hanover, NH, USA 12Centre for Earth Observation Science, University of Manitoba, Winnipeg, Canada 13Earth Sciences Department, University College London, London, UK 14National Snow and Ice Data Center, University of Colorado, Boulder, CO, USA 13Earth Sciences Department, University College London, London, UK 14National Snow and Ice Data Center, University of Colorado, Boulder, CO, USA Correspondence: David N. Wagner (david.wagner@slf.ch) Received: 23 April 2021 – Discussion started: 26 April 2021 Revised: 5 February 2022 – Accepted: 20 May 2022 – Published: 17 June 2022 Received: 23 April 2021 – Discussion started: 26 April 2021 Revised: 5 February 2022 – Accepted: 20 May 2022 – Published: 17 June 2022 tical precipitation sensor, and installed on a railing on the top deck of research vessel Polarstern, was least affected by blowing snow and showed good agreements with SWE re- trievals along the transect. On the contrary, the OTT Pluvio2 pluviometer and the OTT Parsivel2 laser disdrometer were largely affected by wind and blowing snow, leading to too high measured precipitation rates. These are largely reduced when eliminating drifting snow periods in the comparison. ERA5 reveals good timing of the snowfall events and good agreement with ground measurements with an overestima- tion tendency. Retrieved snowfall from the ship-based Ka- band ARM zenith radar shows good agreements with SWE of the snow cover and differences comparable to those of ERA5. Based on the results, we suggest the Ka-band radar- derived snowfall as an upper limit and the present weather detector on RV Polarstern as a lower limit of a cumulative snowfall range. The Cryosphere, 16, 2373–2402, 2022 https://doi.org/10.5194/tc-16-2373-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. The Cryosphere, 16, 2373–2402, 2022 https://doi.org/10.5194/tc-16-2373-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Snowfall and snow accumulation during the MOSAiC winter and spring seasons Based on these ﬁndings, we suggest a cumu- Abstract. Data from the Multidisciplinary drifting Observa- tory for the Study of Arctic Climate (MOSAiC) expedition allowed us to investigate the temporal dynamics of snowfall, snow accumulation and erosion in great detail for almost the whole accumulation season (November 2019 to May 2020). We computed cumulative snow water equivalent (SWE) over the sea ice based on snow depth and density retrievals from a SnowMicroPen and approximately weekly measured snow depths along ﬁxed transect paths. We used the derived SWE from the snow cover to compare with precipitation sensors installed during MOSAiC. The data were also compared with ERA5 reanalysis snowfall rates for the drift track. We found an accumulated snow mass of 38 mm SWE between the end of October 2019 and end of April 2020. The initial SWE over ﬁrst-year ice relative to second-year ice increased from 50 % to 90 % by end of the investigation period. Further, we found that the Vaisala Present Weather Detector 22, an op- 1 Introduction Snow cover on sea ice has many signiﬁcant effects on the ice mass balance and general heat exchange processes be- tween the ocean and the atmosphere (Wever et al., 2020). As snow will cover almost all Arctic sea ice by the beginning of the melt season and with albedo values close to 0.9, a large amount of the incoming solar radiation is reﬂected rather than absorbed into the snowpack. Due to its potentially very high insulating capacity, snow acts as an inhibitor for heat transfer between ocean, sea ice and atmosphere (Holtsmark, 1955; Maykut and Untersteiner, 1971; Sturm et al., 2002b). Depending on the season, accumulation, density and thermal conductivity of the snow, the sea ice growth and melt vary temporally and spatially. For instance, the underlying sea ice might undergo faster (slower) growth in autumn when the snow on top is relatively thin (relatively thick). On the other hand, a thicker (thinner) snow cover might lead to delayed (earlier) sea ice melt in the melt season. Consequently, the small-scale snow distribution – which we deﬁne in the fol- lowing as decimetre- to hectometre-scale snow cover area – affects the ice mass balance on the same scales, as large amounts of snow are accumulated along ridges or dunes, while large areas of level ice experience little snow accumu- lation (Lange and Eicken, 1991; Sturm et al., 1998b; Iacozza and Barber, 1999; Leonard and Maksym, 2011; Trujillo et al., 2016). The snow that has fallen to the ground as fresh precip- itation often gets re-distributed as blowing or drifting snow due to the relatively high average horizontal wind velocities during the Arctic winter. The high snow transport rates are also a result of the relatively low aerodynamic roughness length of sea ice, where z0 is typically lower for ﬁrst-year ice (FYI) than for second- or multi-year ice (SYI or MYI) (Weiss et al., 2011). In addition, large parts of the snow mass can be expected to get blown into leads or undergo sublimation (Déry and Yau, 2002; Déry and Tremblay, 2004; Leonard and Maksym, 2011; Liston et al., 2020), which has recently been shown to be underestimated by current models (Sig- mund et al., 2022). Besides thermodynamic ice growth at its bottom, snow can directly contribute to ice formation on top of the sea ice as snow ice. 1 Introduction Snow-ice formation occurs when snow ﬁrst transforms into slush due to surface ﬂooding of saltwater or direct brine expulsion through thin ice followed by subsequent refreezing (Sturm et al., 1998a; Toyota et al., 2011; Jutras et al., 2016; Sturm and Massom, 2016). The rel- ative mass contribution of snow ice towards sea ice by the dM dt = P ±Es ±Ee −ED −R +B −I −∇·D −L−S, (1) dM dt = P ±Es ±Ee −ED −R +B −I −∇·D −L−S, (1) where dM dt is the rate of change of the mass of the snow cover over the sea ice at one point in kg m−2, which is equivalent to snow water equivalent (SWE) per time unit; P is the snowfall rate; Es is the sublimation rate and Ee is the evaporation rate of the snow cover; ED is the drifting and blowing snow parti- cle sublimation rate; R is runoff; B is brine mass inﬁltration rate into the snow cover from below; I is the snow-ice forma- tion rate; D is the horizontal snow transport rate of blowing and drifting snow; L is the rate of the snow mass blown into leads; and S is the mass of snow pushed or dug under the ice due to sea ice dynamics. Considering a larger area (i.e. above hectometre scale up to a scale of the whole Arctic ice pack), all terms must be considered, while some terms may become zero when considering the equation at one point; e.g. where no open lead is existent at a point, L becomes zero. The ﬁrst and largest source term in Eq. (1) is, depend- ing on the considered area, P . The central Arctic has a dry climate, and depending on location, a yearly average snow- fall of approximately 100 to 350 mm can be expected in this area (Serreze and Hurst, 2000; Chung et al., 2011; McIlhat- tan et al., 2020; Webster et al., 2021). During polar night, the mass decrease in the snow cover by sublimation (Es) and evaporation (Ee) as well as the mass increase due to depo- sition (re-sublimation) and condensation can probably be as- sumed negligible (Liston et al., 2020; Webster et al., 2021). However, sublimation and evaporation terms become larger by the beginning of summer in May and stay relatively large until September. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring season 2374 lative snowfall of 72 to 107 mm and a precipitation mass loss of the snow cover due to erosion and sublimation as between 47 % and 68 %, for the time period between 31 October 2019 and 26 April 2020. Extending this period beyond available snow cover measurements, we suggest a cumulative snow- fall of 98–114 mm. end of the accumulation season depends strongly on loca- tion, with an approximated average of 6 %–10 % for Arctic sea ice and with estimated local peaks of up to 80 % (Merk- ouriadi et al., 2020). As a further term in the snow mass bal- ance, Webster et al. (2021) mention sea ice dynamics. How- ever, we can only imagine that the dynamics, such as ridge formation, can lead to a snow mass decrease when the snow is pushed below the ice or into the water. Considering all effects as snow mass source and mass sink, we can write the mass balance equation of snow over sea ice, modiﬁed from the general mass balance description of snow (e.g. King et al., 2008), as Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2375 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons tation, as they ﬁnd that all saltated mass ﬂux blown towards an open lead vanishes there. They also emphasize that al- though the mass ﬂux within the saltation layer in their model is lower than in the blowing snow column above, the higher frequency of saltation (about 50 % on 23 d in October 2007) compared against blowing snow frequency makes the mass loss due to saltation an important term. However, only a very limited number of studies were carried out that investigate this speciﬁc problem, and the saltation layer with relative large snow mass ﬂux has not been considered in great de- tail so far. Hence, the existing estimates go along with large uncertainties. 179 mm of precipitation was found for the same time period, the blowing snow sublimation mass sink was 6 % of the total cumulative snowfall. During the Canadian Arctic Shelf Ex- change Study (CASES) overwintering campaign, Savelyev et al. (2006) found a relative humidity of over 95 % most of the time and concluded on very low blowing snow sublima- tion rates. Liston et al. (2020); however, suggested a signiﬁ- cant mass reduction of the snow cover by 20 % due to blow- ing snow sublimation based on modelling results. Within the melt season in summer, R can be expected to be the largest mass sink (Webster et al., 2021). Considering brine inﬁltra- tion, B, which is often accompanied by the expression of frost ﬂowers, Nghiem et al. (1997) found a 4 mm slush layer forming beneath frost ﬂowers in indoor experiments. How- ever, when snow falls onto frost ﬂowers or a layer of brine, it gets soaked by brine, transformed into slush and, when cold enough, is often transformed quickly into snow ice. Hence, we assume that brine only can be a positive mass term as long as a certain ambient temperature is not undercut, where the snow begins to transform into snow ice. Regarding mass de- crease due to snow-ice formation I, Merkouriadi et al. (2020) give an average value of less than 0.05 m snow-ice thickness for the central Arctic. It is hard to estimate a precipitated amount of SWE from recalculation from 0.05 m, as the pro- cess of snow-ice formation is complex (Jutras et al., 2016). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons However, when we assume 0.05 m as snow height with an average fresh snow density of 100 kg m−3, we expect around 5 mm of SWE decrease, which would mean only about 3 %, relative to the measured 179 mm during SHEBA. The snow- ice formation rate is expected to be highest in the months of September, October and November (Webster et al., 2021). On one hand, the largest sink term in Eq. (1) is the erosion outside the melting season, represented as D in the mass bal- ance equation, which may make up to 50 % SWE decrease over sea ice of the total precipitated snow mass (Leonard and Maksym, 2011). On the other hand, locally, the eroded mass may deposit at the windward and leeward sides of ridges, on level areas such as dunes, and ﬁll frozen leads – hence lo- cally very often exceeding the precipitated mass. The amount of drifting and blowing snow that is lost and gets melted in open leads L varies strongly depending on location, consid- ered area, ice dynamics and lead properties such as width and orientation relative to the wind. However, the total vanished mass ﬂux from the column of blowing and drifting snow can be locally up to 100 % (Déry and Tremblay, 2004; Leonard and Maksym, 2011). Déry and Tremblay (2004) computed an annual blowing snow loss of 20 mm SWE for a 10 km fetch using the blowing snow model PIEKTUK with a mean lead width of 100 m, an open water fraction of 1 % and a typical lead trap efﬁciency of 80 %. However, in this model setup, saltation mass ﬂux is not considered. Leonard et al. (2008) and Leonard and Maksym (2011) were doing compu- tations with the same model base for Antarctic sea ice, but considering saltation mass ﬂux in addition. They emphasize As we will only consider the accumulation time period, we can omit runoff R from Eq. (1). Further, snow cover evapo- ration and sublimation terms are negligible during this time; hence we can neglect the terms Es and Ee. Then we write the simpliﬁed mass balance equation for winter and early spring as dM dt = P ± D −L −ED −I −S. 1 Introduction Reliable values from literature are hard to determine, but the snow cover decrease as a combination of Es and D (as snow particles that get lifted into suspension) may be up to 50 % (Essery et al., 1999). To estimate the blowing snow sublimation ED, Chung et al. (2011) applied the sophisticated PIEKTUK blowing snow model (used often and in various forms; e.g. Déry et al., 1998; Déry and Yau, 1999, 2002; Déry and Tremblay, 2004; Leonard et al., 2008; Leonard and Maksym, 2011) for a SHEBA (Surface Heat Budget of the Arctic Ocean) ﬁeld experiment (Uttal et al., 2002) site, drifting between 74◦and 81◦N. They computed 12 mm of SWE blowing snow sublimation over a time period of 324 d between November 1997 and September 1998. As https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2.1 Ice conditions and central observatory According to Krumpen et al. (2020), the ﬂoe where RV Po- larstern moored had a size of approximately 2.8 km × 3.8 km and was a loose assembly of pack ice little less than a year old that had survived the 2019 summer melt. Figure 2 shows a map of the ice and snow surface structures and installations by 5 March 2020 of the MOSAiC central observatory. Note that the shown elevation range is only approximate as prob- lems occurred with the inertial navigation system of the laser scanner. This led to tilts, and the single swaths within the map have staggered heights. At present, these uncertainties could not yet be corrected. However, very bright areas in- dicate ridges of around 2 m height, with locally 3 m height and more. The central observatory (all installations in the close vicinity of Polarstern) was distinguished from the dis- tributed network, which consisted of remote autonomous sta- tions at least a few kilometres away from the CO. The de- tailed concept of the central observatory and distributed net- work is explained in Nicolaus et al. (2021b). The core of the ﬂoe consisted mostly of deformed second-year ice (SYI), and the ice surrounding this core mainly consisted of frozen melt ponds (remnant SYI) and partially ﬁrst-year ice (FYI). When the ship moored, the heading of RV Polarstern was about 220◦in October 2019. Signiﬁcant changes in ice conditions occurred the ﬁrst time around 16 November 2019, when a storm led to strong ice deformations in and around the CO. Another signiﬁcant ice deformation event occurred around 11–12 March 2020 and periodically until 7 May 2020. Over time, the ﬂoe rotated anticlockwise and reached a minimum heading of 75◦on 21 March 2020. This paper investigates the snow accumulation period from October 2019 to May 2020, where precipitation is solid, and no signiﬁcant snowmelt was observed. For this period, the intentions in this paper are as follows. – Compute reliable values for SWE evolution along the ﬁxed transect paths that include surface heterogeneities. – Use the computed SWE for periods where no drifting snow occurred to compare with snowfall rates from pre- cipitation gauges installed during the MOSAiC expedi- tion and make a best estimate of total precipitation dur- ing the investigation period. – Evaluate an existing radar reﬂectivity–snowfall (Ze–S) relationship (Matrosov, 2007; Matrosov et al., 2008) for the ship-based Ka-band ARM zenith radar (KAZR). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons – Investigate average snow mass balance and discrepan- cies of computed snowfall rates and SWE on the sea ice and shed light on the processes described in Eq. (2), such as total eroded mass. Scarchilli et al., 2009). Hence, we expect that blowing snow can often be falsely detected as precipitation by snowfall sen- sors (Sugiura et al., 2003). Some issues caused by the wind can be corrected with scaling factors or transfer functions, but these need to be identiﬁed for these speciﬁc conditions (Goodison et al., 1998). Another approach is to measure the snow water equivalent (SWE) of the snow cover. From this, one can derive snowfall rates. However, especially during the polar night, the precipitated snow is dry, and as already indi- cated above, the wind speed is often sufﬁciently high to drift the freshly fallen snow particles away immediately. Hence, single point measurements are not appropriate to estimate snowfall, and horizontal sampling distance and temporal dis- tance between sampling days should be kept as short as pos- sible. This becomes more crucial the windier the location is. Scarchilli et al., 2009). Hence, we expect that blowing snow can often be falsely detected as precipitation by snowfall sen- sors (Sugiura et al., 2003). Some issues caused by the wind can be corrected with scaling factors or transfer functions, but these need to be identiﬁed for these speciﬁc conditions (Goodison et al., 1998). Another approach is to measure the snow water equivalent (SWE) of the snow cover. From this, one can derive snowfall rates. However, especially during the polar night, the precipitated snow is dry, and as already indi- cated above, the wind speed is often sufﬁciently high to drift the freshly fallen snow particles away immediately. Hence, single point measurements are not appropriate to estimate snowfall, and horizontal sampling distance and temporal dis- tance between sampling days should be kept as short as pos- sible. This becomes more crucial the windier the location is. – Investigate average snow mass balance and discrepan- cies of computed snowfall rates and SWE on the sea ice and shed light on the processes described in Eq. (2), such as total eroded mass. Section 2 introduces our methods, followed by Sect. 3, where we show the results. In Sect. 4 we discuss our results, and in Sect. 5 we draw conclusions about our ﬁndings and give an outlook about potential future work. 2 Data and methodology All data used for evaluations in the following were collected during the MOSAiC campaign (Krumpen et al., 2020; Nico- laus et al., 2021b; Shupe et al., 2022) from the beginning of Leg 1 (24 October 2019) until the end of Leg 3 (7 May 2020) (Fig. 1). On 4 October 2019, RV Polarstern moored along an ice ﬂoe that originated in the Siberian shelf (Krumpen et al., 2020). During the year-long Multidisciplinary drifting Observa- tory for the Study of Arctic Climate (MOSAiC) expedition, during which the research vessel (RV) Polarstern (Alfred- Wegener-Institut Helmholtz-Zentrum für Polar- und Meeres- forschung, 2017) served as a base moored on two different ice ﬂoes, data of snow on the ice as well as of in situ snowfall were collected in great detail for almost the whole MOSAiC period (October 2019–October 2020) (Nicolaus et al., 2021b; Shupe et al., 2022). The dataset includes measurements of the penetration force into the snowpack with a SnowMi- croPen (SMP) (Schneebeli and Johnson, 1998; Schneebeli et al., 1999) from which snowpack densities can be esti- mated (Proksch et al., 2015) as well as bulk SWE mea- surements, weekly repeated transects of snow depth mea- surements and a set of precipitation sensors installed on the ice (Vaisala Present Weather Detector 22 (PWD22) (Vaisala, 2004; Kyrouac and Holdridge, 2019), OTT Pluvio2 plu- viometer (Bartholomew, 2020a; Wang et al., 2019b), OTT Parsivel2 (Bartholomew, 2020b; Shi, 2019) and on board RV Polarstern (Vaisala PWD22, OTT Parsivel2). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons (2) (2) To investigate all effects of the snow cover over the ice – the insulating effect, the sea ice mass contribution effect and the albedo effect – light must be shed into the snow processes that are represented, and detailed knowledge of the evolu- tion of total snow mass dM dt , or SWE over time on top of the ice, is required. However, due to logistical challenges, espe- cially for the winter and spring months, snowfall rate and snow accumulation estimates could only be roughly approx- imated so far. The past estimates mostly made use of rare point measurements, or rather old time series (Petty et al., 2018) and satellite remote sensing (Petty et al., 2018; Cabaj et al., 2020), leading to high uncertainties in weather, cli- mate and snow cover models as well as in reanalyses. Ba- trak and Müller (2019), for instance, could show that a 5 to 10 ◦C warm bias of the sea ice surface temperature in weather forecasts and reanalyses is due to a missing snow layer modelled on top of the sea ice. For snowfall rates and mass balance estimations, some general problems occur: lim- ited data about snowfall rates from precipitation gauges cur- rently exist for this region. Buoys that measure snow height with acoustic sensors which record long continuous time se- ries along its drift tracks throughout the central Arctic do exist (Nicolaus et al., 2021a). However, uncertainties with point snow measurements arise in those windy regions due to the snow transport processes described above. If using pre- cipitation sensors, the high average horizontal wind veloci- ties make snowfall rate estimates difﬁcult for both weighing gauges (Goodison et al., 1998) and optical sensors (Wong, 2012). The wind itself may lead to an undercatch for weigh- ing bucket gauges (Goodison et al., 1998), while blowing snow may lead to overestimation for both weighing gauges and optical sensors (Sugiura et al., 2003). Blowing snow typ- ically occurs at heights up to 10 m, while it can even reach several hundreds of metres in altitude (Budd et al., 1966; https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 2376 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring season https://doi.org/10.5194/tc-16-2373-2022 2.2.1 SMP force and SWE measurements We measured snow water equivalent (SWE) with an ETH tube, a SWE sampler that is commonly used in Switzerland (Haberkorn, 2019; López-Moreno et al., 2020), as well as resistance force with the SnowMicroPen (SMP) (Schneebeli and Johnson, 1998; Schneebeli et al., 1999) and snow height at different sites (areas shaded in yellow in Fig. 2). The bulk SWE measurements with the ETH cylinder follow the simple principle where the mass of the snow ﬁtting in a tube with a known cross-sectional area is weighed on a spring scale, which yields the SWE in millimetres or kg m−2. The device is calibrated for low temperatures, which is most important for the steel spring of the scale. López-Moreno et al. (2020) made an intercomparison of various bulk density and SWE samples including the ETH tube and tested for instrumental bias and variability. It can be concluded that from a single ETH tube measurement we might expect a maximum error of 10 %. This value appears high, but given the fact that the average Arctic sea ice snow cover is thin, the absolute error will be low. Given a 20 cm snow depth, the maximum ex- pected error would only be 2 cm. López-Moreno et al. (2020) also argue that particularly light samples may lead to an ad- ditional 10 % of error with respect to the weighing process with the spring scale itself. Nonetheless, a currently non- quantiﬁed error is that during a bulk SWE measurement a sharp transition between snow cover and sea ice often cannot be determined, which is especially valid for an underlying surface scattering layer (SSL) on SYI. However, we use a rel- atively big sample size of n = 195 bulk SWE measurements, and with increasing sample size, uncertainties are expected to be increasingly levelled out. To avoid wind inﬂuence on the measurements, the weighing was conducted in the wind shadow of surrounding objects, surrounding persons or the person measuring. Figure 1. The drift trajectory of RV Polarstern between 24 Octo- ber 2019 and 7 May 2020. Figure 2. Main snow measuring areas on the MOSAiC ﬂoe by 5 March 2020. The bottom layer is a digital elevation map (DEM) from airborne laser scanning (ALS) with the helicopter. The square side length of the underlay grid is 500 m. The transect paths and margins of the shaded measuring areas are based on GPS measure- ments. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Figure 1. The drift trajectory of RV Polarstern between 24 Octo- ber 2019 and 7 May 2020. 2.2.1 SMP force and SWE measurements The legend for elevation is shown in metres. However, the elevation range is only approximate due to issues with the inertial navigation system which could not be corrected as of yet. The SMP is a device which measures the penetration re- sistance force (N) by means of a rod with a conic tip that is slowly driven vertically into the snowpack. A force sensor is connected to the tip which detects the force that is needed to drive into the snowpack with micrometre resolution. The out- put is given as a force–snow depth signal. These penetration resistance force signals can be used to estimate snowpack density and detect the layers in the snowpack (Proksch et al., 2015; King et al., 2020). We used three different sensors, but all SMP version 4, during MOSAiC. Processing of density from SMP force signals is discussed in the next section. Figure 2. Main snow measuring areas on the MOSAiC ﬂoe by 5 March 2020. The bottom layer is a digital elevation map (DEM) from airborne laser scanning (ALS) with the helicopter. The square side length of the underlay grid is 500 m. The transect paths and margins of the shaded measuring areas are based on GPS measure- ments. The legend for elevation is shown in metres. However, the elevation range is only approximate due to issues with the inertial navigation system which could not be corrected as of yet. The map in Fig. 2 shows the ﬂoe state on 5 March 2020, which changed signiﬁcantly due to ice dynamics that started on 11 March 2020. Snow was measured at the different sites as well as along both transect loops. The measurements cover a large area of the ﬂoe, including level, remnant SYI, FYI and deformed SYI. Details about the sampling procedure will fol- low below. Snow 1 was characterized mainly by a mixture 2.1 Ice conditions and central observatory – Evaluate the ERA5 (Hersbach et al., 2020) mean snow- fall rates for the MOSAiC drift track. We describe the measuring setup and the post-processing for all used data streams in the following. https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 https://doi.org/10.5194/tc-16-2373-2022 2377 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons The ex- pected result from this surrounding sea ice characteristic is that during drifting snow events, snow might get caught up- wind of the transect areas, while wind speeds may decrease, potentially leading to ﬂow separation and a bias in the total snow accumulation. We consider this a non-quantiﬁable un- certainty at this point of research. The only way to overcome this problem in the future is to cover larger sampling areas and increase the number of these. To ﬁnd an optimum be- tween technical effort and reliable result, one could decrease these areas until average values are not changing any more signiﬁcantly. The GPS coordinates of the Magnaprobe were trans- formed into coordinates of a local metric coordinate system, called “FloeNavi” (Fig. 3). Furthermore, the transects were partially corrected for shifts within the ice, which was es- pecially the case for an event with strong ice dynamics on 16 November 2019. Thus, the southern loop transects un- til (including) 14 November 2019 are marked as a yellow rectangle in Fig. 3. Note that a part of the northern tran- sect was off the regular transect track on 31 October 2019, which leads to some uncertainties in evaluations. However, we tested how it affected the average when only the off track was cut off versus the whole northern transect. We found an increase of only 0.8 mm when the whole track was consid- ered. Hence, though this leads to some uncertainties, we in- cluded the 31 October 2019 transect for further evaluations. For all other days of sampling, though the transects may de- viate from one another within the FloeNavi coordinate sys- tem, the actual transect path was the same. After the coordi- nate transformation and horizontal correction, for good spa- tial and temporal comparability, clear margins as shown in the rectangles in Fig. 3 were deﬁned for the “southern” and “northern” transect loops. By the overlays, one can recog- nize that the transect loops were not signiﬁcantly impacted by internal differential ice movements. At the measuring locations, SWE, snow height and pen- etration resistance force measurements with the SMP were done. The SMP measurements at the recurring snow pit lo- cations were conducted as follows: ﬁve SMP measurements were performed at a distance of about 20 cm along a line par- allel to the old snow pit wall to account for the spatial het- erogeneity of the snowpack. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2378 of remnant SYI and deformed SYI. In the beginning, Snow 1 was mostly ﬂat, but the surface became rougher over the time of the expedition. At Snow 1 we deployed three snow pit sites, which were maintained until the end of leg 3. The Snow 2 plot was characterized as an open level ﬁeld, mostly on remnant and deformed SYI with a distinct high and long pressure ridge in the centre of the plot. On Snow 2, we main- tained two snow pit locations until the end of leg 3. Both sites had very similar underlying ice conditions. Snow 3 was created at a later point, furthest away from the vessel. In the beginning, it was a very ﬂat area with underlying FYI and was maintained during leg 3 but needed to be abandoned due to ice dynamics in mid-March. Further, weekly snow pit measurements were conducted along the south-westerly section of the northern transect loop. Also, transects were conducted infrequently over ridges, and measurements were conducted weekly at the ice coring sites during Leg 1 (be- yond the map boundaries in Fig. 2, but located north-west of the ship), among other measuring locations. The large vari- ety of locations, their underlying ice types and snow depths allow us to take the spatial heterogeneities of the snow cover into account. However, since we use a bulk approach with the collected SMP and direct SWE data, detailed information on each measuring site is not needed and will not be provided here. sect loop locations can be seen in orange on the MOSAiC ﬂoe map from 5 March 2020 (Fig. 2). The elevations on the southern transect are mostly below around 1 m height. The elevations are generally higher on the northern transect, al- though it does not cover ridges of up to 3 m height or more as they have been observed on the Snow 2 plot. One impor- tant aspect to note is that surrounding elevations of the ice (i.e. ridges) often exceed the height of the transect areas, for instance in the north and the south of the northern transect or in the north and the east of the southern transect. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons On the ridge sites, for instance, SMP measurements were conducted infrequently as transects over ridges. We used these measurements to estimate SWE along the northern and southern transect loops, which will be explained below. For more details about the SMP and SWE collection, we refer to Macfarlane et al. (2021, 2022), Wag- ner et al. (2021) (data publicly accessible after the end of the MOSAiC moratorium in January 2023) and soon-to-be- published data and method papers by MOSAiC participants that describe the MOSAiC snow measurement setup in de- tail. https://doi.org/10.5194/tc-16-2373-2022 https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2379 the user, while the users varied mainly between each leg of MOSAiC. The average distance between measurements was computed after applying the FloeNavi coordinate transfor- mation. Values z < 0.00 m as well as z > 1.40 m (the tech- nically constrained measurable length) were discarded as in- correct data. In this study, we did not account for further cor- rections that may come along with a tip sinking into mate- rial below the snow cover, for instance the surface scattering layer on SYI. Sturm and Holmgren (2018) showed that this error is hard to quantify, as it depends strongly on the ground material and of course the applied force as well – an issue which also occurs for crusts within the snow cover. Similar issues occur for other snow measurements, such as the ETH tube and SMP, as well, which will be mentioned later in the text again. Figure 3. Magnaprobe transect paths with coordinates transformed to the FloeNavi grid corrected for ice drift. The rectangles represent the margins that were used as a deﬁnition for the “northern tran- sect loop” (upper left) and “southern transect loop” (bottom right) for good comparability. The shifts between transect paths within a rectangle originate from corrections and coordinate transformation, though the actual transect paths were the same. The northern loop was sampled from 24 October 2019 to 7 May 2020 on 24 d with an average path length of 954 m. The southern loop was sampled from 31 October 2019 to 26 April 2020 14 times with an average transect path length of 974 m. More in-detail data and instrument description are found in Itkin et al. (2021). Figure 3. Magnaprobe transect paths with coordinates transformed to the FloeNavi grid corrected for ice drift. The rectangles represent the margins that were used as a deﬁnition for the “northern tran- sect loop” (upper left) and “southern transect loop” (bottom right) for good comparability. The shifts between transect paths within a rectangle originate from corrections and coordinate transformation, though the actual transect paths were the same. To take surface roughness (i.e. variability of snow surface height) and potential snow accumulation at surface irregu- larities better into account, we looked at the weekly snow height differences of the transects. With the given average horizontal sampling distance (1.03–1.21 m), no small-scale patterns are considered (sastrugi, for instance) for evalua- tion. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons However, since the extent of ridges and most types of dunes are larger than 1.2 m in all horizontal directions(Filhol and Sturm, 2015), we expect our typical horizontal sampling scale to accurately characterize the spatial distribution of ac- cumulation, which we demonstrate in Sect. 2.3.1. etration force of the SMP (N) for a speciﬁed sliding win- dow; and L is the microstructural length scale (Löwe and Herwijnen, 2012) for the same window. Both ˜F and L are computed for the window size of 2.5 mm with a 50 % over- lap, which is the same as used in Proksch et al. (2015), but contrary to Calonne et al. (2020) (1 mm) and King et al. (2020) (5 mm). King et al. (2020) calibrated the correspond- ing coefﬁcients to snow on Arctic sea ice and found a = 315.61 kg m−3, b = 46.94 N−1, c = −43.94 N−1 mm−1 and d = −88.15 mm−1. The coefﬁcients show a signiﬁcant im- provement in density derivation for snow on sea ice, which is reﬂected by the decrease in the root-mean-square error (RMSE) (Proksch et al. (2015): RMSE = 130 kg m−3; King et al. (2020): RMSE = 41 kg m−3) without removing out- liers, compared against density cutter measurements. Con- sequently, we used the coefﬁcients from King et al. (2020) for the following SWE computations. From the SMP density estimates we can compute 2.3 SMP density retrievals and SWE from transect snow depths Based on how the campaign was planned, we have consid- erably more SMP force measurements available (N = 3007) than bulk SWE weighing measurements (N = 195). Further- more, for each snow pit we made at least n = 5 SMP mea- surements, and the SMP was often used even for ridge tran- sects; these measurements best characterize the spatial het- erogeneity in the snow depth across the sea ice. Not many direct SWE measurements or SMP force measurements are available along the transect path. Hence, we use the di- rect SWE measurements for validation but apply a statistical SWE–snow depth (HS) relationship to estimate SWE along the full path (Sturm et al., 2002a; Jonas et al., 2009). (4) SWE = HS · ρ, (4) 2.2.2 Transects Snow depth transects were conducted weekly with a Mag- naprobe (Sturm and Holmgren, 2018; Itkin et al., 2021), if the atmospheric, ice or overall safety conditions did not prevent it. The transect path was distributed into two loops (Fig. 2): a northern loop, mostly situated on deformed SYI, and a south- ern loop, which was mainly situated on FYI and remnant SYI with underlying frozen melt ponds. A transition zone distinguished these two loops, mostly consisting of frozen melt ponds with a very ﬂat surface without signiﬁcant het- erogeneities. The approximate ice conditions and the tran- However, ice dynamics affected the transects, especially from 11/12 March 2020 on, where leads and cracks opened throughout the paths. Overall, we tried to minimize the inﬂu- ence of these ice deformation events on the transect mea- surements. However, an impact on the time series cannot be excluded. On the transects, snow height measurements were sampled with the Magnaprobe with an average distance between measuring points of 1.1 m. Note that this value is simply an average that contains the uncertainty of GPS lo- calization, coordinate transformation and the step length of https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons well as SWE retrievals from the SMP: well as SWE retrievals from the SMP: SWE = m · HSa, (5) (5) SWE = m · HSa, where m is the ﬁtted slope and a is a ﬁtting coefﬁcient. For the bulk SWE measurements, we found m = 334.61 and a = 1.14 (Fig. 4). For the SMP retrievals we found m = 323.97 and a = 1.07. As we have more SMP measurements avail- able in total, and especially for deep snow depths compared with the ETH tube, we computed SWE based on ﬁtted SMP density–SWE parameters as SWE = 323.97 · HS1.07. (6) (6) From Fig. 4 one can clearly see that the improvement for snow on sea ice of the coefﬁcients found by King et al. (2020) is valid for MOSAiC legs 1–3 SMP data, too (Fig. 4c) and that the coefﬁcients determined by Proksch et al. (2015) and Calonne et al. (2020) appear not appropriate to estimate SWE of snow for this MOSAiC period. Furthermore, the lowest RMSE (expressed here as average error of individually com- puted SWE relative to the regression line for an individual parameter setup) was found for the ﬁtted model with the co- efﬁcients from King et al. (2020) (7.2 mm SWE) compared against 15.4 mm (Proksch et al., 2015) and 9.4 mm (Calonne et al., 2020). However, one should note the following limi- tations in this comparison: ﬁrst, we used a sliding window size of 2.5 mm for all computations, which is the same as in Proksch et al. (2015), while Calonne et al. (2020) used 1 mm and King et al. (2020) 5 mm. However, the strength of the inﬂuence can be at least partially invalidated by the fact that Calonne et al. (2020) state that they tested for sensitivity of three different window sizes of 1, 2.5 and 5 mm and could not ﬁnd a signiﬁcant inﬂuence on the result – which is not quan- tiﬁed in the publication. At least choosing a ﬁxed window for each parameterization – as we did with the 2.5 mm window – increases the comparability. Another limitation might be that the Proksch et al. (2015) calibration was made with a SMP version 2, while we, Calonne et al. (2020) and King et al. (2020) use the newest SMP version 4. Figure 4. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2380 Figure 4. Scatter plots and ﬁtted HS-SWE function of SMP derived SWE and measured SWE with the ETH tube for (a) the density computation coefﬁcients from Proksch et al. (2015), (b) Calonne et al. (2020) and (c) King et al. (2020). well as SWE retrievals from the SMP: SWE = HS · ρ, Snowpack density can be estimated with a statistical model from SMP snow depth–force signal proﬁles (Proksch et al., 2015): where HS (m) is the height of snow over the ice or snow depth, and ρ (kg m−3) is the vertically averaged density of the snowpack. The computed SWE dataset is documented in detail by Wagner et al. (2021). Similar to Jonas et al. (2009), but applying the function directly to SWE, we ﬁtted the fol- lowing function to the available bulk SWE measurements as ρ = a + bln ˜F + cln ˜F L + dL, (3) (3) where a (kg m−3), b (N−1), c (N−1 mm−1) and d (mm−1) are empirical regression coefﬁcients; ˜F is the median pen- https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2381 Figure 5. (a) SMP measurement locations along the Magnaprobe (MP) transect path on 14 (2019-11-14), 21 (2019-11-21) and 28 Novem- ber 2019 (2019-11-28). The GPS coordinates were transformed into local FloeNavi grid coordinates. Panels (b), (c) and (d) show the compar- ison of SWE estimates from direct SMP measurements, direct bulk SWE measurements and SWE derived with the HS-SWE model from the Magnaprobe snow depth measurements along the northern transect as x-axis location on the FloeNavi grid, for 14, 21 and 28 November 2019. Figure 5. (a) SMP measurement locations along the Magnaprobe (MP) transect path on 14 (2019-11-14), 21 (2019-11-21) and 28 Novem- ber 2019 (2019-11-28). The GPS coordinates were transformed into local FloeNavi grid coordinates. Panels (b), (c) and (d) show the compar- ison of SWE estimates from direct SMP measurements, direct bulk SWE measurements and SWE derived with the HS-SWE model from the Magnaprobe snow depth measurements along the northern transect as x-axis location on the FloeNavi grid, for 14, 21 and 28 November 2019. This comparison shows that the modelled SWE matches the derived SWE from SMP retrievals and bulk SWE mea- surements quite well during the three chosen time periods, even for higher SWE estimates, where a higher scatter is expected (Fig. 4). Note that although the time distance be- tween the 3 d of measurements is relatively short, we found that 42 % of the time for drifting snow conditions the thresh- old friction velocity for snow transport was exceeded (the lower snow particle counter (SPC) was not installed yet at this time) from and including 14 November until and includ- ing 21 November 2019. From and including 21 November until and including 28 November 2019 the threshold was exceeded 57 % of the time. This means we can expect re- distributed snow for the 2 d following 14 November. Inter- comparing SMP SWE versus ETH SWE, we ﬁnd a RMSE of 16.3 mm for 14 November, 8.1 mm for 21 November and 3.5 mm for 28 November. However, we must note that the depth of SWE measurements from the ETH tube and SMP has some individual but differing restrictions: ﬁrstly, as the SMP cut-off force signal was set to 40–41◦N (depending on the device), the snow depth was determined whenever one of those values was reached, which is not necessarily the snow– ice interface. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Secondly, during the sampling period, there was no method established to distinguish between surface scat- tering layer (SSL) and snow. Hence, its vertical position was determined visually, which was not always clear. Therefore, a measurement with the ETH tube might or might not in- clude the surface scattering layer which formed during the melt season of 2019. If the SMP was able to penetrate the SSL only partially while it was not measured with the ETH tube, then SWE is overestimated from the SMP measure- ments. Otherwise, if the SMP could not penetrate the SSL while it was partially measured with the ETH tube, the SMP- based SWE computation overestimates actual SWE. How- ever, as the SMP SWE retrievals are often close to the di- rect SWE measurements, one can assume reliable values on the whole. Research to determine exact boundaries between snow and sea ice is ongoing. Furthermore, since the number of measurement points along a transect is large and we do not expect systematic biases, we believe that ﬂuctuations caused by these various sources of uncertainty will largely average out, such that the results from the applied SWE model yield a reasonable estimate along the transect. Under Appendix A we make a comparison with derived SWE over Arctic sea ice during the SHEBA campaign con- ducted by Sturm et al. (2002a) in a similar manner. The com- parison shows the difference between their and our results and underlines the importance of using our approach for MO- SAiC snow cover data. well as SWE retrievals from the SMP: Scatter plots and ﬁtted HS-SWE function of SMP derived SWE and measured SWE with the ETH tube for (a) the density computation coefﬁcients from Proksch et al. (2015), (b) Calonne et al. (2020) and (c) King et al. (2020). different snow layers. Instead, for validation, we compared SMP and ETH tube measurements with transect-computed SWE along a section of the northern transect loop for 14, 21 and 28 November 2019 (Fig. 5). The validation measure- ments were conducted at different positions at each day of measurements and contain drift locations and level ice areas. Note that this quantitative comparison of bulk SWE and SMP SWE versus transect contains uncertainties as the accuracy of GPS measurements (2 m) and the following coordinate trans- formation of the Magnaprobe as well as the SMP coordinates do not allow for centimetre-scale precision. Further, the pits were dug up to a vertical distance of 1 m from the transect path, in order to sample fresh snow that is not disturbed by re- peated transects. For quantitative comparison, SWE compu- tations from direct bulk SWE and SMP measurements along the transects were plotted over SWE model retrievals. Fig- ure 5a shows the measuring locations for each SMP measure- ment along the northern transect loop (ﬁve measurements at each snow pit location), and Fig. 5b–d show the correspond- ing SWE plotted over the x axis of the FloeNavi for different days of measurements. We applied our ﬁtted formula to each snow depth measure- ment with the Magnaprobe along the transect path to obtain the SWE estimates. The SWE was rounded to integers for the following description in the text, except when two values that are compared are very similar. No rounding was conducted before any computations. The average computed SWE will also serve as a reference comparison with snowfall sensors and ERA5 as described in Sect. 2.8. A limitation with this approach is that different snow lay- ers are not distinguished by density, even though a wind- packed layer has a higher density than a depth hoar layer. Hence, when high winds lead to drifting snow deposition that is detected by a snow height increase with the Magnaprobe, the SWE increase is likely to be underestimated, as would be the eroded mass of a drifting snow layer. well as SWE retrievals from the SMP: It is beyond the scope of this study to attempt an approach that distinguishes https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2.3.1 Evaluating the sensitivity of the arithmetic mean with respect to horizontal sampling distance The x axis shows the horizontal tran- sect sampling frequency in relation to the original sampling fre- quency f0, and the y axis shows the ratio of the average SWE of all tested frequencies to the average SWE of the original sam- pling frequency for each day of sampling (31 October 2019 to 26 April 2020). Optical devices evaluated here are the Vaisala PWD22 and the OTT Parsivel2. However, the measurement technique and the process of estimating snowfall rates are different. The Parsivel2 is a laser disdrometer that processes the voltage sig- nal changes due to light extinction when a hydrometeor falls through the laser beam. It has an effective measuring area of 54 cm2 to estimate hydrometeor size and velocity (Löfﬂer- Mang and Joss, 2000). The hydrometeors are classiﬁed into size classes which can be used to investigate the particle size distribution. The precipitation type is determined by device- internal spectral signature comparison, where the spectra are determined empirically. Based on particle size, velocity and estimated precipitation type, device-internal software com- putes a snowfall estimate. No details are known about the exact formula used by the manufacturer for the snowfall es- timate. Its accuracy is given by the manufacturer as ±20 % with an intensity range of 0.001 to 1200 mm h−1 (Table 1). The calibration was conducted in the manufacturer’s labora- tory, and therefore no calibration was needed in the ﬁeld. numbers from using all samples (Magnaprobe average sam- pling distance of 1.1 m) down to considering every 10th sam- ple (about 11 m sample distance) for the average. The pro- cess was conducted for each day of sampling, and the av- erages were normalized against the original sampling fre- quency (Fig. 6). The results show that for sampling frequencies down to one-third of the original frequency (sampling distances rang- ing from 1.1 to 3.4 m) the average mass estimates vary by less than ±1 %. This indicates that a sampling distance up to 3.4 m is mostly robust and that no signiﬁcant undersampling occurred. This also shows that the impact of variations in sampling interval distance that inevitably occurs with differ- ent operators of the Magnaprobe is probably negligible. The larger ﬂuctuations in computed average mass for longer sam- ple interval distances suggests undersampling at those scales and less reliable averages. 2.3.1 Evaluating the sensitivity of the arithmetic mean with respect to horizontal sampling distance As shown by Trujillo and Lehning (2015), a sufﬁciently small sampling interval of point measurements is crucial for estimating representative values of spatially averaged snow depths. We studied the sensitivity of the horizontal sampling interval for average mass estimates by reducing the sample https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2382 Figure 6. Sensitivity of the transect average as a factor of the Mag- naprobe horizontal sampling. The x axis shows the horizontal tran- sect sampling frequency in relation to the original sampling fre- quency f0, and the y axis shows the ratio of the average SWE of all tested frequencies to the average SWE of the original sam- pling frequency for each day of sampling (31 October 2019 to 26 April 2020). diation Measurement (ARM) program. Two sensors inves- tigated here were installed on the railing on the top deck of Polarstern – a Vaisala Present Weather Detector 22 (Vaisala, 2004; Kyrouac and Holdridge, 2019) (referred to as PWD22PS in the following) and an OTT Parsivel2 laser disdrometer (Shi, 2019; Bartholomew, 2020b) (referred to as P2PS in the following) (Table 1). The PWD22PS was in- stalled at 22 m and the P2PS at 24 m above the water line. On the ice, in “met city” (Fig. 2, in the following, referred to as MC), three precipitation sensors were installed: (1) an OTT Parsivel2 (P2MC), installed at 1.5 m nominal height above the snow surface, surrounded by a double-alter shield; (2) a PWD22 (PWD22MC), installed at 2 m nominal height, un- shielded; and (3) an OTT Pluvio2 L (Wang et al., 2019b; Bartholomew, 2020b), shielded by a double-alter shield and installed at 1 m above the snow (referred to as Pluvio2 in the following). Different ARM data levels of the devices are given, where a1 means “calibration factors applied and con- verted to geophysical units” and b1 means “QC checks ap- plied to measurements”. Figure 6. Sensitivity of the transect average as a factor of the Mag- naprobe horizontal sampling. 2.3.1 Evaluating the sensitivity of the arithmetic mean with respect to horizontal sampling distance However, a validation of uncer- tainties that could accompany varying vertical penetration force leading to different measured snow height, e.g. when a crust within the snow is penetrated or not due to varying operators, is not conducted here. The operators were aware of this issue and tried to apply a similar power for the Mag- naprobe sampling. The PWD22 consists of several sensors that are used to compute the snowfall rate: the two core sensors are a transmitter–receiver combination, where the transmitter emits pulses of near-infrared (NIR) light. The receiver on the other side measures the scattered part at 45◦of the light beam from the emitted signal (sampling volume 100 cm3). Rapid changes in the scatter signal between transmitter and receiver are used to compute precipitation intensity. The sam- pling volume allows for the detection of single crystals and aggregates of snow crystals (snowﬂakes). Furthermore, the PWD22 is equipped with a heated RAINCAP rain sensor, which produces a signal proportional to the amount of water on the sensing element. By means of the ratio from sample volume and water content determined with the RAINCAP sensor, precipitation types are distinguished. In the tube be- tween the transmitter and receiver, another temperature sen- 2.4.1 Precipitation gauges Snowfall rates were estimated using standard internal pro- cessing software from ﬁve distinct precipitation gauges op- erated by the US Department of Energy Atmospheric Ra- https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2383 Figure 7. Computed cumulative snowfall for different KAZR range gates and PWD22PS. sor (thermistor) is installed. The detected temperature is used to select the default precipitation type. When frozen precip- itation is detected, the PWD22 software multiplies optical intensity with a scaling factor, determined from RAINCAP and optical intensities from the receiver to estimate snowfall intensity as SWE per time unit (Vaisala, 2004). The manu- facturer does not provide a value for accuracy; however the intensity measuring range is given as 0.00 to 999 mm h−1. There is no calibration principle known for the ﬁeld, but the manufacturer mentions comparisons with close reference gauges as a calibration method. The only device that we compare here that uses a weigh- ing principle is the OTT Pluvio2. The instrument’s core is a sealed load cell that continuously measures the weight of the precipitation falling into the entry of the bucket. The in- stalled variant was an OTT Pluvio2 L Version 400, with a col- lecting area of 400 cm2 and a recording capacity of 750 mm of precipitation. Its accuracy is given by the manufacturer as ± 0.1 mm min−1 or ± 6 mm h−1, or ± 1 %, and its intensity range is given as ±6 mm h−1 or 0.1 to 30 mm h−1. No cali- bration for the OTT Pluvio2 is needed in the ﬁeld as it was delivered calibrated by the manufacturer. However, calibra- tion weights were used to test for accuracy. Figure 7. Computed cumulative snowfall for different KAZR range gates and PWD22PS. The data streams were downloaded from the ARM data archive (https://adc.arm.gov/, last access: 14 June 2022) and scanned for quality control ﬂags. Values with timestamps that correspond to ﬂags indicating maintenance time or sus- picious or incorrect values were discarded. range gates (Fig. 7). The ﬁrst range gate of 100 m did not yield any measurements, while at 130 m reﬂectivities were too low. From Fig. 2.4.1 Precipitation gauges 7 we see that the differences in the cu- mulative snowfall from range gates between 220 and 280 m are the least. The decrease in computed snowfall with height beyond 280 m is probably due to very low cloud heights in winter (Jun et al., 2016), such that snowfall would get under- estimated as these range gates are often at higher elevations within the clouds or even beyond the cloud top. We found the largest snowfall rates for the 280 m range gate; thus we chose it as the range gate from which we extracted the snowfall re- trievals. However, based on this simple analysis, the poten- tial differences in snowfall based on this choice of range gate are on the order of about 10 %. With an instrument elevation of 14 m a.s.l., the elevation of the extracted snowfall rates is 294 m a.s.l. 2.4.2 Snowfall retrievals from the Ka-band ARM zenith radar Snowfall was retrieved from the Ka-band ARM zenith radar (KAZR) (Widener et al., 2012; Lindenmaier et al., 2019) that was installed on a container at the bow of RV Polarstern. Us- ing a radar snowfall retrieval allows us to investigate snowfall continuously and eliminates impacts on gauges such as ac- celeration effects of wind that result in undercatch or overes- timation due to blowing snow particles. The KAZR operated at approximately 35 GHz. We computed the snowfall rate S (mm h−1) according to the power law Ze = a Sb, (7) 2.5 Atmospheric ﬂux station data Ze = a Sb, (7) (7) A meteorological tower of 10 m height was installed on the ice 558 m away from RV Polarstern at about 60◦off the bow of the vessel in the middle of October 2019. How- ever, due to ice dynamics, by the end of leg 3 (begin- ning of May 2020), the distance was only about 334 m while the direction from the ship stayed approximately the same (Fig. 2). At nominal levels z = 2, 6 and 10 m above the snow, three-dimensional wind (u,v,w) and temperature were measured at high frequency with METEK uSonic-3 Cage MP anemometers (METEK GmbH, 2022), while on the same elevation levels, relative humidity and temperature were measured with Vaisala HUMICAP humidity and tem- where Ze (mm6 m−3) is the radar equivalent reﬂectivity fac- tor, and a and b are empirical coefﬁcients. We chose a = 56 and b = 1.2 as these were found to be good average val- ues for dry snowfall at this radar frequency, and no signiﬁ- cant riming was observed (Matrosov, 2007; Matrosov et al., 2008). Near-ﬁeld radar measurements can suffer from a variety of issues, such that snowfall retrievals typically must be applied to radar signals that are elevated above the surface. To ﬁnd an appropriate KAZR range gate to extract snowfall rates, we plotted the cumulative sums of SWE based on KAZR- derived snowfall from reﬂectivity measurements at different https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 gner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2384 Table 1. Summary of validated installed precipitation sensors and radar during MOSAiC as well as details about the ERA5 reanalysis (PS: RV Polarstern, MC: met city, DA: double-alter shield, WL: water line, DFIR: double fence intercomparison reference). (7) Device/reanalysis Loc Abbreviation Nominal height Shield Data reference ARM Accuracy Intensity range Selected existing snowfall validations/ reference setup Calibration Snow/WL (m) data level Vaisala PWD22 PS PWD22PS 22 (WL) – Kyrouac and Holdridge (2019) b1 – 0.00–999 mm h−1 −32 % from median/DFIR; (Wong, 2012) Reference comparison +33 % from median/- manual (Boudala et al., 2016) OTT Parsivel2 PS P2PS 24 (WL) – Shi (2019) b1 ±20 % 0.001–1200 mm h−1 +46 %/+54 % from median/DFIR (Wong, 2012); Manufacturer +24 %/+29 % from median/DFIR (Wong et al., 2012) KAZR PS KAZR 14 (WL) – Lindenmaier et al. (2019) a1 – – 23 % relative bias/ shielded Nipher gauge (Matrosov et al., 2008) – Vaisala PWD22 MC PWD22MC 2 (Snow) – Kyrouac and Holdridge (2019) b1 – 0.00–999 mm h−1 See above Reference comparison OTT Parsivel2 MC P2MC 1.5 (Snow) DA Shi (2019) b1 ±20 % 0.001–1200 mm h−1 See above Manufacturer OTT Pluvio2 MC Pluvio2 1 (Snow) DA Wang et al. (2019b) a1 ±6 mm h−1 6–1800 mm h−1 −63 % from median (max)/DFIR; Manufacturer/ ±0.1 mm min−1 −14 % from median (min)/DFIR calib. weights ±1 % (Wong, 2012) ERA5 – – – – Hersbach et al. (2020) – – – +62.8 mm cum. SWE/snow buoys (Wang et al., 2019a); – slight underestimate in summer/CloudSat (Cabaj et al., 2020); overestimate in other months osphere, 16, 2373–2402, 2022 https://doi.org/10.5194/tc-16-2373-2 2.6 Drifting and blowing snow mass ﬂux On the meteorological tower described under Sect. 2.5, two snow particle counters (SPCs) (Sato et al., 1993) were in- stalled. The devices continuously detect number and sizes of snow particles which are transported through a laser beam. The devices rotate with very low friction on a vertical axis, and mounted wind vanes at the back of the sensor keep the laser beam 90◦towards the wind. One SPC was installed at about 0.1 m (SPC1104) and one at 10 m (SPC1206) above the snow. The lower SPC1104 ran with only a few inter- ruptions from 2 December 2019 until 7 May 2020 (data availability for this period 96.6 %). The upper SPC1206 ran with only a few interruptions from 14 October 2019 until 7 May 2020 (data availability for this period 94.9 %). One bigger data gap for the SPC1206 was between 17 Novem- ber 2019, 03:05:00 UTC and 18 November, 11:15:00 UTC because there was a power interruption due to sea ice dynam- ics resulting in broken power lines. Note that the SPC1206 data at 10 m are still under quality control, and the absolute mass ﬂux values have some error yet to be quantiﬁed. Re- gardless, the comparison between the two SPCs yields an or- der of magnitude of the mass ﬂux and its relative change at 10 m compared to near the surface at 0.1 m. The mass decrease computed with the HS-SWE function from the transect is temporally compared against computed cumulative snow mass ﬂux from the snow particle counters. Note that the cumulative horizontal mass ﬂux is only an indi- cator for the strength of the erosion but cannot be translated into actual eroded mass. To distinguish in the text between computed SWE decrease in the snow cover and cumulative mass ﬂux and to avoid confusion, we keep the designation SWE for the snow cover but use kg m−2 for cumulative mass ﬂux in the following, although SWE has the same units. for snow particles was calculated as (Bagnold, 1941) for snow particles was calculated as (Bagnold, 1941) for snow particles was calculated as (Bagnold, 1941) perature HMT330 sensors (Vaisala, 2009). The University of Colorado/NOAA surface ﬂux team carried out the post- processing and computed turbulent ﬂuxes, such as momen- tum ﬂux and turbulent heat ﬂuxes, mixing ratio, or friction velocity. Wind vectors were corrected; i.e. processed wind directions are according to geographic true north. We used wind velocity, wind direction, computed latent heat ﬂux, fric- tion velocity, relative humidity, the temperature at 2 m and temperature of the snow surface from the described dataset (Cox et al., 2021). u∗t = A · rρice −ρair ρair gd, (9) u∗t = A · rρice −ρair ρair gd, (9) where A is a threshold parameter and is here assumed to be 0.18 as found by Clifton et al. (2006) for drifting snow initiation, ρice = 917 kg m−3 is the density of ice, ρair is the density of air, g = 9.81 m s−1 is gravity acceleration on earth and d is an average particle diameter, which we assumed to be 260 µm, which was found as the lowest particle diameter on the surface where snow transport was observed by Clifton et al. (2006). ρair could be retrieved from the meteorological tower data. The computed thresholds were applied to com- puted u∗from the tower. If u∗> u∗t, particles begin to get lifted from the ground, and drifting snow ﬂux is initiated. 2.7 ERA5 mean snowfall rates For the drift track coordinates of RV Polarstern, shown in Fig. 1, we extracted ERA5 (Hersbach et al., 2020) mean snowfall rates, which are the sum of the convective and large- scale snowfall in ERA5. While the large-scale snowfall is generated from the cloud scheme in the ECMWF Integrated Forecasting System (IFS) (IFS Documentation CY47R1 – Part IV, 2020), the convective snowfall is generated from the IFS convection scheme. The resolution for ERA5 over the sea is 0.28125◦× 0.28125◦, which is about 31 km × 31 km. Hence, the extracted snowfall rate from ERA5 for the drift track does not refer to points but represents an averaged value over these grid cells closest to the drift track coordinates. The purpose here is to compare the ERA5 mean snowfall against snow cover SWE and sensors in this study. To determine periods where snow transport and erosion have occurred, horizontal mass ﬂux (kg m−2 s−1) for both SPCs was computed as (Sugiura et al., 2009) QSPC = π ρp 6 64 X n=1 Sn Nn D3 n, (8) (8) Manufacturer The Cryosphere, 16, 2373–2402, 2022 2385 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 3.1 Snow mass accumulation and decrease Figure 8a shows the derived SWE evolution as box-and- whisker plots for the northern and southern transects. The initial average SWE values for the northern loop (66 mm on 31 October) are naturally higher than for the southern loop (32 mm on 31 October), as the northern transect was situated mostly on deformed SYI. The value for the northern loop decreased to 65 mm until 5 December, while average SWE for the southern loop increased by 29 mm to a similar value of 66 mm between 14 November and 5 December 2019. The SWE on both transect loops increased to 92 mm on the north- ern loop and 80 mm on the southern loop, between 5 De- cember 2019 and 20 February 2020. From then on, there was a decrease observed in both loops, with a minimum of 79 mm on the northern loop on 6 April 2020 and a minimum of 73 mm on the southern loop on 5 March 2020. On both transects, SWE increased afterward, to 90 mm on the north- ern loop by 24 April 2020 and 81 mm on the southern loop by 26 April 2020. Hence, even though the initial SWE over the remnant SYI and FYI (southern loop) was approximately only half of the value on the northern loop, it reached 90 % of the snow mass of the northern loop by the end of the ac- cumulation period. This average computed snow cover SWE serves then as our reference for the precipitation sensors and ERA5 snow- fall. Note that for the averaging process, data were discarded when only the northern or southern transect loop was mea- sured on 1 d, except when the temporal distance between the measurement of northern and southern loops was short and there was no snowfall in between. This is only the case for 24 (northern) and 26 (southern) April. Hence, the transect av- eraged SWE starts on 31 October 2019 and ends on 24 and 26 April 2020, with a signiﬁcant reduction of days of sam- pling, compared to all days of transect sampling available (Table 2). RMSE between snow cover SWE and sensor- and reanalysis-estimated SWE was computed for the time period where no SWE decrease in between the days of the transect sampling has been detected by means of the computed snow cover SWE, which is all days before and including 20 Febru- ary 2020. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2386 be representative for the MOSAiC ice ﬂoe. Indeed it is im- possible to determine at this point with this dataset we made use of whether only the SWE derived from the northern or southern or the average of loops, is the best choice to evalu- ate snowfall. A snow height difference dataset based on laser scanners of an area that includes both northern and southern transects and an area beyond that could be used to validate transect snow depth. However, we do not make use of such a dataset here. We will demonstrate in the coming sections that initial average SWE on the northern loop is about twice the value of the average SWE on the southern loop. sect SWE time series for evaluation; hence n = 6 d, which are the days before and after the drifting snow events, were left over for comparison with sensors and ERA5. This results in n = 5 pairs for error calculation. The drifting-snow-free periods are marked as yellow areas in the graph of Fig. 11f. Note that due to the strong cumulative aspect (i.e. we com- pare snowfall that is always accumulated between the days of transect measurements), the difference is naturally reduced when reducing the sample number. The reduction of valida- tion pairs reduces the signiﬁcance of the comparison; how- ever, we additionally inter-compare the error change of the sensors and ERA5, which may strengthen the signiﬁcance considering the role of wind. g p The SWE increase until January 2020 is much faster on the southern loop; hence, we see a different accumulation rate depending on whether we measure snow depth on SYI (northern loop) or FYI (southern loop). Indeed, what “most representative” means also strongly depends on the horizon- tal extent of snowfall and wind patterns, i.e. the total accu- mulated snow mass that has fallen over a certain area but is re-deposited due to wind. This is a problem we are not able to consider in this study but that can potentially be solved by computing snow mass based on the difference of airborne or terrestrial laser-scan-derived heights. The reason why we decided to use an SWE average of the northern and southern loops as reference is that the MOSAiC ice ﬂoe consisted in large parts of these two ice types. 2.8 Sensor and reanalysis comparison method where ρp is the density of a drifting snow particle, which we assumed here to be the density of ice ρp = 917 kg m−3; Sn is the shape factor of snow particles of the nth class, which we assumed to be 1 here; Nn is the particle ﬂux of the nth class (m−2 s−1), which is the number of particles per class passing the SPC sensor area As in a second; and Dn is the diameter of a drifting snow particle of the nth class (m). We computed the average SWE for the northern and southern loops (Table 2) as we expect this combination of deformed SYI, remnant SYI and FYI is more representative for an over- all snow accumulation estimate than choosing a snow depo- sition for one of these ice types. Additionally, in Sect. 3 and Fig. 9 we will show that change of average SWE along a sec- tion of the whole transect several hundreds of metres long is over 200 % from the average SWE along the whole north- ern transect, compared before and after a drifting snow event. This conﬁrms the need for as long of transect sections as pos- sible to ﬁnd an average snow mass value for an area that can It is likely that the distance between sensor and snow cover varied over time since installation on 2 December 2019 due to deposition of new snow. In any case, given these uncer- tainties, to determine potential drifting snow periods for pe- riods where the SPC might fail, the critical friction velocity https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 3.1 Snow mass accumulation and decrease Here we used n = 10 d for subtracting SWE, which results in n = 9 d for error comparison. The RMSE is com- puted as millimetres and always refers to the precipitation sum between days of transect sampling. In the following, we present detailed results about snow mass decrease. Notably, we ﬁnd a net mass decrease, com- puted with the HS-SWE function, of 9.5 mm for the north- ern transect loop between 27 February and 20 March. The mass increased again to 90 mm between 20 March and 24 April 2020. The maximum on the northern loop is reached with 94 mm on 7 May 2020. This, however, is not compa- rable with the southern loop as the southern transect time series only last until the end of April 2020. The SWE maximum on the southern loop is found to be 81 mm on 26 April 2020. The time from 20 February to 20 March falls exactly into the period where (1) the discrepancy be- tween cumulative SWE from the precipitation sensors and SWE from the transect becomes large (Figs. 10b, 11a) and To discuss the erosion inﬂuence on potential discrepan- cies of snow cover SWE and sensor-estimated snowfall in more detail, in addition, RMSE was computed for days af- ter time periods where no signiﬁcant amounts of horizontal mass ﬂux were detected with the SPCs, i.e. where no erosion between 2 d of transect sampling was expected. These de- tected drifting snow periods until 20 February were 3–5 De- cember, 19 December 2019, 30 January–2 February and 18– 20 February 2020. Hence, in this case, 5 December 2019, 6 February and 20 February 2020 were discarded from tran- https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2387 Table 2. Used transect days of sampling and speciﬁcations: transect N–S refers to the northern–southern transect; averaged for validation refers to the days when the transect SWE was averaged as shown in Fig. 8; RMSE with drifting snow describes all days when SWE was subtracted from the respective day before, while all days where subtracted in a row; and RMSE without drifting snow/pair refers to the days used for validation when no or low drifting snow mass ﬂux was detected in between while the respective days after which subtraction was performed consecutively are listed as well. The corresponding RMSE values including regression lines are shown in Fig. 12. The comparison with bulk SWE and SMP refers to the dates where direct comparisons are made with SWE along the transect derived from the HS-SWE (Fig. 5). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Date Transect Averaged for validation RMSE with RMSE without drifting Comparison with N–S drifting snow snow/pair bulk SWE and SMP 24 Oct 2019 N – – – – 31 Oct 2019 N–S yes yes (start) – – 7 Nov 2019 N–S yes yes yes (start)/14 Nov 2019 – 14 Nov 2019 N–S yes yes yes/7 Nov 2019 yes 21 Nov 2019 N – – – yes 28 Nov 2019 N – – – yes 5 Dec 2019 N–S yes yes – – 19 Dec 2019 N – – – 2 Jan 2020 N–S yes yes yes/5 Dec 2019/9 Jan 2020 – 9 Jan 2020 N–S yes yes yes/2 Jan 2020/16 Jan 2020 – 16 Jan 2020 N–S yes yes yes/9 Jan 2020/30 Jan 2020 – 30 Jan 2020 N–S yes yes yes (end)/16 Jan 2020 – 6 Feb 2020 N–S yes yes – – 20 Feb 2020 N–S yes yes (end) – – 27 Feb 2020 N–S yes – – – 5 Mar 2020 N–S yes – – – 20 Mar 2020 N – – – – 26 Mar 2020 N – – – – 30 Mar 2020 S – – – – 6 Apr 2020 N – – – – 16 Apr 2020 N – – – – 24 Apr 2020 N yes (avg with 26 Apr 2020) – – – 26 Apr 2020 S yes (avg with 24 Apr 2020) – – – 30 Apr 2020 N – – – – 7 May 2020 N – – – – (2) where about 45 % (1.977 × 106 kg m−2) of the total cu- mulative horizontal snow mass ﬂux at 0.1 m above the sur- face over the whole measuring period of the SPC1104 has occurred (Fig. 11f). That means over 45 % of drifted snow mass appeared on only 19 % (30 out of 158) of the days from the whole measuring time of the lower SPC. The period is marked as green shaded areas in Fig. 11. Most distinct in this period was the event on 24–25 February (marked as red shaded areas in Fig. 11), during which 1.014 × 106 kg m−2 of cumulative mass ﬂux was detected with the lower SPC – which is 23 % of the total detected cumulative mass ﬂux on 1.3 % of the days the device was running. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons During this storm, a maximum peak of around 11 m s−1 was detected in the 1 h averaged wind speed data at 2 m above the ice, which means that the measured peak at shorter time inter- vals must have been higher. We computed the sum of the detected mass decrease that occurred between days of sam- pling and that is driven by erosion (but does not reﬂect the to- tal eroded mass) for the northern loop as 1SWE = −24 mm (2) where about 45 % (1.977 × 106 kg m−2) of the total cu- mulative horizontal snow mass ﬂux at 0.1 m above the sur- face over the whole measuring period of the SPC1104 has occurred (Fig. 11f). That means over 45 % of drifted snow mass appeared on only 19 % (30 out of 158) of the days from the whole measuring time of the lower SPC. The period is marked as green shaded areas in Fig. 11. Most distinct in this period was the event on 24–25 February (marked as red shaded areas in Fig. 11), during which 1.014 × 106 kg m−2 of cumulative mass ﬂux was detected with the lower SPC – which is 23 % of the total detected cumulative mass ﬂux on 1.3 % of the days the device was running. During this storm, a maximum peak of around 11 m s−1 was detected in the 1 h averaged wind speed data at 2 m above the ice, which means that the measured peak at shorter time inter- vals must have been higher. We computed the sum of the detected mass decrease that occurred between days of sam- pling and that is driven by erosion (but does not reﬂect the to- tal eroded mass) for the northern loop as 1SWE = −24 mm (between 31 October 2019–26 April 2020) and for the south- ern loop as 1SWE = −16 mm (between 31 October 2019– 24 April 2020). Figure 9 shows the SWE for the same section of the northern loop transect as in Fig. 5 (268 m length), for 20 February and 5 March 2020, which are before and after a drifting snow event. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons As the section on 5 March had twice the average horizontal Magnaprobe sampling distance compared to 20 February, and for better illustration, a simple moving average with a window of n = 4 was applied to the section on 20 February while a moving average with a window of n = 2 was applied to the data from 5 March. A signiﬁcant re-distribution due to wind is recognizable. During the same period, the average decrease for this section was 12 mm SWE (from 82.6 to 70.1 mm – a relative value of 15 %), while for the whole northern loop the average decrease was 8 mm (from 87.3 to 79.5 mm – a relative value of 9 %). (between 31 October 2019–26 April 2020) and for the south- ern loop as 1SWE = −16 mm (between 31 October 2019– 24 April 2020). Figure 9 shows the SWE for the same section of the northern loop transect as in Fig. 5 (268 m length), for 20 February and 5 March 2020, which are before and after a drifting snow event. As the section on 5 March had twice the average horizontal Magnaprobe sampling distance compared to 20 February, and for better illustration, a simple moving average with a window of n = 4 was applied to the section on 20 February while a moving average with a window of n = 2 was applied to the data from 5 March. A signiﬁcant re-distribution due to wind is recognizable. During the same period, the average decrease for this section was 12 mm SWE (from 82.6 to 70.1 mm – a relative value of 15 %), while for the whole northern loop the average decrease was 8 mm (from 87.3 to 79.5 mm – a relative value of 9 %). – which is 23 % of the total detected cumulative mass ﬂux on 1.3 % of the days the device was running. During this storm, a maximum peak of around 11 m s−1 was detected in the 1 h averaged wind speed data at 2 m above the ice, which means that the measured peak at shorter time inter- vals must have been higher. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons We computed the sum of the detected mass decrease that occurred between days of sam- pling and that is driven by erosion (but does not reﬂect the to- tal eroded mass) for the northern loop as 1SWE = −24 mm Finally, we present results of the average SWE of the northern and southern loop (Fig. 8b). The average initial SWE value on 31 October 2019 was 48 mm and increased https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 gner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2388 Figure 8. (a) Box-and-whisker plots for SWE estimates along the northern (“Nloop”) and southern (“Sloop”) transect. (b) Box-and-whisker plots for averages of the northern and southern transect loops. Horizontal lines show the median, green triangles the average, the boxes show the interquartile ranges (IQR) (25 %–75 %), and the whiskers represent 1.5 times the upper and lower values of the IQR. The dots represent outliers that are beyond 1.5 times the IQR. Note the different dates between (a) and (b) as data where only data for one loop were available were discarded for computation of (b). The exception is 24 and 26 April 2020 as the temporal distance was so close that these were averaged, too. Figure 8. (a) Box-and-whisker plots for SWE estimates along the northern (“Nloop”) and southern (“Sloop”) transect. (b) Box-and-whisker plots for averages of the northern and southern transect loops. Horizontal lines show the median, green triangles the average, the boxes show the interquartile ranges (IQR) (25 %–75 %), and the whiskers represent 1.5 times the upper and lower values of the IQR. The dots represent outliers that are beyond 1.5 times the IQR. Note the different dates between (a) and (b) as data where only data for one loop were available were discarded for computation of (b). The exception is 24 and 26 April 2020 as the temporal distance was so close that these were averaged, too. to 86 mm on 24 and 26 April 2020. Hence, we estimate the total mass increase over time as about 38 mm. Over the tran- sect average, the SWE decrease during the snow transport event was 5.5 mm on 24–25 February 2020. ability and wide spread between these two identical systems operating at different locations, we no longer consider the P2MC. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons The PWD22PS shows the lowest cumulative snow- fall with 97.6 mm, while the highest is estimated by the P2PS (290.3 mm). It also stands out that the cumulative sum of ERA5 (110 mm) by 7 May 2020 is very similar to that of the snowfall estimated from the KAZR (114 mm). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons For this case the PWD22PS is most similar to the SWE (RMSE = 2.01 mm), followed by ERA5 (3.33 mm), the KAZR (4.65 mm), Pluvio2 (6.7 mm), PWD22MC (8.72 mm) and P2PS (26.39 mm). Figure 10. (a) Cumulative snowfall for different installed precipita- tion sensors during MOSAiC from 31 October 2019 to 7 May 2020. (b) Sensors, ERA5 estimates and SWE of the snow cover. The red dots show the days on which both transect loops were sampled. The red shading shows the time period of the strong drifting snow event on 24–25 February 2020. The green shaded area marks the strong drifting snow period where 45 % of all cumulative horizontal mass ﬂux was detected (Fig. 11). largest difference of the sensors relative to the snow cover is found for P2PS (RMSE = 3.12 mm). Note that due to the strong cumulative aspect (i.e. we com- pare snowfall that is always accumulated between the days of transect measurements), the difference is naturally reduced when reducing the sample number. Nonetheless, the fact that there is an overall tendency towards a decrease in the ap- parent overestimation of the sensors relative to the SWE in- dicates that erosion likely did occur before the days elimi- nated in Fig. 12b. For the PWD22PS, the RMSE is only re- duced by about 2.5 %, for ERA5 by 13 % and the KAZR by 23.7 % while for P2PS the RMSE was reduced by 88 % and for PWD22MC reduced by 69 %. For the Pluvio2, the differ- ence was reduced to 26 % of its initial value. While the ap- parent overestimation of the sensors in Fig. 12a is likely due to erosion (and hence strongly biased), these different magni- tudes of RMSE reduction (Fig. 12b) suggest that PWD22PS is less affected by overestimation due to high wind speeds that accompany blowing snow, compared to PWD22MC or Pluvio2, both of which were installed near the surface, but However, in the second case (n = 6 d), the differences are reduced signiﬁcantly for all devices and ERA5, too. In this case, Pluvio2 shows the closest comparison to the SWE (RMSE = 1.72 mm). PWD22PS shows good agree- ment (RMSE = 1.96 mm) with a tendency towards un- derestimation. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2389 D. N. Wagner et al.: Snowfall and snow accumulation durin Figure 9. Retrieved SWE from same Magnaprobe section as shown in Fig. 5 on 20 February before a strong drifting snow event that occurred on 24–25 February and mass distribution on 5 March 2020 after the drifting snow event. The superscript numbers in brackets in the legend correspond to the count of numbers of the moving median window used for plotting. Figure 10. (a) Cumulative snowfall for different installed precipita- tion sensors during MOSAiC from 31 October 2019 to 7 May 2020. (b) Sensors, ERA5 estimates and SWE of the snow cover. The red dots show the days on which both transect loops were sampled. The red shading shows the time period of the strong drifting snow event on 24–25 February 2020. The green shaded area marks the strong drifting snow period where 45 % of all cumulative horizontal mass ﬂux was detected (Fig. 11). Figure 9. Retrieved SWE from same Magnaprobe section as shown in Fig. 5 on 20 February before a strong drifting snow event that occurred on 24–25 February and mass distribution on 5 March 2020 after the drifting snow event. The superscript numbers in brackets in the legend correspond to the count of numbers of the moving median window used for plotting. stagnated, while the sensors indicate periodic snowfall. Thus, the discrepancy between sensor snowfall rates and snowpack SWE became larger. RMSE with respect to SWE difference is shown for snow- fall sensors, KAZR and ERA5 (Fig. 12). We consider the case ﬁrst where snowdrift time periods are included in the evaluation (i.e. all days of sampling until and including 20 February 2020). For this period, a SWE increase in the snow cover of about 37 mm was detected (Fig. 12a, cumu- lated within the intervals in between n = 9 d). Figure 12b, in contrast, shows RMSE computed only for days when no drifting or low drifting snow occurred (n = 6 d). For this time period, an increase of 13.7 mm SWE was detected for the transect. Considering the ﬁrst case (n = 9 d), all sen- sors appear to overestimate. However, as expected, this in- dicates that erosion occurred in the time periods between the days the transects were sampled, which leads to a systematic positive bias of the sensors. 3.2 Precipitation sensor and radar snowfall retrieval comparisons Figure 10b compares the northern, southern and average transect loop SWE values with the uncorrected cumulative snowfall from a subset of the sensors and the ERA5 mean snowfall. The PWD22PS is well in line with SWE from the snow cover until the middle to end of February 2020. Af- terwards, more snow over the ice was eroded, which is indi- cated by high horizontal drifting and blowing snow mass ﬂux measured with both SPCs (Fig. 11f). After mid-February, the snow cover SWE did not increase signiﬁcantly and instead To compare with the different estimates of snowfall, the cu- mulative SWE values were examined for each approach. The plot of cumulative snowfall between 31 October 2019 and 7 May 2020 without any corrections applied can be seen in Fig. 10a. The snowfall rates deviate heavily from one another between precipitation data source and locations. The P2PS shows the highest cumulative snowfall, while the P2MC shows the lowest, although with limited data availabil- ity (Bartholomew, 2020b). As a result of the limited avail- https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2390 2390 D. N. Wagner et al.: Snowfall and snow ac Figure 11. Time series from 31 October 2019 to 7 May 2020 for (a) estimated SWE from the transect and HS-SWE model as well as cumulative snowfall from ERA5, cumulative snowfall from PWD22 on Polarstern and KAZR-derived snowfall rates. (b) Wind direction at 2 m, (c) air temperature at 2 m above the snow, (d) wind speed at 2 m height, (e) computed friction velocity threshold for snow trans- port after Bagnold (1941) and (f) cumulative horizontal mass ﬂux with the snow particle counter at 0.1 m above the snow and at 10 m height. The green shaded areas mark the strong drifting snow period where 45 % of all cumulative horizontal mass ﬂux was detected. The vertical blue dashed lines mark the days where both transect loops were sampled. The yellow shaded areas mark the time peri- ods when no to very low mass was detected by means of the snow particle counters. cumulation during the MOSAiC winter and spring seasons Figure 11. Time series from 31 October 2019 to 7 May 2020 for (a) estimated SWE from the transect and HS-SWE model as well as cumulative snowfall from ERA5, cumulative snowfall from PWD22 on Polarstern and KAZR-derived snowfall rates. (b) Wind direction at 2 m, (c) air temperature at 2 m above the snow, (d) wind speed at 2 m height, (e) computed friction velocity threshold for snow trans- port after Bagnold (1941) and (f) cumulative horizontal mass ﬂux with the snow particle counter at 0.1 m above the snow and at 10 m height. The green shaded areas mark the strong drifting snow period where 45 % of all cumulative horizontal mass ﬂux was detected. The vertical blue dashed lines mark the days where both transect loops were sampled. The yellow shaded areas mark the time peri- ods when no to very low mass was detected by means of the snow particle counters Figure 12. (a) RMSE (mm) of the sensors and ERA5 with respect to snow cover SWE, for the time period before 20 February 2020 with n = 9 d of sampling, including days when drifting snow was detected. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Panel (b) is as (a) but without days when drifting snow was detected before (n = 6 d). The lines show the linear regression for each sensor. SWE, while especially P2PS, PWD22MC and Pluvio2 ap- pear to be most negatively affected by high wind speeds. Taken together, we detected ﬁve signiﬁcant snowfall events. If we use PWD22PS as reference, we ﬁnd the follow- ing for 3 5 December 2019: ≈5 5 mm 30 January 3 Febru Figure 12. (a) RMSE (mm) of the sensors and ERA5 with respect to snow cover SWE, for the time period before 20 February 2020 with n = 9 d of sampling, including days when drifting snow was detected. Panel (b) is as (a) but without days when drifting snow was detected before (n = 6 d). The lines show the linear regression for each sensor. Figure 11. Time series from 31 October 2019 to 7 May 2020 for (a) estimated SWE from the transect and HS-SWE model as well as cumulative snowfall from ERA5, cumulative snowfall from PWD22 on Polarstern and KAZR-derived snowfall rates. (b) Wind direction at 2 m, (c) air temperature at 2 m above the snow, (d) wind speed at 2 m height, (e) computed friction velocity threshold for snow trans- port after Bagnold (1941) and (f) cumulative horizontal mass ﬂux with the snow particle counter at 0.1 m above the snow and at 10 m height. The green shaded areas mark the strong drifting snow period where 45 % of all cumulative horizontal mass ﬂux was detected. The vertical blue dashed lines mark the days where both transect loops were sampled. The yellow shaded areas mark the time peri- ods when no to very low mass was detected by means of the snow particle counters. Figure 12. (a) RMSE (mm) of the sensors and ERA5 with respect to snow cover SWE, for the time period before 20 February 2020 with n = 9 d of sampling, including days when drifting snow was detected. Panel (b) is as (a) but without days when drifting snow was detected before (n = 6 d). The lines show the linear regression for each sensor. SWE, while especially P2PS, PWD22MC and Pluvio2 ap- pear to be most negatively affected by high wind speeds. Taken together, we detected ﬁve signiﬁcant snowfall events. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons It reveals that ERA5 also performs well (RMSE = 2.88 mm), but with an overestimation tendency, as all validation pairs were positively biased with an average of 2.0 mm. Besides ERA5, the KAZR (RMSE = 3.55 mm), sys- tematically positively biased with 2.4 mm, shows the least RMSE decrease compared to the case using n = 9 d, which is very likely the result of the fact that both ERA5 and KAZR are not wind-vulnerable in contrast to the other sensors. The https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 also compared against P2PS, which is a device known for wind vulnerability towards overestimation of snowfall. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons that the Pluvio2 (Fig. 13b) and the PWD22MC (Fig. 13d) strongly overestimate snowfall relative to PWD22PS, while P2PS (Fig. 13a) and KAZR (Fig. 13d) stay largely unaffected and only measure trace precipitation of 0.1 to 0.2 mm h−1. This becomes even clearer when we look at snowfall rates of Pluvio2 (Fig. 14a) and PWD22MC (Fig. 14b) versus the horizontal mass ﬂux detected by the SPCs. that the Pluvio2 (Fig. 13b) and the PWD22MC (Fig. 13d) strongly overestimate snowfall relative to PWD22PS, while P2PS (Fig. 13a) and KAZR (Fig. 13d) stay largely unaffected and only measure trace precipitation of 0.1 to 0.2 mm h−1. This becomes even clearer when we look at snowfall rates of Pluvio2 (Fig. 14a) and PWD22MC (Fig. 14b) versus the horizontal mass ﬂux detected by the SPCs. For the evaluation of the SWE increase and decrease, note that the net decrease generally includes eroded mass in ad- dition to incoming precipitated mass; hence the eroded mass was often larger than the precipitated mass. These two quan- tities can only be determined separately when considering snowfall events and no drifting snow events at the same time in between days of sampling. The constant value of SWE from 20 February on raises the question of a saturation mass of snow that can accumulate in this windy environment, where the roughness of the surface might be a limiting factor. Possibly, this is due to smoothing of the initially rough sea ice surface due to deposited drifting snow. The less rough the surface, the less snow can accumu- late. This may also be related to the faster increase in SWE on FYI compared to SYI and the fact that the initial 50 % of SWE over FYI reached 90 % of the SWE over SYI by the end of the investigation period. One hypothesis might be that over SYI the snow mass is rather saturated, as the snow had time to smooth the ridges already over one season before, while FYI might be more rough as long as fresh ridges are existent. Weiss et al. (2011) found in general higher aero- dynamic roughness lengths for SYI. With higher roughness length we expect more ﬂow separation and more accumula- tion. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons However, the study is more than 10 years old, and given the fact that average ice thickness is thinning in the context of global warming, we might see less stable FYI by the year 2019/2020, resulting in more ice motion and ridge formation – and leading to larger aerodynamic roughness lengths. Scatter plots for the whole period (31 October 2019– 7 May 2020) for different sensors versus horizontal mass ﬂux reveal the inﬂuence of drifting and blowing snow, too (Fig. 15). Pearson correlation coefﬁcients show medium pos- itive correlations for mass ﬂux and snowfall from Pluvio2 at SPC height at 0.1 m (Fig. 15b, r = 0.58) and PWD22MC (Fig. 15d, r = 0.55) while a weak negative correlation is observed for P2PS (Fig. 15a, r = −0.2) and for PWD22PS (Fig. 15c, r = 0.26). These results indicate that instruments collocated on the ice at about 1–1.5 m height are much more affected by drifting and blowing snow than instruments in- stalled on Polarstern at 22 m height. We compare ERA5 mean snowfall rates against the snow cover SWE and against PWD22PS. As described above, when we consider the comparisons illustrated in Fig. 12b, ERA5 shows reasonable results with a relatively low RMSE of 2.9 mm and an overestimation tendency. As- suming PWD22PS as reference, ERA5 shows an overall good timing of the snowfall events (Figs. 10b, 11a). As for the transect SWE validation, it overestimates snowfall rela- tive to PWD22PS, too, in this case systematically and with an acceleration of the positive bias from the end of February on. This leads to an overestimation (relative to PWD22PS) of the total accumulation of almost 22 mm (+25 %) by the end of the investigation period. We computed the RMSE for the snowfall rate relative to PWD22PS as 0.06 mm h−1 for the whole time period from 31 October to 7 May 2020. g g y g g It also remains to be investigated why most drifting snow and erosion occurred from 20 February 2020 on, as indicated by transect SWE decrease and horizontal mass ﬂux. Indeed, snow mass ﬂux rates are high until 27 February, but on the sampling day itself, the wind speed as well as the mass ﬂux rate dropped (Fig. 11). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Between 27 February and 5 March, the probability for snow transport was very low, indicated by both SPC mass ﬂux and computed threshold friction veloc- ity. One might consider that lower horizontal sampling dis- tance (D5 Mar = 1.6 m) could have led to an underestimation of the accumulated snow. However, from Fig. 6 one can see that reducing the sampling frequency by half has no signif- icant effect on the average. One can only see a signiﬁcant ﬂuctuation from reducing the frequency to one-third and be- low. Hence, a reduced sampling frequency does not explain the mass decrease. Ice dynamics that affected the transect oc- curred from 11 and 12 March 2020 and thereafter. Hence, no ice dynamics inﬂuenced the sampling on 5 March 2020. Nev- ertheless, an impact on the time series cannot be excluded from 12 March 2020 on. Looking at horizontal mass ﬂux and decreased computed snow mass together, we cannot say for sure that erosion was largely responsible for the mass decrease between 27 February and 5 March. The threshold friction velocity was still exceeded on 27 February, indicat- ing snow transport, while low recorded mass ﬂux indicates low transport. The computed threshold friction velocity af- ter Bagnold (1941) (Fig. 11e) does not indicate much more D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons If we use PWD22PS as reference, we ﬁnd the follow- ing for 3–5 December 2019: ≈5.5 mm, 30 January–3 Febru- ary 2020: ≈10 mm, 18–21 February 2020: ≈8.5 mm, 16– 21 April 2020: ≈16.5 mm and 4–7 May 2020: ≈14 mm. Hence, about 54 mm of snow fell during events, while the other 33 mm fell in between, e.g. as trace precipitation or di- amond dust. also compared against P2PS, which is a device known for wind vulnerability towards overestimation of snowfall. In summary, if we only consider time periods with- out drifting snow, Pluvio2 and PWD22PS compare most favourably with SWE, however with reasonable results for ERA5, KAZR, PWD22MC and P2PS, as well. When also considering high wind speeds and blowing snow, the PWD22PS still appears to compare most favourably with To better illustrate the blowing snow inﬂuence on sen- sors that is already suggested by Fig. 12, we made scatter plots of snowfall rates from different sensors with respect to the PWD22PS. Figure 13 shows a scatter plot for the short time of 2 d between 24 and 25 February, where high drifting snow mass ﬂuxes were detected. We can clearly see https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 2391 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 4.1 Snow mass balance With the ﬁtted HS-SWE function, we were able to retrieve the SWE of snow cover over the ice for the transect loops. We could show that comparing the average SWE of a north- ern loop section with 268 m length (Fig. 9) with the average SWE for the whole northern loop, the SWE change due to a drifting snow event was different by more than 100 %. This shows the need for sampling with large spatial extents which was one reason – besides including both characteristic ice types for the ice ﬂoe during MOSAIC, SYI and FYI – why we decided to use the SWE average of both northern and southern loops as a reference for snowfall sensor and reanal- ysis comparison. Nonetheless, a snow height difference com- parison of terrestrial laser scanning (TLS) or airborne laser scanning (ALS) digital elevation models and transect snow depth would be desirable. https://doi.org/10.5194/tc-16-2373-2022 https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2392 Figure 13. Scatter plots of PWD22PS snowfall rates vs. different sensor snowfall rates for the drifting snow event on 24–25 February 2020 for (a) P2PS, (b) Pluvio2, (c) KAZR and (d) PWD22MC. Figure 13. Scatter plots of PWD22PS snowfall rates vs. different sensor snowfall rates for the drifting snow event on 24–25 February 2020 for (a) P2PS, (b) Pluvio2, (c) KAZR and (d) PWD22MC. or less drifting snow compared to other time periods. How- ever, the formula after Bagnold (1941) is rather simple and neglects varying temperatures and therefore varying bond strengths of snow particles at the top of the snowpack. The bond strength depends strongly on weather history. The pre- ceding long period of air and snow surface temperatures of- ten below −30 ◦C (Fig. 11c) could have inhibited sintering, which is strongly temperature-dependent and develops more slowly at lower snow temperatures (Colbeck, 1997, 1998; Blackford, 2007). The Bagnold formula does not consider splash entrainment (Comola et al., 2017; Comola and Lehn- ing, 2017) or surface roughness and atmospheric stability, which largely affects the near-surface wind ﬁeld. Further- more, the used bond strength parameter of A = 0.18 we used in Eq. (9) was found by Clifton et al. (2006) in a wind tunnel, with an ambient temperature range of −16 to 0 ◦C. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2) and the wind direction around 24–25 February (between north and north-east) (Fig. 11b), it is evident that the northern transect was likely to be partially wind-shadowed by higher surface structures on the upwind side. This wind shadowing can lead to less wind transport downwind but maybe also to less ero- sion due to lower surface friction. This is suggested by our ﬁndings, too, as we found more substantial erosion over the northern transect loop. During the drifting snow event, the southern transect was often in the wind shadow of the ves- sel and other installations (Fig. 2), leading to deceleration and less surface friction that could have led to snow trans- port or erosion. In the case of more deposition upwind, the mass over the sampled area would decrease even if the fric- tion velocity behind the obstacles is decreased, which leads to less erosion. The computed SWE would not be represen- tative for the whole ﬂoe in this case. While potentially in- teresting, the method used in our study does not allow the investigation of these balances. Another reason for a com- puted SWE decrease during the later part of the observations might be wind-induced compaction of the upper snowpack. With our approach, we assume almost a linear relationship between snow depth and SWE. However, snowdrifts con- sist of densely packed, rounded grains (Fierz et al., 2009). Hence, in this case, we probably under-estimate the increase (decrease) of the SWE in the case of drifting snow depo- sition (erosion). A thorough investigation is recommended but beyond the scope of this study. Further studies based on SMP measurements are planned. Another reason for un- derestimating snow depth with spatio temporal sampling ap Figure 14. Scatter plots of sensor snowfall rates (y axis) vs. SPC mass ﬂux (x axis) for the drifting snow event on 24–25 Febru- ary 2020 for (a) Pluvio2 and (b) PWD22MC. the total snowfall would then be at least about 98 mm in 189 d. If we assume the estimated snowfall from the KAZR to be the upper limit for cumulative snowfall (as it was demonstrated that Pluvio2 and PWD22MC were affected by wind and blowing snow towards overestimation), we ﬁnd about 114 mm. This value is comparable with the cumula- tive snowfall from ERA5 (110 mm). D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Studies of Déry and Tremblay (2004), Leonard et al. (2008), and Leonard and Maksym (2011) indicate that, be- sides sublimation, large parts of drifting and blowing snow will drift into the open water of leads. However, for the drift- ing snow event on 24–25 February, there was no signiﬁcant open water area in the vicinity. Thus, without such a local sink, we would expect that even though snow was eroded, more snow mass would be delivered from the upwind side at approximately the same amount. However, the given mass of drifting snow from the upwind side depends on the low- pressure system’s extent and trajectory associated with the high wind speeds. In Fig. 11 we see that after the event, some atmospheric parameters have changed signiﬁcantly and rapidly (a temperature drop, a decrease in wind speed). The rapid change of atmospheric conditions could explain why no “new” snow has been transported from the windward side. Another important factor could be that the area the transect covers has a relatively low surface roughness, with ridges that were generally shorter than in the surrounding region. Ero- sion might be large over these relatively smooth areas that the transect covers, and the delivered drifting snow from the up- wind side could have been deposited at higher ridges upwind. But even a just frozen lead will be ﬁlled by drifting snow after a while, which would lead to a mass sink on the upwind side, inhibiting further snow transport downwind. Considering the ALS-based digital elevation map of the ﬂoe (Fig. 2) and the wind direction around 24–25 February (between north and north-east) (Fig. 11b), it is evident that the northern transect was likely to be partially wind-shadowed by higher surface structures on the upwind side. This wind shadowing can lead to less wind transport downwind but maybe also to less ero- sion due to lower surface friction. This is suggested by our ﬁndings, too, as we found more substantial erosion over the northern transect loop. During the drifting snow event, the southern transect was often in the wind shadow of the ves- sel and other installations (Fig. 2), leading to deceleration and less surface friction that could have led to snow trans- port or erosion. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons In the case of more deposition upwind, the mass over the sampled area would decrease even if the fric- tion velocity behind the obstacles is decreased, which leads to less erosion. The computed SWE would not be represen- tative for the whole ﬂoe in this case. While potentially in- teresting, the method used in our study does not allow the investigation of these balances. Another reason for a com- puted SWE decrease during the later part of the observations might be wind-induced compaction of the upper snowpack. With our approach, we assume almost a linear relationship between snow depth and SWE. However, snowdrifts con- sist of densely packed, rounded grains (Fierz et al., 2009). Hence, in this case, we probably under-estimate the increase (decrease) of the SWE in the case of drifting snow depo- sition (erosion) A thorough investigation is recommended Studies of Déry and Tremblay (2004), Leonard et al. (2008), and Leonard and Maksym (2011) indicate that, be- sides sublimation, large parts of drifting and blowing snow will drift into the open water of leads. However, for the drift- ing snow event on 24–25 February, there was no signiﬁcant open water area in the vicinity. Thus, without such a local sink, we would expect that even though snow was eroded, more snow mass would be delivered from the upwind side at approximately the same amount. However, the given mass of drifting snow from the upwind side depends on the low- pressure system’s extent and trajectory associated with the high wind speeds. In Fig. 11 we see that after the event, some atmospheric parameters have changed signiﬁcantly and rapidly (a temperature drop, a decrease in wind speed). The rapid change of atmospheric conditions could explain why no “new” snow has been transported from the windward side. Another important factor could be that the area the transect covers has a relatively low surface roughness, with ridges that were generally shorter than in the surrounding region. Ero- sion might be large over these relatively smooth areas that the transect covers, and the delivered drifting snow from the up- wind side could have been deposited at higher ridges upwind. But even a just frozen lead will be ﬁlled by drifting snow after a while, which would lead to a mass sink on the upwind side, inhibiting further snow transport downwind. Considering the ALS-based digital elevation map of the ﬂoe (Fig. 4.1 Snow mass balance However, this temperature range was undershot most of the time during our investigation period of MOSAiC (Fig. 11c). some periods between the transect sampling days. Hence, the amount of erosion is not quantiﬁable based on the available transect data. Nonetheless, days where it is likely that no ero- sion occurred since the previous transect sampling prior to 20 February 2020 could be detected by means of snow parti- cle counters. The cumulative transect SWE of these time pe- riods was then compared against cumulative SWE from the sensors and ERA5 for the same time periods, which will be discussed in the next section. The validity of the SWE time series decreased with increasing ice dynamics from 11 and 12 March 2020 on and with decreasing temporal sampling frequency on both the northern and southern transect loops. q y p If we assume, based on the ﬁndings, that PWD22PS is least affected by blowing snow and provides a reasonable estimate of snowfall, then the snowfall between 31 Octo- ber 2019 and 26 April 2020 was about 72 mm. We know that the PWD22PS showed the lowest cumulative snowfall and a systematic negative bias compared against ground truth (Fig. 12). Further, it has been suggested that PWD22PS tends to underestimate snowfall, with values of down to 30 % less compared to a Geonor gauge within a DFIR (Wong (2012), Table 1). Until 7 May 2020 as the end of MOSAiC leg 3, In any case, we expect the transect time series until and including 5 March 2020 to be mostly valid. However, the usage of the transect SWE for sensor and reanalysis com- parisons is limited, as erosion has very likely occurred over https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation duri Figure 14. Scatter plots of sensor snowfall rates (y axis) vs. SPC mass ﬂux (x axis) for the drifting snow event on 24–25 Febru- ary 2020 for (a) Pluvio2 and (b) PWD22MC. 2393 agner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Figure 15. Scatter plots of sensor-computed snowfall rates (y axes) vs. SPC mass ﬂux (x axes) for the whole time period for (a) P2PS, (b) Pluvio2, (c) PWD22PS and (d) PWD22MC. Figure 15. Scatter plots of sensor-computed snowfall rates (y axes) vs. SPC mass ﬂux (x axes) for the whole time period for (a) P2PS, (b) Pluvio2, (c) PWD22PS and (d) PWD22MC. of RV Polarstern at 24 m height appears to be well protected against blowing snow (Fig. 15a), the overestimation of P2PS rather appears to be due to wind itself as also found in earlier studies. proaches might be that parts of the snow are caught in the porous parts of ridges, depending on wind direction and on the age of the ridges. This is something that is not covered by this study. Furthermore, for the erosion, the upper snow- pack’s microstructural composition plays a role, especially the strength of the bonds associated with sintering and tem- perature. Regarding Vaisala PWD22 and snowfall, Boudala et al. (2016) found that in comparison with manual measurements, it overestimated snowfall by about 33 % due to detected snow crystals not observed by the human observer. Wong et al. (2012) instead found good agreements with two Vaisala VRG 101 (double-alter shielded) and two OTT Pluvio (Tretyakov shielded) gauges. Compared to a Geonor gauge in a dou- ble fence intercomparison reference (DFIR), Wong (2012) found an underestimation for solid precipitation of 32 % for the PWD22. Hence, we expect and also can conﬁrm an un- derestimation of snowfall during MOSAiC. However, with 1.1 mm the RMSE was the lowest compared against all other validated sensors. Wong (2012) found little wind inﬂuence on the PWD22 measurements, which we can conﬁrm, as the RMSE relative to SWE estimates was reduced least (as rel- ative value) compared against all other sensors after drifting snow periods were eliminated from the comparison. Com- pared against SPC mass ﬂux, we demonstrated that the instal- D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Hence, on 189 d, we have as the best estimate that the total snowfall was between 98 and 114 mm. With the total mass increase of 38 mm for the transect SWE during the full observation time, we can approximately compute the minimum total eroded mass as 34 mm until 26 April 2020. With almost 50 % of eroded snow mass, we ﬁnd magnitudes comparable to those of Leonard and Maksym (2011), although they investigated snow over Antarctic sea ice and their time period for investigation was only about 1 month. However, our ﬁndings also compare well to results from Essery et al. (1999). Further sensor assess- ment discussions are made in the next section. Sublimation of snow crystals during trace precipitation or diamond dust could have led to snowfall detection in the opti- cal sensors but no SWE increase in the snow cover. However, the study design does not currently allow investigation, but it is recommended to investigate in detail. Earlier studies sug- gest that blowing snow sublimation may be responsible for about 6 % for the mass sink (Chung et al., 2011). The rela- tively low sublimation rates also arise from the high relative humidities found over sea ice, inhibiting further sublimation due to a quick saturation of the air. Snow cover sublimation can generally be expected to be negligible during polar night (Webster et al., 2021). https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 gner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2394 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Further, from our study we see that we cannot draw a conclusion from the computed SWE of the snow cover alone on the precipitated sum as the erosion appears to be large, even when measured over larger areas. Nonetheless, the results from Wang et al. (2019a) also indicate that a lot of snow mass gets eroded over time, which is even more evident as we as well as Cabaj et al. (2020) ﬁnd an overestimation tendency for ERA5 snowfall. We must further notice that ERA5 does not consider blowing snow sublimation in their computations. Although Orsolini et al. (2019) do not ﬁnd a mass effect of including blow- ing snow sublimation in ERA5, Chung et al. (2011) for in- stance computed a larger effect of blowing snow sublima- tion of 12 mm yr−1 over sea ice over 324 d. The non-existent blowing snow particle sublimation may be a reason for the overestimation tendency found in our study. Recent results with a new model of drifting snow sublimation (CRYOWRF; Sharma et al., 2021) indicate that it may be more important than previously estimated. We plan to address this particular problem in our future work. The undercatch of snowfall due to the wind for weighing bucket gauges is well known (Goodison et al., 1998; Boudala et al., 2016; Kochendorfer et al., 2017). However, we ob- served a strong positive bias in the cumulative snow mass for the Pluvio2 gauge when comparing with PWD22PS. This bias was largely due to a strong drifting snow event at the end of February, probably leading to blowing snow being lifted from the ground and landing in the bucket (Figs. 14, 15). Available transfer functions (e.g. Goodison et al., 1998; Boudala et al., 2016; Kochendorfer et al., 2017, 2018) cannot correct for this type of blowing snow event, as they correct the underestimation with increasing wind speed. However, we suggest that these events can be detected using snow par- ticle counters and removed before applying other corrections. The snowfall retrieval from the KAZR using a Ze–S re- lationship with coefﬁcients determined by Matrosov (2007) shows an overestimation relative to the snow cover SWE but performs comparably as well as ERA5. The difference to ERA5 in cumulative snowfall was only about 5 mm by the end of the investigation period. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2395 comparison with the cumulative sum of the KAZR (Fig. 10b) rather speaks against this theory as the ERA5 cumulative snowfall is always below the KAZR snowfall. Exact reasons with respect to ERA5 should be thoroughly investigated. Cabaj et al. (2020) also found a general positive bias (in daily snowfall rates) comparing ERA5 with CloudSat data. How- ever, CloudSat is only available up to 82◦N, while MOSAiC was north of 84◦N most of the time and for large parts even above 87◦N (Fig. 1) between October 2019 and May 2020. Further, in Cabaj et al. (2020), they found a decrease in the positive bias towards the spring months compared with the winter months of December, January and February while we ﬁnd an increased apparent bias during this time when com- paring with PWD22PS. Wang et al. (2019a) compared ERA5 snowfall with data from several snow buoys for a drift track in the Arctic Ocean comparable to that of MOSAiC and a time span comparable to that of our validation, from autumn to spring in the following year. Although they do not give de- tails about the method, they mention a positive bias of ERA5 cumulative snowfall of about 63 mm compared with snow buoys by the end of the investigation period. They partially found a negative bias and very varying results but on aver- age a positive bias. However, as already pointed out, a con- clusion cannot be drawn from a few point measurements on the overall accumulated snow on a larger area. Further, from our study we see that we cannot draw a conclusion from the computed SWE of the snow cover alone on the precipitated sum as the erosion appears to be large, even when measured over larger areas. Nonetheless, the results from Wang et al. (2019a) also indicate that a lot of snow mass gets eroded over time, which is even more evident as we as well as Cabaj et al. (2020) ﬁnd an overestimation tendency for ERA5 snowfall. We must further notice that ERA5 does not consider blowing snow sublimation in their computations. Although Orsolini et al. (2019) do not ﬁnd a mass effect of including blow- ing snow sublimation in ERA5, Chung et al. (2011) for in- stance computed a larger effect of blowing snow sublima- tion of 12 mm yr−1 over sea ice over 324 d. 4.3 ERA5 performance ERA5 performed reasonably well, with a weak overestima- tion when compared with snow cover SWE. Overall, the tim- ing of the snowfall events is represented well by ERA5. Rel- ative to PWD22PS, the cumulative ERA5 snowfall would have been overestimated by about 25 % by 7 May 2020. However, as already discussed, due to the light underesti- mation of the PWD22PS, the true snowfall probably lies between PWD22PS and ERA5 or the KAZR. Relative to PWD22PS, ERA5 appears to perform better before about March 2020, although the available data do not allow us to prove that it generally performs worse before. As the ERA5 performance depends on input of measurements and numer- ical weather data, we can at least point out that there was a substantial decrease in the air-based (minus 50 %–75 % be- tween March and May 2020; Chen, 2020) and ship-based observations, which may lead to a worsening of the per- formance. Chen (2020) found a worsening of temperature forecasts of up to 2 ◦C globally in the time period March– May 2020, compared against February 2020. Nonetheless, a D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Considering the known low bias in PWD22 measurements, and the fact that the radar snowfall retrievals are not affected by wind or blowing snow, it is likely that this radar-based estimate provides reasonable results. However, such radar-based retrievals are dependent on the inherent properties of the snowfall observed in the datasets from which they are derived. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons The non-existent blowing snow particle sublimation may be a reason for the overestimation tendency found in our study. Recent results with a new model of drifting snow sublimation (CRYOWRF; Sharma et al., 2021) indicate that it may be more important than previously estimated. We plan to address this particular problem in our future work. lation on the railing on the top deck of RV Polarstern has pro- tected the sensor from blowing snow. Conversely, the same device installed in met city on the ice showed a relative over- estimate, suggesting an inﬂuence from blowing snow. Com- parisons with the KAZR-derived snowfall, which is also un- affected by blowing snow, further support the notion that the PWD22PS was not signiﬁcantly impacted by blowing snow. comparison with the cumulative sum of the KAZR (Fig. 10b) rather speaks against this theory as the ERA5 cumulative snowfall is always below the KAZR snowfall. Exact reasons with respect to ERA5 should be thoroughly investigated. Cabaj et al. (2020) also found a general positive bias (in daily snowfall rates) comparing ERA5 with CloudSat data. How- ever, CloudSat is only available up to 82◦N, while MOSAiC was north of 84◦N most of the time and for large parts even above 87◦N (Fig. 1) between October 2019 and May 2020. Further, in Cabaj et al. (2020), they found a decrease in the positive bias towards the spring months compared with the winter months of December, January and February while we ﬁnd an increased apparent bias during this time when com- paring with PWD22PS. Wang et al. (2019a) compared ERA5 snowfall with data from several snow buoys for a drift track in the Arctic Ocean comparable to that of MOSAiC and a time span comparable to that of our validation, from autumn to spring in the following year. Although they do not give de- tails about the method, they mention a positive bias of ERA5 cumulative snowfall of about 63 mm compared with snow buoys by the end of the investigation period. They partially found a negative bias and very varying results but on aver- age a positive bias. However, as already pointed out, a con- clusion cannot be drawn from a few point measurements on the overall accumulated snow on a larger area. 4.2 Snowfall sensor estimates Snowfall rates from precipitation gauges show large dif- ferences among each other. Battaglia et al. (2010) already showed that OTT Parsivel instruments overestimate the num- ber of large particles. Further, the uncertainty they found regarding fall velocity was high. Wong (2012) also found a large overestimation of snowfall for the OTT Parsivel of about 50 % (Table 1), while the overestimation became larger during high wind speeds. The relatively low accuracy of ± 20 % for snowfall as given by the manufacturer compared to other instruments does make strong overestimations not surprising. Observations examined here appear to conﬁrm this overestimation. Although the installation on the top deck https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Appendix A: SWE comparison with Sturm et al. (2002a) We can compare the HS-SWE function (SWE = 323.97 · HS1.07, with HS in metres) with the ﬁtted function from Sturm et al. (2002a) (SWE = 0.348·HS, with HS in centime- tres), derived for sea ice snow cover during the SHEBA cam- paign (Perovich et al., 1999; Uttal et al., 2002). It is shown that the ﬁt in this paper (hereby called “W2021”) generally delivers lower SWE values for the same snow height com- pared against Sturm et al. (2002a) (hereby called “S2002”). The difference becomes larger the higher the snow depth is, which is partially a result of the different gradient (323.97 vs. 348). However, there is also a faster change between 0 and 200 mm, which is a result of the slight non-linearity of the W2021 function. The overall difference is signiﬁcant, as the deviation of the overall average from S2002 (86.5 mm) to W2021 (74 mm) is +17 %, while the RMSE is 13 mm. Although the unquantiﬁed eroded snow mass limits the potential for inter-comparisons, we found that the Vaisala Present Weather Detector 22 optical precipitation sensor in- stalled on Polarstern showed the smallest differences with the estimated SWE during periods without indication of drift- ing snow. We demonstrated that this PWD22 was mostly protected from blowing snow inﬂuences while this blowing snow led to high correlations between horizontal mass ﬂux and precipitation rates of the pluviometer and the present weather detector at met city. We assume that the high snow- fall rates detected with the ice-based pluviometer originate signiﬁcantly from blowing snow that was blown into the gauge opening. Similar to PWD22 on Polarstern, the KAZR snowfall retrievals are likely not affected by wind or blowing snow. The snowfall retrieved with this Ka-band radar shows a relatively high bias relative to the snow cover SWE, and it appears to overestimate snowfall. This overestimation how- This deviation is relevant, especially when comparing ground truth with snowfall sensors in such a dry environ- ment as the Arctic, where average yearly snowfall rates are typically very small. These points justify the application of a speciﬁc ﬁt for the winter and early spring during MOSAiC, instead of using values from literature. Differences in com- parison with Sturm et al. (2002a) stand out very quickly. They found an average snow depth of 0.337 m and an av- erage density of 340 kg m−3 resulting in 166 mm SWE. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2396 depth sources, such as snow height differences from terres- trial laser scans, could be used with this function to compute SWE. ever can be reduced when doing more research on selecting range gates and ﬁtting of the coefﬁcients. The OTT Parsivel2 laser disdrometer showed a strong tendency towards overes- timation. For the Vaisala Present Weather Detector 22, in- stalled on Polarstern, we ﬁnd a total cumulative snowfall of 98 mm over 189 d. We suggest the KAZR snowfall as a pos- sible upper cumulative limit of 114 mm. Snowfall rates from the ERA5 reanalysis showed a reasonable performance, with a good timing of snowfall events but a relatively safe ten- dency towards overestimation. This light overestimation may arise from non-existing blowing snow sublimation. However, to our knowledge, we present the ﬁrst validation of ERA5 snowfall for the high Arctic, based on a combination of re- peating snow depth transects and a set of in situ precipitation sensor data. One particular ﬁnding of our work is that SWE differences between snow on deformed SYI and snow on remnant SYI and FYI decrease until the end of the snow accumulation season at the beginning of May. The SWE on remnant SYI and FYI, while starting out at only about 50 % in late Oc- tober 2019, reached almost 90 % of the value for snow on deformed SYI by the end of April. Since SWE also did not increase much after 20 February, the observations raise the question as to whether there is a saturation point for snow mass accumulation. We suggest a range of 34 mm (47 %) to 69 mm (68 %) of precipitation that has been eroded over time, with the PWD22PS as a lower limit and the KAZR as an upper limit of cumulative snowfall. There was a remark- able snow erosion event between 24 and 25 February 2020, which we decided to have a closer look into as snow cover SWE decreased during this time. The fate of the eroded snow is unclear, but it is likely that a signiﬁcant amount was de- posited around higher ridges or ﬁlled in the gaps of frozen leads, limiting its transport to the areas covered by the tran- sects. However, as transects were conducted approximately weekly, processes in the snow that have occurred in the meantime are not detected. D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons More snow erosion accompanies the temporal adjustment of the surface roughness between remnant SYI and deformed SYI. Besides temporal sampling frequency of the transect, another limitation of the study is that layering of the snowpack was not considered for estimat- ing deposited and eroded snow mass. However, validation measurements at different points in time suggest that the im- pact of this effect might be small. A thorough investigation of layer density is recommended. Further research, connecting snow microstructure with snow transport rates, for instance, investigating sintering and bond strengths of snow grains that depend on temperature, could help to elucidate high Arctic snow processes. This is also important as the eroded snow has inﬂuenced several sensors’ measurements. This study sets the base for future snow mass balance re- search for MOSAiC and further general snow research for Arctic sea ice. We are better aware of snow mass quantities that accumulate and erode over the central Arctic sea ice sur- face for almost the whole accumulation season, including the polar night. The data can be used to improve numerical snow cover, sea ice, and weather and climate models and for more detailed process-understanding research across disciplines. Initial sensor validations were conducted, which allow for more speciﬁed, more thorough research. 5 Conclusions We ﬁtted a snow depth–SWE function to computed SWE values from SnowMicroPen force signals and applied the model to snow depths from Magnaprobes for the northern and southern transect loops of MOSAiC, for the winter and spring periods between October 2019 and May 2020. We show that the SWE reconstructions compared well against di- rect SWE measurements. Besides transect paths, other snow https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons Appendix A: SWE comparison with Sturm et al. (2002a) As discussed above, the av- erage SWE change due to a snow transport event can be very different when we compare one transect section SWE against the whole transect SWE. Hence, comparability is al- ways subject to great uncertainties. Also note that Sturm et al. (2002a) found a 25 % higher value of snow cover SWE, com- pared against snowfall measurements with a Nipher shielded gauge, even after applying a 2.5 factor as precipitation cor- rection against undercatch. The lower SWE on the snow cover is in any case very contrary to our results, as we es- timate at least 56 % less snow mass on the ice compared against measured snowfall. A larger impact on the results could be due to the fact that the measuring during our inves- tigation period did mostly occur between the 85th and 83th latitude, while during SHEBA, the camp started at around 75◦N, 142◦W and drifted up to 75◦N, 162◦W. We can spec- ulate that a less dry climate further away from the pole led to more snowfall, or that certain weather patterns led to no- table differences over longer time spans in snowfall, wind conditions or temperature, which we cannot determine at this point. Given that ﬁtting the HS-SWE function is highly spe- ciﬁc for given snow conditions and considering the substan- tial different average snow depth and snow density, we con- Author contributions. DNW took the lead in writing the manuscript and implemented comments, improvements and ﬁndings from dis- cussions with co-authors. DNW, JR, NK, ES, MO, IR, MS, MJ, DK, ARM and SA conducted the snow pit measurements. DNW and ARM processed the snow pit data. The principal investigator for snow pits is MS. SH, MO, PI, MJ, SA, JS and RR conducted transect measurements. The principal investigator for the transect is PI. Transect data were post-processed by DNW. MDS, OGP, TU, MMF, AK and CC were responsible for the met tower measure- ments, including SPC. The principal investigator for the tower is MDS. The University of Colorado/NOAA ﬂux team processed the met tower data. The principal investigator for the SPC is MMF. DNW and MMF post-processed the SPC data. Team ICE created ﬂoe maps during the cruise. SH provided the code to correct the co- ordinate transformation from global to FloeNavi coordinates. Appendix A: SWE comparison with Sturm et al. (2002a) We found an average snow depth of 0.249 m and an average den- sity of 286.5 kg m−3. Hence, both values are lower in our https://doi.org/10.5194/tc-16-2373-2022 The Cryosphere, 16, 2373–2402, 2022 agner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2397 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons g S Figure A1. Scatter plot of derived SWE after ﬁtted function in this paper and function by Sturm et al. (2002a). The orange line marks where r = 1.0. clude that these are the main reasons for different ﬁtting pa- rameters that are found for our study. Nonetheless, the mea- suring setup and instrumentation itself might lead to differ- ences and cannot be easily quantitatively compared. Data availability. KAZR (https://doi.org/10.5439/1498936; Lin- denmaier et al., 2019), PWD22 (https://adc.arm.gov/discovery/#/ results/instrument_code::pwd, last access: 14 June 2022; Kyrouac and Holdridge, 2019), Parsivel2 (https://doi.org/10.5439/1498731; Shi, 2019) and Pluvio2 data (https://adc.arm.gov/discovery/#/ results/datastream::moswbpluvio2S3.a1, last access: 14 June 2022 Wang et al., 2019b) are publicly available in the ARM data archive. The following data sets will be publicly accessible by 1 January 2023 on the PANGAEA data publisher. Derived SWE from the SnowMicroPen is available at https://doi.pangaea. de/10.1594/PANGAEA.92746 (Wagner et al., 2021). Raw SMP data are available at https://doi.org/10.1594/PANGAEA.935554 (Macfarlane et al., 2021). Bulk SWE measurements are avail- able at https://doi.org/10.1594/PANGAEA.940199 (Macfarlane et al., 2022). Magnaprobe snow depths are available at https://doi.org/10.1594/PANGAEA.937781 (Itkin et al., 2021). Raw met tower data are available at the Arctic Data Center (https://doi.org/10.18739/A2VM42Z5F; Cox et al., 2021). Data will be available at the UK Polar Data Centre by 15 July 2022 (https: //www.bas.ac.uk/data/uk-pdc/). Figure A1. Scatter plot of derived SWE after ﬁtted function in this paper and function by Sturm et al. (2002a). The orange line marks where r = 1.0. study, resulting in an average SWE of 71.2 mm. This is in- deed less than the half found by Sturm et al. (2002a). Note that the timing and setup of the snow measurements of Sturm et al. (2002a) are indeed comparable to ours, as they con- ducted snow transect measurements along transects for the accumulation period from October to May, as well. How- ever, they used different instrumentation and also sampled over different horizontal extents. https://doi.org/10.5194/tc-16-2373-2022 D. N. Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 2398 Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Bagnold, R. A. (Ed.): The physics of blown sand and desert dunes, Methuen & Co., London, 1941. Bartholomew, M.: Weighing Bucket Rain Gauge Instrument Hand- book., Tech. rep., edited by: Stafford, R., ARM user facility. DOE/SC-ARM-TR-232, 2020a. Acknowledgements. Data used in this paper were produced as part of the international Multidisciplinary drifting Observatory for the Study of the Arctic Climate (MOSAiC) with the tag MO- SAIC20192020 and the project ID: AWI_PS122_00. KAZR reﬂec- tivities and precipitation data were obtained from the Atmospheric Radiation Measurement (ARM) user facility, a U.S. Department of Energy (DOE) Ofﬁce of Science user facility managed by the Bi- ological and Environmental Research Program. We thank Sergey Matrosov for valuable discussions about snowfall and for helping with his radar expertise. 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Wagner et al.: Snowfall and snow accumulation during the MOSAiC winter and spring seasons 730965) for berth fees associated with the participa- tion of the DEARice project and the WSL Institute for Snow and Avalanche Research SLF (grant no. WSL_201812N1678). Met tower data were supported by the National Science Foundation (grant no. OPP-1724551) and by NOAA’s Physical Sciences Lab- oratory (PSL) and Global Ocean Monitoring and Observing Pro- gram (GOMO). Matthew D. Shupe was supported by the DOE At- mospheric System Research Program (grant nos. DE-SC0019251 and DE-SC0021341). Polona Itkin was supported by the National Science Foundation (grant no. NSF-1820927) and by the Research Council of Norway (grant no. RCN-287871). Stefanie Arndt, Stefan Hendricks, Daniela Krampe, Marcel Nicolaus, Robert Ricker and Julia Regnery were supported through the Alfred Wegener Insti- tute funds AWI_SNOW, AWI_ICE and AWI_ROV. 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https://openalex.org/W4384130875	https://www.preprints.org/manuscript/202305.1794/v1/download	English	null	Adjuvant Oligonucleotide Vaccine Increases Survival and Improves Lung Tissue Condition of B6.Cg-Tg (K18-ACE2)2 Transgenic Mice	Scientia pharmaceutica	2,023	cc-by	6,080	Adjuvant oligonucleotide vaccine increases survival of transgenic mice [B6.Cg-Tg (K18-ACE2)2] Abstract: The main problem in creating anti-coronavirus vaccines that target mainly proteins of the outer membrane of the virus remains the rapid variability of the RNA genome of the pathogen that encodes these proteins. In addition, the introduction of technologies that can provide affordable and fast production of flexible vaccine formulas that easily adapt to the emergence of new subtypes of SARS-CoV-2 is required. Universal oligonucleotide vaccine can take into account the dynamics of rapid changes in the virus genome, as well as be synthesized on automatic DNA synthesizers in large quantities in a short time. In this brief report, the effectiveness of four phosphorothioate constructs of the La-S-so type oligonucleotide vaccine will be evaluated for the first time on transgenic mice [B6.Cg-Tg (K18-ACE2)2]. In our primary trials, the oligonucleotide vaccine increased the survival rate of animals infected with SARS-CoV-2 and also reduced the destructive effects of the virus on the lung tissue of mice. The obtained results show the perspective of the development of vaccine constructs of the La-S-so type for the prevention of coronavirus infections, including those caused by SARS-СoV-2. Keywords: oligonucleotide vaccines, SARS-CoV-2, phosphorothioate oligonucleotides, innate immunity, adaptive immunity Keywords: oligonucleotide vaccines, SARS-CoV-2, phosphorothioate oligonucleotides, inn immunity, adaptive immunity Brief Report Brief Report Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 doi:10.20944/preprints202305.1794.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprin doi:10.20944/preprints202305.1794.v1 2 of 10 2 of 10 Preprints.org In this brief report, for the first time, we will try to answer the question of the viability of the idea of oligonucleotide vaccines using a practical example. For this study, we will use the transgenic mice [B6.Cg-Tg (K18-ACE2)2] and four ‘La-S-so’ constructs containing an antigen-presenting ‘head’ with a specific sequence in order to activate adaptive immunity, a tail with CpG islands to activate innate immunity, and ‘neck’, ‘connecting ‘head’ and 'tail'. In addition, in the article we will consider two routes of administration: subcutaneous and intranasal. 1. Introduction As of March 10, 2023 according to the Johns Hopkins University Coronavirus Resource Center, more than 676 million people had fallen ill, and more than 6.8 million had died, more than 13.3 billion vaccine doses have been administered worldwide [1]. The COVID-19 pandemic is now considered to be over. [2]. However, outbreaks of the disease continue to be recorded throughout the world. [3, 4, 5]. Obviously, the microevolution of SARS-CoV-2 will continue, and new subtypes will arise that can eventually lead to the next pandemic. Due to the lack of specific drugs that can provide reliable protection, vaccines are an effective tool for preventing COVID-19 in this situation. To date, there are three main platforms for vaccines: inactivated [6], adenoviral vectors [7], and mRNA vaccines [8]. However, all three platforms have side effects on human health. For example, the inactivated CoronaVac vaccine causes pain at the injection site, headache, fatigue, muscle and joint pain [9]. The adenovirus vaccine, using VaxZevria as an example, was manifested by frequent systemic reactions in the form of fatigue, myalgia, headache, fever, and atypical thrombosis [10, 11]. Humans have reported glomerulonephritis as one of the most serious side effects following Moderna injection [12]. In addition, all of the listed vaccine platforms do not provide the long operational half-life associated with the rapidly changing SARS-CoV-2 RNA genome. One of the promising ways to create anti- coronavirus vaccines is to use conservative regions of the coronavirus genome to create universal vaccines; however, this issue is still under study. Thus, from the point of view of improving the safety and duration of action of vaccines and the search for new platforms is relevant. Of interest is the postgenomic platform for creating vaccines [13], which is based on the use of nucleotide sequences of RNA viruses as antigens and not just adjuvants [14]. The avalanche accumulation of SARS-CoV-2 genomic sequences contributes to this direction of the search. © 2. Materials and Methods 2.1. Design, synthesis and purification of a La-S-so type oligonucleotide vaccine 2.1. Design, synthesis and purification of a La-S-so type oligonucleotide vaccine We used phosphorothioate oligonucleotide constructs for our experiments: La-S-so-1 5'- (CCCCCGGGGG)’neck’(GCAGAGACAGAAGAAACAGCAAAC)’head’(CCCCCGGGGG)’neck’(AACGC CAACGCC)’tail’-3'; La-S-so-2 5'- (CCCCCGGGGG)’neck’(AGGCACAACAACAAGGCCAAAC)’head’(CCCCCGGGGG)’neck’(AACGCCA ACGCC)’tail’-3'; La-S-so-3 5'- (CCCCCGGGGG)’neck’(AACAAGACAAAAACACCCAAGAAG)’head’(CCCCCGGGG)’neck’(AACGCC AACGCC)’tail’-3'; La-S-so-4 5'- (CCCCCGGGGG)’neck’(CACCGAGGCCACGCGGAG)’head’(CCCCCGGGGG)’neck’(AACGCCAACGCC )’tail’-3'. We used phosphorothioate oligonucleotide constructs for our experiments: L S 1 We used phosphorothioate oligonucleotide constructs for our experiments: 2.5. Mice infestation SARS was modeled by intranasal administration of a suspension of virus particles. Biomaterial from 15 patients (2 µl each) with PCR-confirmed SARS-CoV-2 was mixed with a 0.9% NaCl solution, after which it was injected into mice in an amount of 30 µl of liquid, and thus all animals received relatively equal doses in terms of virulence. When using the Polivir SARS-CoV-2 Express kit (Litekh, Russia), according to the instructions, Cq in these patients was always in the range from 17 to 18 cycles. For RNA isolation, 100 µL of a swab in 0.9% NaCl solution was used, and then 3.5% of the isolated RNA was used for RT-PCR. 2.6. PCR procedure The infection of mice was confirmed by PCR. A swab for PCR analysis was taken from the oral cavity of mice. The resulting smear was placed in a test tube with a 0.9% NaCl solution. RNA was isolated using the "Polivir SARS-CoV-2 Express" kit (Litekh, Russia) according to the instructions. PCR was performed with a kit for polymerase chain reaction with reverse transcription "Polivir SARS-CoV-2 Express" kit (Litekh, Russia) according to the instructions. The work was carried out on a real-time cycler CFX96 Touch (Bio-Rad, USA). 2.3.Vaccination The oligonucleotide vaccine was administered intranasally and subcutaneously twice, at weekly intervals, in an amount of 30 μl at a concentration of 1000 ng/μl. Injections were carried out using an insulin syringe, and in the case of intranasal administration, the vaccine was administered intranasally with a pipette dispenser. During intranasal administration, mice were immersed in anesthesia and the vaccine was added to the nasal passages for further reflex inhalation and the vaccine entering the respiratory tract. 2.4. Weighing The weight of mice was measured every day on an Ad100 scale (Axis, Poland) with a precision of 0.001 g. 2.2. Animals and experimental groups Transgenic mice [B6.Cg-Tg (K18-ACE2)2] (The Jackson Laboratory, USA) at the age of 7 weeks were randomly divided into 4 groups: 1) intact, 2) with SARS modeling without treatment, 3) two intranasal injections of the vaccine with an interval of a week, two weeks before the SARS modeling, 4) subcutaneous administration of the vaccine with an interval of a week, two weeks before the SARS modeling. There were six mice in each group. The animals were kept in specific pathogen free cages inside laminar flow hoods of the 2nd class of protection with free access to water and food. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 3 of 10 Preprints.org Preprints.org CCCCCGGGGG)’neck’(CACCGAGGCCACGCGGAG)’head’(CCCCCGGGGG)’neck’(AACGCCAACG 3' The vaccine was designed using SARS-CoV-2 genome sequences, the GenBank database was used to search for conserved regions, and the ClustalW 2.0.3 programs were used to align oligonucleotide sequences [15]. In our ‘La-S-so’ construct, we placed the CpG motifs in the ‘tail’ region in the 5′-purine-purine-unmethylated deoxycytosine-deoxyguanosine-pyrimidine- pyrimidine-3′ position [16]. The ‘neck’ region of the construct features a double-stranded DNA region stabilized by 30 hydrogen bonds between 10 cytosines and 10 guanines. The double-stranded DNA ‘neck’ makes it possible to form a loop in the region of the antigen-presenting ‘head’. The general look of the La-S-so type vaccine was represented in [17]. Only adenines, cytosines, and guanines are present in the construct, which makes it possible to activate pattern recognition receptors interacting with both ‘non-self’ DNA and ‘non-self’ RNA. Since the PS backbone is the basis of the construct, the presence of deoxyribose will be of little importance and everything will depend on the nucleotide sequence of the construct [18]. Also, CpG motifs in nuclease-resistant PS backbones have been found to dramatically enhance B cell stimulatory properties [19, 20]. The developed vaccine was synthesized on the ASM-800 DNA synthesizer (BIOSSET, Russia) using standard phosphoramidite synthesis. The synthesis was carried out in the direction from the 3’ to the 5’ end. After completion of all cycles of synthesis, the target oligonucleotide is removed from the solid-phase carrier; the removal of the protective groups was carried out overnight at 55°C in a concentrated ammonia solution [21]. Purification of the synthesized vaccine was performed by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Jupiter 5µm C18 300 Ǻ column (4.6 mm × 250 mm) using a preparative HPLC system (Azura P6.1 L, UVD 2.1S detector). Buffers were used for purification: (A) 0.1 M triethylammonium acetate in water, (B) 50% MeCN/buffer A. Gradient 30%. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 4 of 10 Preprints.org Preprints.org 2.8. Immunohistochemical assay Dehydration and paraffin impregnation were carried out in a Logos microwave histoprocessor (Milestone, Italy). Tissue blocks were sectioned 4 µm thick and stained with hematoxylin and eosin. Reagents for fixation, dehydration, wiring and staining manufactured by Biovitrum (Russia) were used: formalin 10%, isopropyl alcohol, paraffin, o-xylene, a set of dyes hematoxylin and eosin, recommended for histological studies. Stained sections were scanned on an Aperio CS2 scanner (Leica, USA) for morphometric analysis of changes in the lungs, 10 fields of view in each section were selected in the Aperio ScanScope program, the relative areas occupied by the lumen of the alveoli and bronchi, interalveolar septa, vessels, areas of edema and fibrosis were calculated. 2.9. Statistical data processing Statistical processing was carried out using the program STATISTICA 10. The normality of the distribution of the trait was determined by the Shapiro-Wilk method. The mean values of indicators, standard deviation, and error of the mean, upper and lower quartiles were calculated. The Kruskal- Wallis method was used to compare scores between groups. Differences were considered significant at p < 0.05. The study was approved by the ethics committee of the Federal State Autonomous Educational Institution of Higher Education V.I. Vernadsky Crimean Federal University. Vernadsky” dated October 01, 2021 protocol No. 25/21 When conducting experimental studies, the principles and provisions of the Guide for the Care and Use of Laboratory Animals (USNIH, No. 85-23), international rules “Guide for the Care and Use of Laboratory Animals” (2009), taking into account the Council of Europe Convention for the Protection of Vertebrate Animals used for Experimental or other Scientific Purposes (Strasbourg, 1986). 2.7. Autopsy of model animals Animals from the intact group and those receiving the vaccine were euthanized by intramuscular injection of chloral hydrate at a dose of 200 mg/kg of live weight, after which they were decapitated. The autopsy was carried out in several stages: fixation of the limbs of the animal with dissection needles on the dissecting tray with its back down. Next, the skin was captured and lifted with tweezers; a longitudinal incision was made along the white line of the abdomen from the inguinal region to the region of the lower jaw. The abdominal wall must remain intact. The skin was pushed back along the edges of the abdominal wall and fixed. An incision was made in the abdominal wall from the inguinal region to the midline of the abdomen, and an incision was made in the chest with scissors. After that, the lungs were carefully removed and the target organs were placed in histological cassettes. Organs were stored in 10% buffered formalin for 12 hours to prepare for immunohistochemical analysis. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 reprints.org 5 of Figure 1. Change in the mass of mice in groups: a) negative and positive controls; b) negative control and subcutaneous administration; c) negative control and intranasal administration. significant difference compared to the negative control group (p < 0.05) 5 of 10 Figure 1. Change in the mass of mice in groups: a) negative and positive controls; b) negative control and subcutaneous administration; c) negative control and intranasal administration. significant difference compared to the negative control group (p < 0.05) Histochemical studies showed a picture similar to organometric indicators. The analysis of morphometric data showed significant changes in the area of various parts of the lung (Figure 2). The dynamics were revealed in the study of three parameters of tissue sections: areas of edema, vessels, and lumen of the alveoli. Interalveolar and perivascular edema in all groups becomes significant on the 5th day, but decreases on the 10th day and subsides by the 30th day, its area becomes significantly lower relative to the early stages of the disease. Congestion and vascular area are most pronounced in the group with intranasal administration of the vaccine. At the beginning of the disease on the 5th day in all groups, the greatest dilatation of capillaries is observed. On the 10th day in the group without treatment and with treatment, alveolar-hemorrhagic syndrome was noted in about half of the animals. The area of the lumen of the alveoli in animals is significantly reduced due to distlectasis and atelectasis on the 10th day of illness in all groups, except for the group with intranasal administration of the vaccine. It is this indicator that is the key, in our opinion, since it is most pronounced in the group without treatment and is clinically accompanied by lethality, noted only in this group. In the mice from the group without the use of the vaccine, on the 5th day after infection in the lung parenchyma, significant areas of dyslectasis and atelectasis, thickening of the interalveolar septa, edema and lymphocyte infiltration of the alveoli and bronchi, and desquamation of the epithelium of the bronchioles were detected. By the 10th day, proliferation of type II alveolocytes developed, fibrin clots appeared in the lumen of the vessels, hemorrhages into the lumen of the alveoli, and single hemosiderophages. 3. Results In the course of the studies, a decrease in the weight of animals of the infected control was found on the 8th day of the experiment compared with individuals from the groups where vaccination was carried out (Figure 1). Weight loss is a nonspecific sign in viral diseases, including COVID-19 [22-25]. It can be concluded that the applied four constructs of the La-S-so type oligonucleotide vaccine were able to save the animals from weight loss, which is consistent with the data of the organometric parameters of the lungs of mice (Table 1). p g In groups using the oligonucleotide vaccine, the timing of the onset of the disease did not differ, the manifestation of the disease was pronounced, however, on average, hyperthermia was lower by 1.2℃, and the clinical condition of the animals recovered faster. On the second day, there was no significant difference in weight between the positive and negative controls, in contrast to the experimental groups, where a significant difference was already observed on the second day of the experiment. On the 4th day, a linear increase in body weight was noted in the positive group and in the experimental groups, where the weight increased every second day, compared with the negative control group, where the weight was in a plateau state throughout the experiment (p < 0.05). On the 8th day, the weight of the negative control decreased by 0.89±0.19 g; by the 10th day, the weight of the animals increased by 0.86±0.09 g, which corresponds to the weight of the animals on the 6th day of the disease. This suggests that the administration of the vaccine (intranasal and subcutaneous) prevents weight loss when infected with COVID-19. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 6 of 10 Preprints.org 6 of 10 Table 1. Organometric parameters of the lungs of mice with simulated SARS in different groups of the experiment Area % Stroma Edema Vessels Lumen of the alveoli Fibrosis Group Intact (i) 40,0 [34.0;42.0] 0 [0.0;0.0] 12,50 [10.0;14.0] 47,50 [43.0;50.0] 0 [0.0;0.0] Intranasal vaccine (n) 5 day 10 day 30 day 30,50+ [27.5;34.0] 40,00□ [37.0;45.0] 41,50□ [39.0;45.5] 7,50+ [50;9.0] 5,00 [3.0;7.0] 2,00□ [1.0;4.0] 21,50* [14.0;28.5] 19,50 [13.0;31.0] 13,50 [9.5;17.0] 37,00 [31.5;44.5} 30,50* [25.0;41.0] 42,00 [35.0;45.0] 1,50 [1.0;3.0] 1,00 [0.0;1.0] 1,00 [0.0;1.0] Subcutaneous vaccine(s) 5 day 10 day 30 day 35,00 [32.0;38.5] 45,50 [42.0;48.0] 39,50 [36.0;47.5] 8,00+ [6.0;12.5] 8,00 [8.30;13.0] 3,00□ [1.5;4.0] 17,00 [12.0;21.5] 17,50 [17.0;23.0] 16,50 [15.5;22.5] 39,50 [34.5;42.5] 25,50* [24.0;29.0] 37,50□ [32.5;41.0] 1,00 [1.0;2.0] 2,00 [2.0;3.0] 1,00 [1.0;1.0] Model without treatment (c) 5 day 10 day 30 day 40,50 [37.5;46.5] 43,50 [38.0;50.0] х 5,00 [4.0;8.5] 8,00 [5.0;10.0] х 15,00 [11.0;27.0] 25,00 [21.0;28.0] х 32,50 [22.0;41.5] 20,00* [19.0;25.0] х 1,00 [0.0;1.5] 1,50 [1.0;2.0] х Note: differences between groups were determined by comparing many independent samples using the Kruskal-Wallis method and were considered significant at p < 0.05; * – differences from the intact group; + - differences from the group without treatment at the corresponding time of the experiment; □ – differences within the group at different periods of the experiment (compared to 5 days); X – for this variant, the study was not carried out due to lack of information. Table 1. Organometric parameters of the lungs of mice with simulated SARS in different groups of the experiment 1. Organometric parameters of the lungs of mice with simulated SARS in different groups of Note: differences between groups were determined by comparing many independent samples using the Kruskal-Wallis method and were considered significant at p < 0.05; * – differences from the intact group; + - differences from the group without treatment at the corresponding time of the experiment; □ – differences within the group at different periods of the experiment (compared to 5 days); X – for this variant, the study was not carried out due to lack of information. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 Between the 10th and 30th days of the disease, all animals from this group died at night, which did not allow the revealed changes in the structure of the lung to be interpreted as associated with the development of SARS, since autolysis additionally destroyed tissues. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 reprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 7 of 10 7 of 10 Preprints.org Figure 2. Paraffin sections of the lungs of SARS model mice (except 1A). Stained with hematoxylin and eosin. SW. 20x. 1 A(i) - intact animals, 2 A - SARS model 5 days, 3 A - SARS model 10 days, 1 B (n) - group with intranasal administration of the vaccine, 5 days after infection, 2 B - group with intranasal administration of the vaccine , 10 days after infection, 3 C - group with intranasal administration of the vaccine, 30 days after infection. 1 B (s) - group with intramuscular administration of the vaccine, 5 days after infection, 2 B - group with intramuscular administration of the vaccine, 10 days after infection, 3 B - group with intramuscular injection of the vaccine, 30 days after infection. Figure 2. Paraffin sections of the lungs of SARS model mice (except 1A). Stained with hematoxylin and eosin. SW. 20x. 1 A(i) - intact animals, 2 A - SARS model 5 days, 3 A - SARS model 10 days, 1 B (n) - group with intranasal administration of the vaccine, 5 days after infection, 2 B - group with intranasal administration of the vaccine , 10 days after infection, 3 C - group with intranasal administration of the vaccine, 30 days after infection. 1 B (s) - group with intramuscular administration of the vaccine, 5 days after infection, 2 B - group with intramuscular administration of the vaccine, 10 days after infection, 3 B - group with intramuscular injection of the vaccine, 30 days after infection. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 Note: differences between groups were determined by comparing many independent samples using the Kruskal-Wallis method and were considered significant at p < 0.05; * – differences from the intact group; + - differences from the group without treatment at the corresponding time of the experiment; □ – differences within the group at different periods of the experiment (compared to 5 days); X – for this variant, the study was not carried out due to lack of information. Note: differences between groups were determined by comparing many independent samples using the Kruskal-Wallis method and were considered significant at p < 0.05; * – differences from the intact group; + - differences from the group without treatment at the corresponding time of the experiment; □ – differences within the group at different periods of the experiment (compared to 5 days); X – for this variant, the study was not carried out due to lack of information. In mice of the group with subcutaneous application of the vaccine, on the 5th day of the development of the disease, edema of the interalveolar septa, areas of emphysema, extensive areas of lymphoid infiltration, hemorrhages, plethora of large vessels and capillaries were observed. On the 10th day, the area of the lumen of the alveoli decreased due to atelectasis and proliferation of alveolocytes, the vessels remained full-blooded, areas of fibrosis and fibrin fibers appeared in the lumen of the vessels. By the 30th day, the area of the lumen of the alveoli slightly increased due to compensatory emphysema, the state of the parenchyma as a whole remained the same. The use of intranasal vaccine showed the best effect in assessing the morphology of the lungs of experimental animals. At the initial stages (up to 5 days), the course of the disease turned out to be more acute than in other groups and was accompanied by significant edema in the interalveolar septa and paravasal region, lymphoid infiltration, and areas of atelectasis. On the 10th day, the edema became less noticeable, but a focal complete or partial collapse of the alveoli was observed. The vessels still remained full-blooded, single hemorrhages were revealed. By the 30th day, no edema was detected, however, the interalveolar septa remained thickened due to lymphoid infiltration and proliferation of lining cells. 5. Conclusions This manuscript for the first time offers for consideration the results of studies with the constructions of an oligonucleotide vaccine of the La-S-so type. The data obtained indicate a positive effect of the La-S-so type oligonucleotide vaccine on the weight of transgenic mice, as well as on the organometric parameters and cellular structure of the lung tissue. The observed effects, in our opinion, cannot be explained only by the presence of an inline adjuvant (CpG islands) in the vaccine design, although they undoubtedly make a significant contribution to the observed effects. In the absence of developed test systems capable of assessing the formation of antibodies to the administered oligonucleotide vaccine, the data obtained indirectly indicate the prospects for the development of this vaccine platform. The synthesis of oligonucleotide vaccines can be automated relatively easily and prepare us for the next encounter with the SARS-CoV-2 coronavirus pandemic, unfortunately, there is no doubt that it will happen again. Further research will be aimed at proving 1) the existence antibodies capable of penetrating cells and attacking the unique nucleic acid sequences of RNA viruses, and 2) antigen presentation ability of dendritic cells with respect to nucleic acids. In our opinion, even oligonucleotide vaccines do not yet fit within the framework of a modern textbook on immunology, they have a great potential in prophylaxis of COVID-19 and other diseases caused by coronaviruses. Author Contributions: Conceptualization, V.V.O.; methodology, V.V.O., K.A.Y., and T.P.M; software, I.A.N.; formal analysis, V.V.O. and K.V.L.; investigation, V.V.O., K.A.Y., and T.P.M.; resources, I.A.N., A.V.K; data curation, V.V.O. and K.V.L.; writing—original draft preparation, V.V.O.; writing—review and editing, V.V.O., K.A.Y., O.A.A., and A.I.B.; visualization, V.V.O. and K.A.Y.; supervision, V.V.O.; project administration, V.V.O. and K.V.L. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, V.V.O.; methodology, V.V.O., K.A.Y., and T.P.M; software, I.A.N.; formal analysis, V.V.O. and K.V.L.; investigation, V.V.O., K.A.Y., and T.P.M.; resources, I.A.N., A.V.K; data curation, V.V.O. and K.V.L.; writing—original draft preparation, V.V.O.; writing—review and editing, V.V.O., K.A.Y., O.A.A., and A.I.B.; visualization, V.V.O. and K.A.Y.; supervision, V.V.O.; project administration, V.V.O. and K.V.L. All authors have read and agreed to the published version of the manuscript. Funding: The research results are obtained within the framework of as state assignment V.I. Vernadsky Crimean Federal University for 2021 and the planning period of 2022–2023 No. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 8 of 10 Preprints.org Preprints.org living cells [30], obviously that this is not always the case. Numerous investigations conducted mostly in cultured cells over the years have demonstrated that it is possible to facilitate the cellular internalization of antibodies [31]. The potential for in vivo therapeutic advantages of a nuclear- penetrating lupus anti-DNA autoantibody have also been shown in a number of studies [32, 33]. Thus, the ability of oligonucleotide vaccines will be determined by the ability of dendritic cells to make antigen presentation of coronavirus oligonucleotide sequences, as well as the ability of antibodies to attack unique coronavirus sequences inside infected host cells. 5. Conclusions FZEG-2021-0009 (‘Development of oligonucleotide constructs for making selective and highly effective preparations for medicine and agriculture’, registration number 121102900145-0). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 6. Agrawal, A.S.; Tao, X.; Algaissi, A.; Garron, T.; Narayanan, K.; Peng, B.H.; Couch, R.B.; Tseng, C.T. Immunization with inactivated Middle East Respiratory Syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus. Hum. Vacci.n Immunother. 2016, 0, 1-6. doi: 10.1080/21645515.2016.1177688. p j p Del Rio, C.; Malani, P.N. COVID-19 in 2022-The Beginning of the End or the End of the Beginning? JAMA 2022, 327, 2389-2390. doi: 10.1001/jama.2022.9655. 4. Discussion Scientists have conducted experiments over the past decades and have collected sporadic but convincing data on the possibility of using nucleic acids as an active immunogen [26, 27]. Still, there are two serious questions that should be addressed. Scientists have conducted experiments over the past decades and have collected sporadic but convincing data on the possibility of using nucleic acids as an active immunogen [26, 27]. Still, there are two serious questions that should be addressed. First, the question of whether dendritic cells can present an antigen fragment of an oligonucleotide vaccine containing a unique sequence of coronavirus RNA genomes remains insufficiently studied. Leukocytes and dendritic cells are able to penetrate into all parts of the body, since they have the qualities of absorption, transport, processing and presentation of antigens to T lymphocytes [28]. Dendritic cells move to secondary lymphoid organs to provide delivery of antigens to T lymphocytes and this, in turn, contributes to the manifestation of a powerful antigen-specific immune response. If B cells are affected when an antigen is detected, then this also applies to T cells, since B cells cannot be involved on alone in immune response [29]. This suggests that if B cells are activated, then so are T cells, whose full activation depends on dendritic cells. Currently, there is no single antigen that can activate B cells without activating T cells. However, in systemic lupus erythematosus, B cells are activated and antibodies are produced that attack the DNA of cells of a sick person [26, 27], but activation of T cells by dendritic cells due to antigen presentation of nucleic acid fragments has not yet been shown. To date, this issue has not yet been closely studied yet. Second, we assume that the human body can contain antibodies that can enter human cells during viral infection and target particular fragments of nucleic acids in RNA viruses. While generally speaking, antibodies do not pass easily through intact cellular or subcellular membranes in Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 9 of 10 Preprints.org Preprints.org 7. Voysey, M.; Clemens, S.A.C.; Madhi, S.A.; Weckx, L.Y.; Folegatti, P.M.; Aley, P.K.; Angus, B.; Baillie, V.L..; Barnabas, S.L.; Bhorat, Q.E.; et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 2021, 1-13. doi: 10.1016/S0140-6736(20)32661-1. 8. Sekar, A.; Campbell, R.; Tabbara, J.; Rastogi, P. ANCA glomerulonephritis after the Moderna COVID-19 vaccination. Kidney Int. 2021, 100, 473-474. doi: 10.1016/j.kint.2021.05.017. 9. Mallapaty, S. China COVID vaccine reports mixed results—what does that mean for the pandemic? Nature 2021. doi: 10.1038/d41586-021-00094-z. 10. Gras-Champel, V.; Liabeuf, S.; Baud, M.; Albucher, J.F.; Benkebil, M.; Boulay, C.; Bron, A.; El Kaddissi, A.; Gautier, S.; Geeraerts, T. et al. Atypical thrombosis associated with VaxZevria® (AstraZeneca) vaccine: data from the French Network of Regional Pharmacovigilance Centres. Therapies 2021, 76, 369-373. doi: 10.1016/j.therap.2021.05.007. 1. European Medicines Agency. Available online: https://www.ema.europa.eu/en/documents/assessm report/covid-19-vaccine-astrazeneca-epar-public-assessment-report_en.pdf (accessed on 29 January 20 12. Lee, L.A.; Franzel, L.; Atwell, J.; Datta, S.D.; Friberg, I.K.; Goldie, S.J.; Reef, S.E.; Schwalbe, N.; Simons, E.; Strebel, P.M.; Sweet, S.; Suraratdecha, C.; Tam, Y.; Vynnycky, E.; Walker, N.; Walker, D.G.; Hansen, P.M. The estimated mortality impact of vaccinations forecast to be administered during 2011–2020 in 73 countries supported by the GAVI Alliance. Vaccine 2013, 31, B61–B72. doi:10.1016/j.vaccine.2012.11.035. 13. Oberemok, V.V.; Andreeva, O.A.; Laikova, K.V.; Novikov, I.A.; Kubyshkin, A.V. Post-genomic platform for development of oligonucleotide vaccines against RNA viruses: diamond cuts diamond. Inflamm. Res. 2022, 71, 729–739. doi: 10.1007/s00011-022-01582-2. 14. Lai, C.Y.; Yu, G.Y.; Luo, Y.; Xiang, R.; Chuang, T.H. Immunostimulatory Activities of CpG- Oligodeoxynucleotides in Teleosts: Toll-Like Receptors 9 and 21. Front. Immunol. 2019, 10. doi: 10.3389/fimmu.2019.00179. 15. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. doi:10.1093/nar/22.22.4673. 16. Krieg, A.M.; Wagner, H. Causing a commotion in the blood: immunotherapy progresses from bacteria to bacterial DNA. Immunol. Today 2000; 21:521–6. doi:10.1016/S0167-5699(00)01719-9. 17. Oberemok, V.V.; Laikova, K.V.; Yurchenko, K.A.; Marochkin, N.A.; Fomochkina, I.I.; Kubyshkin, A.V. SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in ‘lasso’ for the multi-faced virus. Inflamm. Res. 2020, 69, 801–812. doi:10.1007/s00011-020-01377-3 f 18. Haas, T.; Metzger, J.; Schmitz, F.; Heit, A.; Müller, T.; Latz, E.; Wagner, H. 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Pisetsky, D.S.; Reich, C.F. The binding of anti-DNA antibodies to phosphorothioate oligonucleotides solid phase immunoassay. Mol. Immunol. 1998, 35, 1161–70. doi: 10.1016/s0161-5890(98)00108-4. 27. Pisetsky, D.; Vrabie, I. Antibodies to DNA: infection or genetics? Lupus 2009, 18, 1176-1180. doi:10.1177/0961203309106492. 28. Dieli, F. Dendritic cells and the handling of antigen. Clin. Exp. Immunol. 2003, 134, 178–80. doi:10.1046/j.1365-2249.2003.02279.x. j 29. Langelaar, J.; Rijvers, L.; Smolders J.; Luijn, M.M. B and T cells driving multiple sclerosis: identity, mechanisms and potential triggers. Front Immunol. 2020, 11. doi:10.3389/fimmu.2020.00760. 30. Muller, S.; Zhao, Y.; Brown, T.L.; Morgan, Al.C.; Kohler, H. TransMabs: cell-penetrating antibodies, the next generation. Expert Opin. Biological. Ther. 2005, 5, 237–41. doi:10.1517/14712598.5.2.237. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 25 May 2023 doi:10.20944/preprints202305.1794.v1 10 of 10 Preprints.org 31. Lackey, C.A.; Press, O.W.; Hoffman, A.S.; Stayton, P.S. A biomimetic pH-responsive polymer directs endosomal release and intracellular delivery of an endocytosed antibody complex. Bioconjug. Chem. 2002, 13, 996–1001. doi: 10.1021/bc010053l. 32. Noble, P.W.; Young, M.R.; Bernatsky, S.; Weisbart, R.H.; Hansen, J.E. A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells. Sci. Rep. 2014, 4. doi: 10.1038/srep05958. p p 33. Weisbart, R.H.; Chan, G., Jordaan, G.; Noble, P.W.; Liu, Y.; Glazer, P.M.; Nishimura, R.N.; Hansen, J.E. DNA-dependent targeting of cell nuclei by a lupus autoantibody. Sci. Rep. 2015, 5. doi: 10.1038/srep12022. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4380686537	https://journal.kdpu.edu.ua/ecolog/article/download/7655/7105	Ukrainian	null	ТЕРИТОРIАЛЬНА ДИФЕРЕНЦIАЦIЯ РОСЛИННОГО ПОКРИВУ СТАРОВIКОВИХ ВIДВАЛIВ КРИВБАСУ	Ekologìčnij vìsnik Krivorìžžâ	2,023	cc-by	5,437	Екологiчний Вiсник Криворiжжя. 2022. Випуск 7. C. 44–59 Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7. P. 44–59 ISSN 2664–505X (print) ISSN 2664–5068 (online) Екологiчний Вiсник Криворiжжя. 2022. Випуск 7. C. 44–59 Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7. P. 44–59 ISSN 2664–505X (print) ISSN 2664–5068 (online) https://doi.org/10.31812/eco-bulletin-krd.v7i0. Corresponding author. E-mail addresses: kras.kbs.17@gmail.com ТЕРИТОРIАЛЬНА ДИФЕРЕНЦIАЦIЯ РОСЛИННОГО ПОКРИВУ СТАРОВIКОВИХ ВIДВАЛIВ КРИВБАСУ О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Анотацiя. Старовiковi вiдвали мають суттєву наукову цiннiсть щодо збереження iнформацiї про перебiг пiзнiх стадiй сукцесiйного розвитку рослинностi та гiрничопромислових ландшафтiв загалом. Дослiдження просторового розподiлу рослинностi на залiзорудних вiдвалах Криворiзького басейну наразi має фрагментарний характер, тому вiдомостi про територiально-структурний аспект органiзацiї рослинного покриву таких об’єктiв iндустрiальної спадщини є наступним кроком у пiзнаннi шляхiв їх генезису. Метою роботи є виявлення закономiрностей територiальної диференцiацiї рослинностi залiзорудних вiдвалiв Кривбасу на прикладi дрiбних вiдвалiв у балцi Пiвнiчнiй Червонiй i старовинних рудникiв «Дубова балка» i «Рахманiвський». В основу роботи покладенi матерiали польових дослiджень, проведених у 2016–2021 роках; використано 80 геоботанiчних описiв i створено 5 великомасштабних картосхем рослинного покриву. За результатами дослiдження територiальної диференцiацiї фiтоструктур встановлено, що вона обумовлена неоднорiднiстю екотопiв i характеризується дрiбноконтурною мозаїчнiстю. За бiльш нiж сторiчний перiод саморозвитку цих постмайнiнгових ландшафтiв iз рослинного покриву повнiстю елiмiнувалися рудеральнi ценоструктури, натомiсть сформувалися «квазiстеповi» та петрофiтнi угруповання, подiбнi до природних за флористичним складом. У територiальнiй структурi вiдвалiв на пiвночi регiону частка лiгнозної рослинностi є вищою, порiвняно з вiдвалами центральної частини; водночас у пiвнiчнiй частинi Криворiжжя спостерiгається переважання в ценоструктурах представникiв аборигенної флори. У мiру змiщення вiдвалiв на широтному градiєнтi в пiвденному напрямку у складi лiгнозної рослинностi зростає участь посухостiйких адвентивних видiв, а самi контури рослинностi змiщуються з плато на схили зi сприятливiшими мiкроклiматичними умовами. Закономiрнiстю територiальної диференцiацiї є наявнiсть на крутих схилах усiх дослiджених вiдвалах значних площ екотопiв без рослинностi. Подальший етап їх заростання ми пов’язуємо з розвитком деревно-чагарникових угруповань. Перспективи подальших дослiджень полягають у використанi їх в органiзацiї системи екологiчного монiторингу зон техногенезу Криворiзького регiону. Ключовi слова: вiдвали, мозаїчнiсть, трав’яна та деревно-чагарникова рослиннiсть. Вступ. Розробка корисних копалин Криворiзького залiзорудного басейну здiйснюється понад 130 рокiв. Освоєння природних ресурсiв Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 45 iз залученням потужної технiки зумовлює виникнення техногенних новоутворень, у яких видозмiненi, а то й повнiстю зруйнованi природно обумовленi зв’язки, якi властивi зональним бiологiчним системам. Водночас такi змiни довкiлля настiльки iстотнi, що доводиться говорити про виникнення не лише нових техногенних бiогеоценозiв, але й техногенних неоландшафтiв [4]. Дестабiлiзацiя природних екосистем пiд дiєю антропогенних чинникiв здiйснюється через порушення природних механiзмiв саморегуляцiї, якi б забезпечували пiдтримку рiвноваги та спроможнiсть структурно-функцiонального розвитку як на рiвнi окремих компонентiв, так i системи загалом [23]. iз залученням потужної технiки зумовлює виникнення техногенних новоутворень, у яких видозмiненi, а то й повнiстю зруйнованi природно обумовленi зв’язки, якi властивi зональним бiологiчним системам. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Водночас такi змiни довкiлля настiльки iстотнi, що доводиться говорити про виникнення не лише нових техногенних бiогеоценозiв, але й техногенних неоландшафтiв [4]. Дестабiлiзацiя природних екосистем пiд дiєю антропогенних чинникiв здiйснюється через порушення природних механiзмiв саморегуляцiї, якi б забезпечували пiдтримку рiвноваги та спроможнiсть структурно-функцiонального розвитку як на рiвнi окремих компонентiв, так i системи загалом [23]. Однак, досвiд природничих дослiджень Кривбасу показав, що з часом, трансформованi гiрничовидобувними роботами, землi стають ареною формування вторинних екосистем, розвиток яких пiдпорядковується загальним природним закономiрностям [7]. Рослинний покрив у постмайнiнгових ландшафтах (таких, що виникли пiсля завершення видобування та збагачення корисних копалин) стає потужним фiтомелiоративним фактором. Дослiдження процесiв формування рослинностi на постмайнiнгових територiях Кривбасу проводяться у двох основних напрямах: фiторекультивацiя та спонтанне заростання. Ґрунтовний ретроспективний аналiз науково-практичних робiт iз фiторекультивацiйної проблематики спiвробiтникiв кафедри ботанiки й екологiї Криворiзького державного педагогiчного унiверситету здiйснено Е. О. Євтушенком [30]. Пiдсумки лiсової рекультивацiї вiдвальних ландшафтiв Кривбасу пiдведенi Ф. М. Бровком та О. Ф. Бровко [4]. Бiотехнологiя рекультивацiї залiзорудних вiдвалiв за допомогою створення стiйких трав’янистих рослинних угруповань розроблена колективом авторiв Криворiзького ботанiчного саду НАН України [15]. Щодо розвитку спонтанної рослинностi автори публiкацiй останнiх рокiв акцентують на екологiчнiй зумовленостi формування деревної та чагарникової рослинностi природним шляхом [22], процесi колонiзацiї поверхнi вiдвалiв Betula pendula Roth. [13], самопiдтриманнi популяцiй трав’яних рослин (Crambe pontica Steven ex Rupr., Hyssopus officinalis L.), якi впродовж трьох десятилiть пiсля проведення рекультивацiйних експериментiв опинилися на шляху саморозвитку [1, 19]. Дослiдження просторового розподiлу рослинностi на залiзорудних вiдвалах Криворiзького басейну наразi має фрагментарний характер. Так, О. М. Сметаною зi спiвавторами доведено, що використання ISSN 2664–505X Екологiчний вiсник Криворiжжя. 2022. Вип. 7 46 тривимiрного моделювання в дослiдженнi ґрунтового та рослинного покривiв iндустрiальних об’єктiв є бiльш ефективним, порiвняно з планiметричними методами [24]. Доведено, що застосування таких об’ємних моделей необхiдне для прогнозування динамiки та шляхiв прискорення самозаростання техногенних об’єктiв, а також для оцiнки якостi виконання нанесення родючих i потенцiйно родючих порiд на поверхню техногенно порушених територiй [22]. Т. С. Коптєвою обґрунтовується наявнiсть висотної диференцiацiї рослинного покриву гiрничопромислових ландшафтних комплексiв, зокрема, вiдвалiв Бурщицького, Шиманiвського та Степового, яка має вираз у приуроченостi певних фiтоструктур до висотно-ландшафтних мiкросмуг [12]. На територiї Кривбасу збереглися 64 вiдвали вiком понад 100 рокiв [17]. Якщо цi об’єкти iндустрiальної спадщини не використовують для вторинної переробки, перепрофiлювання на смiтники, дачнi дiлянки, сади, городи, вони стають цiнними носiями iнформацiї щодо сукцесiйного розвитку гiрничопромислових ландшафтiв [29]. З’ясування специфiки просторового розподiлу елементiв рослинної мозаїки на поверхнi старих залiзорудних вiдвалiв є наступним кроком у пiзнаннi шляхiв їх генезису. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Мета роботи — виявлення закономiрностей територiальної диференцiацiї рослинностi старовiкових залiзорудних вiдвалiв Кривбасу на прикладi кар’єрно-вiдвальних комплексiв у балцi Пiвнiчнiй Червонiй, «Дубова балка» та «Рахманiвський». Матерiали та методи. В основу роботи покладенi матерiали польових дослiджень, проведених у 2016–2021 роках на п’яти вiдвалах у пiвнiчнiй, центральнiй i пiвденнiй частинах Криворiжжя. Геоботанiчнi описи (всього 80) виконувались згiдно iз сучасними методичними рекомендацiями [27]. Картування рослинностi вiдвалiв здiйснено з використанням ортофотокарт (Google Maps) [14]. Латинськi назви рослин подано за номенклатурним зведенням С. Л. Мосякiна та М. М. Федорончука [16]. Результати. Усi старi гiрничопромисловi ландшафти, порiвняно iз сучасними, вiдрiзняються незначними розмiрами. Зокрема, висоти вiдвалiв обмежуються 10–20 м за ширини й довжини в межах 15–100 м. Вiдвали мають витягнуту, або округлу подушкоподiбну форму [10]. Саме такi параметри притаманнi вiдвалам у балцi Пiвнiчнiй Червонiй i залишкам одноярусного насипу дореволюцiйного рудника «Дубова балка» (рис. 1). Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 47 Рис. 1. Картосхема рослинного покриву вiдвалiв: а — у балцi «Пiвнiчна Червона»; b — рудника «Дубова балка» Figure 1. Map scheme of vegetation cover of dumps: а — in the gully “Pivnichna Chervona”; b — in mine “Dubova Balka” Умовнi позначення (угруповання з домiнуванням або переважанням): 1 — Stipa capillata; 2 — Festuca valesiaca; 3 — Koeleria cristata; 4 — Bromopsis inermis; 5 — Poa compressa; 6 — Melica transsilvanica; 7 — Carex praecox; 8 — Teucrium chamaedrys; 9 — Medicago romanica; 10 — Galatella villosa; 11 — Galium ruthenicum; 12 — Cephalaria uralensis; 13 — Hieracium umbellatum; 14 — Euphorbia agraria; угруповання зi спiвдомiнуванням: 15 — Festuca valesiaca + Pilosella echioides, 16 — Poa compressa + Poterium polygamum, 17 — угруповання Koeleria cristata з лишайниковим наземним покривом; Рис. 1. Картосхема рослинного покриву вiдвалiв: а — у балцi «Пiвнiчна Червона»; b — рудника «Дубова балка» На вiдвалах балки Пiвнiчної Червоної деревно-чагарниковi заростi займають найбiльшу частку загальної площi. На вiдмiну вiд iнших вiдвалiв основу лiгнозних угруповань тут складають аборигеннi кущi — Crataegus fallacina Klokov, Swida sanquinea (L.) Opiz.; кiлькiсть ISSN 2664–505X Екологiчний вiсник Криворiжжя. 2022. Вип. 7 48 адвентивних видiв — Padellus mahaleb (L.) Vassilcz, Cotinus coggygria Scop., Elaeagnus angustifolia L. значно менша. Поодинокi дерева представленi здебiльшого Armeniaca vulgaris Lam. та Ulmus minor Mill. адвентивних видiв — Padellus mahaleb (L.) Vassilcz, Cotinus coggygria Scop., Elaeagnus angustifolia L. значно менша. Поодинокi дерева представленi здебiльшого Armeniaca vulgaris Lam. та Ulmus minor Mill. Трав’яна рослиннiсть на округлому вiдвалi, розташованому пiвденнiше (рис. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна 1а), репрезентована угрупованнями на вiдсипцi кварцитового щебеню; такi ценоструктури подiбнi до поширених на природних оголеннях кристалiчних гiрських порiд. У їх складi злаки вiдiграють другорядну роль; представниками цiєї родини є Poa bulbosa L., P. compressa L., Melica transsilvanica Schur. Серед характерних петрофiтiв мiсцевої флори тут вiдмiченi Cephalaria uralensis (Murr.) Roem. et Schult., Poterium polygamum Waldst. et Kit., Chondrilla juncea L., Thymus × dimorphus Klokov et Des.- Shost. Трав’яна рослиннiсть на округлому вiдвалi, розташованому пiвденнiше (рис. 1а), репрезентована угрупованнями на вiдсипцi кварцитового щебеню; такi ценоструктури подiбнi до поширених на природних оголеннях кристалiчних гiрських порiд. У їх складi злаки вiдiграють другорядну роль; представниками цiєї родини є Poa bulbosa L., P. compressa L., Melica transsilvanica Schur. Серед характерних петрофiтiв мiсцевої флори тут вiдмiченi Cephalaria uralensis (Murr.) Roem. et Schult., Poterium polygamum Waldst. et Kit., Chondrilla juncea L., Thymus × dimorphus Klokov et Des.- Shost. На пiвнiчнiшому вiдвалi, що має витягнуту форму, частина платоподiбних поверхонь вiдсипана суглинками. Тому, окрiм петрофiтниих, тут близько 20% площi займають угруповання, що за флористичним складом i видовим багатством подiбнi до степових. У природних межах таких фiтоценозiв налiчується до 45 видiв вищих судинних рослин; серед них понад 60% — степанти. Обидва вiдвали є мiсцями зростання созологiчно цiнних видiв — Astragalus dasyanthus Pall. (включений до Червоної книги України [6], та Свiтового червоного списку [9]); A. pallescens M. Bieb., Goniolimon besserianum (Schult.) Kusn., Thymus × dimorphus (включенi до Червоної книги Днiпропетровської областi (ЧКДО) [5]. Два одноярусних вiдвали дореволюцiйного рудника «Дубова балка» вiком близько 130 рокiв, очевидно, є залишками єдиного геоморфологiчного утворення, вiдсипаного кварцитами з високим умiстом залiза. Схили характеризуються екстремальними умовами: великi площi їх не заростають протягом десятилiть; на їх поверхнях зустрiчаються лише окремi особини вищих рослин (переважно Oberna cserei (Baumg.) Ikonn. За останнi роки розширилася площа оголених схилiв унаслiдок розробки цих вiдвалiв як вторинних техногенних родовищ. У верхнiй частинi захiдного схилу зберiгся локалiтет iз досить щiльним травостоєм iз Hieracium umbellatum L. Деревно-чагарниковi куртини з Ulmus minor Mill., U. pumila L., Acer negundo L., Scop., Elaeagnus angustifolia L., Cotinus coggygria Scop. здебiльшого також зосередженi на схилах. Центральна частина пiвнiчного вiдвалу являє собою нешироку перемичку. У субстратi цього екотопу запаси вологи iнтенсивно витрачаються через бiчний стiк, тому проєктивне покриття (ПП) квiткових рослин становить лише 45%, решта поверхнi вкрита кiркою лишайникiв Cladonia sp. 49 Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 На пласких вершинах вiдвалiв сформувалися примiтивнi кам’янистi ґрунти. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Суттєву частку рослинного покриву тут утворюють злаковники з домiнуванням Stipa capillata L., Koeleria cristata (L.) Pers., Festuca valesiaca Gaudin. Проєктивне покриття таких фiтоценозiв становить 50–85%. Рiзнотрав’я в їх складi представлене Seseli campestre Besser, Medicago romanica Prodan, Galium ruthenicum Willd., Euphorbia seguieriana Neck., Pilosella echioides (Lumn.) F. Schultz et Sch. Bip. Вiдмiченi також цибулиннi ефемероїди: Gagea bulbifera (Pall.) Sallisb. i G. podolica Schult. et Schult. f. Кiлькiсть видiв у цих угрупованнях сягає 20–25. Дiлянки площин iз нерозвиненими кам’янистими ґрунтами займають угруповання з переважанням Melica transsilvanica й участю Asperula montana Waldst. et Kit., Leontodon biscutellifolius DC., Minuartia leiosperma Klokov, Thymus × dimorphus, Chondrilla juncea, Sedum acre L. Своєрiдностi петрофiтним угрупованням надає суттєва участь Kohlrauschia prolifera (L.) Kunth (вiд 3–4 до 10% ПП). Цей стенотопний вид, що зрiдка зустрiчається на природних кристалiчних вiдслоненнях, включено до офiцiйного перелiку регiонально рiдкiсних рослин Днiпропетровської областi [5]. Висока ценотична рiзноманiтнiсть рослинностi одного з вiдвалiв старовинного рудника «Рахманiвський», очевидно, пояснюється його розташуванням в зонi ландшафтного екотону на межi Приднiпровської височини та Причорноморської низовини й безпосередньому контакту iз природною рослиннiстю балки Галаганової. Вiдвал одноярусний, проте поверхня його вершинного плато має ускладнений мiкрорельєф за рахунок пагорбiв, здебiльшого вiдсипаних вапняками (рис. 2). Деревно-чагарникова рослиннiсть, складена переважно Padellus mahaleb, Ulmus minor, Armeniaca vulgaris, зосереджена на крутому схилi захiдної експозицiї, який переходить у борт затопленого кар’єру. Участь лiгнозної рослинностi на вершинному плато вкрай незначна: до невеликої западини в пiвденнiй частинi вiдвалу приурочена куртина Prunus stepposa Kotov, а у верхiв’ї ерозiйного рiвчака в пiвнiчнiй частинi зростає декiлька старих кущiв Padellus mahaleb. На сформованих примiтивних ґрунтах вершинного плато поширенi «квазiстеповi» угруповання, подiбнi до формацiй природної рослинностi: Agripyroneta pectinate, Festuceta valesiacae, Jurineeta brachycephalae, Stipeta capillatae, Stipeta lessingianae, Koelerieta cristatae. Як видно з рис. 2, саме ценози з домiнуванням Koeleria cristata займають найбiльшу площу в територiальнiй структурi рослинного покриву. ISSN 2664–505X Екологiчний вiсник Криворiжжя. 2022. Вип. 7 50 Рис. 2. Картосхема рослинного покриву вiдвалу кар’єрно-вiдвального комплексу «Рахманiвський» Figure 2. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Map scheme of vegetation cover of a dump of quarry-dump complex “Rakhmanivskyi” мовнi позначення (угруповання з домiнуванням або переважанням): 1 — Koeleria cristata; 2 — Agropyron pectinatum; 3 — Stipa capillata; 4 — Stipa lessingiana; 5 — Festuca valesiaca; 6 — Elytrigia intermedia; 7 — Poa gustifolia; 8 — Jurinea brachycephala; 9 — Galatella villosa; 10 — Tanacetu millefolium; 11 — Convolvulus lineatus; 12 — Ajuga chia; угруповання зi спiвдомiнуванням: 13 — Agropyron pectinatum + Koeleria cristata; 14 — Koeleria cristata + Artemisia santonica; 15 — Agropyron pectinatum + Medicago romanica, 16 — Kochia prostrata + Agropyron pectinatum, 17 — Agropyron pectinatum + Cephalaria uralensis; 18 — Elytrigia repens + Poa Рис. 2. Картосхема рослинного покриву вiдвалу кар’єрно-вiдвального комплексу «Рахманiвський» Figure 2. Map scheme of vegetation cover of a dump of quarry-dump complex “Rakhmanivskyi” Рис. 2. Картосхема рослинного покриву вiдвалу кар’єрно-вiдвального комплексу «Рахманiвський» Рис. 2. Картосхема рослинного покриву вiдвалу кар’єрно-вiдвального комплексу «Рахманiвський» Figure 2. Map scheme of vegetation cover of a dump of quarry-dump complex “Rakhmanivskyi” Figure 2. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Map scheme of vegetation cover of a dump of quarry-dump complex “Rakhmanivskyi” quarry-dump complex “Rakhmanivskyi” Умовнi позначення (угруповання з домiнуванням або переважанням): 1 — Koeleria cristata; 2 — Agropyron pectinatum; 3 — Stipa capillata; 4 — Stipa lessingiana; 5 — Festuca valesiaca; 6 — Elytrigia intermedia; 7 — Poa angustifolia; 8 — Jurinea brachycephala; 9 — Galatella villosa; 10 — Tanacetum millefolium; 11 — Convolvulus lineatus; 12 — Ajuga chia; угруповання зi спiвдомiнуванням: 13 — Agropyron pectinatum + Koeleria cristata; 14 — Koeleria cristata + Artemisia santonica; 15 — Agropyron pectinatum + Medicago romanica, 16 — Kochia prostrata + Agropyron pectinatum, 17 — Agropyron pectinatum + Cephalaria uralensis; 18 — Elytrigia repens + Poa angustifolia; 19 — деревно-чагарниковi заростi; 20 — мiкроценоз Sempervivum ruthenicum; 21 — суглинистий субстрат без рослинностi; 22 — вапняковий субстрат без рослинностi q y p p y Умовнi позначення (угруповання з домiнуванням або переважанням): 1 — Koeleria cristata; 2 — Agropyron pectinatum; 3 — Stipa capillata; 4 — Stipa lessingiana; 5 — Festuca valesiaca; 6 — Elytrigia intermedia; 7 — Poa angustifolia; 8 — Jurinea brachycephala; 9 — Galatella villosa; 10 — Tanacetum millefolium; 11 — Convolvulus lineatus; 12 — Ajuga chia; угруповання зi спiвдомiнуванням: 13 — Agropyron pectinatum + Koeleria cristata; 14 — Koeleria cristata + Artemisia santonica; 15 — Agropyron pectinatum + Medicago romanica, 16 — Kochia prostrata + Agropyron pectinatum, 17 — Agropyron pectinatum + Cephalaria uralensis; 18 — Elytrigia repens + Poa angustifolia; 19 — деревно-чагарниковi заростi; 20 — мiкроценоз Sempervivum ruthenicum; 21 — суглинистий субстрат без рослинностi; 22 — вапняковий субстрат без рослинностi Суттєву частку площi вершинного плато займають дiлянки карбонатопетрофiльної рослинностi на вапняковiй вiдсипцi. Характерними видами тут є Jurinea brachycephala Klokov, Haplophyllum suaveolens (DC.) G. Don. f., Convolvulus lineatus L. Найбiльшi площi серед них займають розрiдженi угруповання Cephalaria uralensis. 51 Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 Уламки кристалiчних порiд на цьому вiдвалi вiдсипались у невеликiй кiлькостi. Наразi вiдбувається їх гiпергенез iз вивiльненням фiтотоксичних солей, що спричинює локальну появу галофiльного виду Artemisia santonica L. Щодо силiкопетрофiтних ценоструктур на вiдсипцi кварцитiв, то вони представленi єдиним мiкроценозом, сформованим Sempervivum ruthenicum Schnittsp. et C. B. Lehm. Мезоксерофiтнi злаковники з домiнуванням Elytrigia intermedia та Poa angustifolia зосередженi в пiвденнiй частинi вiдвалу, де штучно створений укiс переходить у схил пiвденної експозицiї балки. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Сприятливiший для розвитку ксеромезофiтних угруповань гiдротермiчний режим створюється на схилi пiвнiчної експозицiї та на днищi одного з ярiв, де спостерiгається початок задернiння субстрату Elytrigia repens i Poa angustifolia. Схил схiдної експозицiї вiдсипаний лесовидними суглинками, якi досить легко розмиваються дощовими i талими снiговими водами, унаслiдок чого вся його поверхня вкрита ерозiйними рiвчаками. Гребенi мiж рiвчаками частково закрiпленi вiд розмиву фрагментарними заростями Kochia prostrata (L.) Schrad. та Agropyron pectinatum (M. Bieb) P. Beauv.: така рослиннiсть характерна для природних лесових оголень пiвдня степової зони України. Созологiчно цiннi рослиннi раритети на цьому вiдвалi представленi 8 видами, включеними до ЧКДО [20]. Окрiм ценозоутворювачiв Stipa capillata, S. lessingiana (на їх належнiсть до «червоних спискiв» вищих рангiв вказано вище) i Jurinea brachycephala, у трав’яних угрупованнях часто зустрiчаються Сentaurea orientalis L., Convolvulus lineatus, Haplophyllum suaveolens (DC.) G. Don. f., Sempervivum ruthenicum. Щодо останнього виду — це єдина вiдома нам знахiдка його в постмайнiнговому ландшафтi. На обласному рiвнi охороняється також Rosa bordzilowskii Chrshan. — кущ, що розсiяно зростає на всiй поверхнi вiдвалу. у Обговорення. Провiдним чинником диференцiацiї рослинностi в умовах степової зони, як вiдомо, є гiдротермiчний режим [3]. Однак на вiдвалах детермiнуючий вплив на розподiл рослинних структур чинить екотопiчна неоднорiднiсть ландшафту, зокрема складний рельєф, рiзний рiвень зволоження, лiтохiмiчна строкатiсть [25]. На старовiкових вiдвалах вiдбувається формування специфiчних структур ґрунтового покриву. Кiнцевою стадiєю ґрунтоутворення є ґрунтовi тiла, за будовою подiбнi до природних дерново-степових. У їх приповерхневому шарi спостерiгається значний умiст гумусу, що зумовлено помiтним надходженням на поверхню органiчного ISSN 2664–505X Екологiчний вiсник Криворiжжя. 2022. Вип. 7 52 опаду вiд рослинностi, активною деструкцiєю органiки, слабкою риючою дiяльнiстю фауни тощо [8]. Оскiльки територiальна структура рослинностi є «вiдбитком» iз матрицi ландшафту, за таких екотопiчних умов вона набуває дрiбноконтурної мозаїчностi. Вважається, що швидкiсть формування рослинного покриву вiдвалiв значною мiрою обумовлюються наявнiстю або вiдсутнiстю безпосереднього контакту їх територiй iз природними ландшафтами [11, 18]. Однак, отриманi нами данi свiдчать, що це ствердження не завжди є справедливим. Якщо пояснення високого видового та ценотичного рiзноманiття рослинностi постмайнiнгових ландшафтiв, прилеглих до територiй балок Пiвнiчної Червоної та Галаганової, цiлком зрозумiле, то подiбне явище для вiдвалiв рудника «Дубова балка» з цим положенням не узгоджується. Адже цi вiдвали «затиснутi» мiж двома залiзничними колiями; на пiвнiч вiд них на кiлька кiлометрiв простягається житлова забудова, а на пiвдень — гiрничi техногеннi ландшафти. Вiдповiдь на це питання, iмовiрно, знаходиться у площинi виявлення механiзмiв мiґрацiї дiаспор. О. О. Красова, А. О. Павленко Криворiзький ботанiчний сад НАН України, м. Кривий Рiг, Україна Ранiше нами було показано, що в постмайнiнгових ландшафтах за типами дiаспорохорiї домiнують «облiгатнi» балiсти (37,5% видiв); «облiгатнi» анемохори посiдають друге мiсце (25%), а частка зоохорiв складається лише iз 7,5% видового складу рослинностi [20]. Перевагу у приживаннi мають види, у яких поєднується кiлька можливих способiв поширення, тобто тi, яким притаманнi змiшанi типи дiаспорохорiї. Антропiчним фактором впливу на територiальну структуру рослинностi виступає рекультивацiя, зокрема лiсова. Новим викликом для iснування лiсової рослинностi у вододефiцитних девастованих ландшафтах є сучаснi змiни клiмату [2]; згiдно з цим поглядом, деревно-чагарникову рослиннiсть старовiкових вiдвалiв доцiльно розглядати як «адаптованi лiсовi структури». Слiд зазначити, що деякi автори цiлком слушно розглядають спонтанне деревне заростання вiдвалiв як альтернативу штучному залiсненню [26, 31]. Проведене нами детальне картування рослинного покриву старовiкових вiдвалiв дозволило виявити, що в територiальнiй структурi вiдвалiв на пiвночi регiону (за сприятливого гiдротермiчного режиму) частка лiгнозної рослинностi є вищою, порiвняно з вiдвалами центральної частини. Водночас тут спостерiгається переважання в ценоструктурах представникiв аборигенної флори. У мiру змiщення вiдвалiв на широтному градiєнтi в пiвденному напрямку у складi лiгнозної рослинностi зростає участь посухостiйких адвентивних видiв, а самi контури рослинностi змiщуються з плато на схили зi сприятливiшими мiкроклiматичними умовами. Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 53 Сукцесiйнi процеси в межах екотопiв зi специфiчними ґрунтами, подiбними до дерново-степових (переважно на горизонтальних площинах), призвели до формування трав’яних ценоструктур, що мають ознаки степової стадiї розвитку. На субстратах, якi не зазнали суттєвих ґрунтотвiрних процесiв, наявна петрофiтна рослиннiсть, що наближена до природних аналогiв. Тому ми цiлком погоджуємося iз твердженням С. В. Яркова, що вiдвальнi геосистеми Кривбасу вже є i в майбутньому можуть стати рефугiумами (сховищами) для зональної й азональної флори та рослинностi Криворiзького регiону [28]. Висновки. Отже, територiальний розподiл рослинних структур на вiдвалах старовинних рудникiв Криворiжжя, обумовлений неоднорiднiстю екотопiв, характеризується дрiбноконтурною мозаїчнiстю. За бiльш нiж сторiчний перiод саморозвитку цих постмайнiнгових ландшафтiв iз рослинного покриву повнiстю елiмiнувалися рудеральнi ценоструктури, натомiсть сформувалися «квазiстеповi» та петрофiтнi угруповання, подiбнi до природних за флористичним складом. У територiальнiй структурi вiдвалiв на пiвночi регiону частка лiгнозної рослинностi є вищою, порiвняно з вiдвалами центральної частини; водночас у пiвнiчнiй частинi Криворiжжя спостерiгається переважання в ценоструктурах представникiв аборигенної флори. У мiру змiщення вiдвалiв на широтному градiєнтi в пiвденному напрямку у складi лiгнозної рослинностi зростає участь посухостiйких адвентивних видiв, а самi контури рослинностi змiщуються з плато на схили зi сприятливiшими мiкроклiматичними умовами. 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Chy mozhe industrial’nyi turyzm zminyty oblychchia Kryvoho Rohu? [Whether can industrial tourism change the face of Kryvyi Rih?] In: Urbanistychna Ukraina: v epitsentri prostorovykh zmin [Urbanistic Ukraine: at the epicenter of spatial changes : monograph]. Eds. K. Mezentsev, Ya. Oliynyk, N. Mezentseva. Kyiv, “Fenix”, 378–393. (in Ukrainian). 18. Pavlenko, A. O., Krasova, O. O., & Korshykov, I. I. (2017). Synhenetychni protsesy na zalizorudnykh vidvalakh pivnichnoi chastyny Kryvorizhzhia [Syngenesis processes on iron ore dumps in the northern part of Kryvyi Rih area]. Ukrainskyi botanichnyi zhurnal [Ukrainian botanical journal], 74 (4), 360–372. https://doi.org/10.15407/ukrbotj74.04.360 (in Ukrainian). 19. Pavlenko, A., & Krasova, O. (2021). Stan introduktsiynoi populiatsii Crambe pontica Steven ex Rupr. References na zalizorudnomu vidvali (Kryvyi Rih) [The state of introduced population of Crambe pontica Steven ex Rupr. on an iron ore waste dump (Kryvyi Rih)]. Ekolohichni nauky [Ecological Sciences], 2 (35), 54–59. https://doi.org/10.32846/2306- 9716/2021.eco.2-35.9 (in Ukrainian). 20. Pavlenko, A. O., Krasova, O. O., Korshykov, I. I., & Baranets, M. O. (2020). Sozofity u postmayninhovykh landshaftakh Kryvbasu [Sozophytes in postmining landscapes of Kryvyi Rih basin]. Visnyk Odes’koho natsional’noho universytetu. Biolohiya [Odesa National University Herald. Biology], 25, 1 (46), 23–41. https://doi.org/10.18524/2077-1746.2020.1(46).205802 (in Ukrainian). 21. Prisyazhnyuk, T. M., Dolina, O. O., & Bondarenko, A. M. (2019). Otsinka stupeniu vidnovlennia industrial’nykh landshaftiv na osnovi biomasovykh kharakterystyk roslynnosti ta tryvymirnoho modeliuvannia gruntovoho pokryvu [Estimation of the degree of industrial landscapes restoration based on biomass vegetation characteristics and three-dimensional soil cover modeling]. Chornomorski Bot. J., 15 (4), 351–361. https://doi.org/10.32999/ksu1990-553X/2019-15-4-4 (in Ukrainian). 22. Savosko, V., Lykholat, Yu., Domshyna, K., & Lykholat, T. (2018). Ekolohichna ta heolohichna zumovlenist’ poshyrennia derev i Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 57 chaharnykiv na devastovanykh zemliakh Kryvorizhzhia [Ecological and geological determination of trees and shrubs dispersal on the devastated lands at Kryvorizhya]. Journal of Geology, Geography and Geoecology, 27 (1), 116–130. https://doi.org/10.15421/111837 (in Ukrainian). 23. Shapar, A. G., & Mikheyev, A. V. (2018). Kontseptual’ni pidkhody do rozuminnia protsesiv antropohennoi destabilizatsii ekolohichnykh system [Conceptual approaches to understanding of processes of anthropogenic destabilization of ecological systems]. Visnyk of the National Academy of Sciences of Ukraine, 3, 56–66. https://doi.org/10.15407/visn2018.03.056 (in Ukrainian). 24. Smetana, A., Dolina, A., & Yaroschuk, Y. (2013). 3-D models using in the soil and plant covers topological organization analyse [Ispolzovanie 3-D modeley v analize topologicheskoy organizatsii pochvennogo i rastitelnogo pokrova]. Bulletin of DSAEU [Visnyk DDAEU], 2 (32), 12–18. (in Russian). 25. Smetana, A. N., Dolina, A. A., & Yaroschuk, Y. V. (2013). Dyferentsiatsiya ekotopiv posttekhnohennykh landshaftiv (hihro- ta litokhimichnyi aspekt) [Differentiation of post-industrial landscape ecotopes (humidity and lithochemical aspect)]. Pytannia bioindykatsii ta ekolohii [Problems of bioindications and ecology], 18 (1), 9–13. (in Ukrainian). 26. Wo´zniak, G., Chmura, D., Dyderski, M. K., B lo´nska, A., & Jagodzi´nski, A. M. (2022). How different is the forest on post-coal mine heap regarded as novel ecosystem? Forest Ecology and Management, 515. https://doi.org/10.1016/j.foreco.2021.118951 27. Yakubenko, B. Ie., Popovych, S. Iu., Ustymenko, P. M., Dubyna, D. V., & Churilov, A. M. (2018) Geobotany: methodical aspects of researches. Text-book. Kyiv : Lira K, 316. (in Ukrainian). 28. Yarkov, S. V. (2007). References Hirnychopromyslovi landshafty Kryvbasu yak refuhiumy zonal’noi roslynnosti [Mining landscapes of Kryvbas as refugia of zonal vegetation]. Heohrafichni doslidzhennia Kryvbasu: materialy kafedral’nykh naukovo-doslidnyts’kykh tem [Geographical studies of Kryvyi Rih Iron Ore Basin: materials of cathedral scientific research topics], issue 2. Kryvyi Rih, Vydavnychyi dim, 27–35. (in Ukrainian). 29. Yarkov, S. V. (2013). Rozvytok mishanykh za substratom 20-40- richnykh vidval’nykh landshaftiv Kryvorizhzhia [Development of mixed- substratum 20-40-year-old dump landscapes of Kryvorizhzhia]. Naukovi 29. Yarkov, S. V. (2013). Rozvytok mishanykh za substratom 20-40- richnykh vidval’nykh landshaftiv Kryvorizhzhia [Development of mixed- substratum 20-40-year-old dump landscapes of Kryvorizhzhia]. Naukovi 58 ISSN 2664–505X Екологiчний вiсник Криворiжжя. 2022. Вип. 7 zapysky Ternopi’skoho natsional’noho pedahohichnoho universytetu. Seriya Heohrafiya [Scientific notes of Ternopil’ National Pedagogical University. Series Geography], 2 (35), 23–30. (in Ukrainian). 30. Yevtushenko, E. O. (2020). History of phytoremediation research and project activity in department of botany and ecology at Kryvyi Rih state pedagogical university [Istoriya fitorekul’tyvatsiynykh doslidzhen’ i proektnoi diyal’nosti kafedry botaniky ta ekolohii Kryvoriz’koho derzhavnoho pedahohichnoho universytetu]. Ekolohichnyi visnyk Kryvorizhzhia [Ecological Bulletin of Kryvyi Rih District], 5, 13–30. https://doi.org/10.31812/eco-bulletin-krd.v5i0.4351 (in Ukrainian). 31. Zapata-Carbonell, J., B´egeot, C., Carry, N., Choulet, F., Delhautal, P., Gillet, F., Girardclos, O., Mouly, A., & Chalot, M. (2019). Spontaneous ecological recovery of vegetation in a red gypsum landfill: Betula pendula dominates after 10 years of inactivity. Ecological Engineering, 132, 31– 40. https://doi.org/10.1016/j.ecoleng.2019.03.013 Красова О. О., Павленко А. О. Територiальна диференцiацiя рослинного покриву старовiкових вiдвалiв Кривбасу. Екологiчний Вiсник Криворiжжя. 2022. Вип. 7. C. 44–59. Kryvyi Rih Botanical Garden of the National Academy of Sciences of Ukraine, Kryvyi Rih, Ukraine Kryvyi Rih Botanical Garden of the National Academy of Sciences of Ukraine, Kryvyi Rih, Ukraine Abstract. Old age dumps have significant scientific value in terms of preserving information about the course of the late stages of successional development of vegetation and mining landscapes in general. The study of the spatial distribution of vegetation on the iron ore dumps of the Kryvyi Rih Iron Ore Basin is currently fragmentary, so information on the territorial and structural aspect of the vegetation cover organization such objects of industrial heritage is the next step in learning the ways of their genesis. The aim of the work is to identify the patterns of territorial differentiation of iron ore dumps vegetation in Kryvyi Rih Iron Ore Basin using the examples of small dumps in the gully Pivnichna Chervona and the olden mines “Dubova balka” and “Rakhmanivskyi”. The work is based on the materials of field research conducted in 2016–2021; 80 geobotanical descriptions were used and 5 large-scale vegetation maps were created. The results of the territorial differentiation research of phytostructures, it was established that it is caused by the heterogeneity of ecotopes and is characterized by a fine-contour mosaic. Over a hundred-year period of self-development of these post-mining landscapes, ruderal cenostructures were completely eliminated from the vegetation, instead, “quasi-steppe” and petrophytic communities, similar to natural ones in terms of floral composition, were formed. The share of woody vegetation is higher in the territorial dumps structure in the north of the region, compared to the dumps in the central part; at the same time, in the northern part of Kryvyi Rih there is a predominance of representatives of the aboriginal flora in the cenostructures. The presence of drought-resistant adventitious species in the woody vegetation increases as the dumps move south along the 59 Ecological Bulletin of Kryvyi Rih District. 2022. Issue 7 latitudinal gradient and the contours of the vegetation themselves shift from the plateau to the slopes with more favourable microclimatic conditions. The regularity of territorial differentiation is the presence of significant areas of ecotopes without vegetation on the steep slopes of all the investigated dumps. We associate the further stage of their overgrowth with the development of tree-shrub communities. Prospects for further research are to use them in the organization of the ecological monitoring system of technogenesis zones of the Kryvyi Rih region. Key words: dumps, mosaic, coenostructures, herbal and tree- shrub vegetation. ДСТУ 8302:2015 Kryvyi Rih Botanical Garden of the National Academy of Sciences of Ukraine, Kryvyi Rih, Ukraine Citation as: APA Krasova, O. O., & Pavlenko, A. O. (2022). Terytorialna dyferentsiatsiia roslynnoho pokryvu starovikovykh vidvaliv kryvbasu Territorial differentiation of vegetation cover of ancient Kryvbas dumps [Territorial differentiation of Kryvbas olden dumps vegetation cover]. Ekolohichnyi visnyk Kryvorizhzhia [Ecological Bulletin of Kryvyi Rih District], 7, 44–59. https://doi.org/10.31812/eco-bulletin- krd.v7i0. Citation as: APA Krasova, O. O., & Pavlenko, A. O. (2022). Terytorialna dyferentsiatsiia roslynnoho pokryvu starovikovykh vidvaliv kryvbasu Territorial differentiation of vegetation cover of ancient Kryvbas dumps [Territorial differentiation of Kryvbas olden dumps vegetation cover]. Ekolohichnyi visnyk Kryvorizhzhia [Ecological Bulletin of Kryvyi Rih District], 7, 44–59. https://doi.org/10.31812/eco-bulletin- krd.v7i0. ДСТУ 8302:2015 Красова О. О., Павленко А. О. Територiальна диференцiацiя рослинного покриву старовiкових вiдвалiв Кривбасу. Екологiчний Вiсник Криворiжжя. 2022. Вип. 7. C. 44–59.
https://openalex.org/W4382653077	https://www.mdpi.com/2073-4441/15/13/2429/pdf?version=1688134770	English	null	An Assessment of the Effects of Light Intensities and Temperature Changes on Cyanobacteria’s Oxidative Stress via the Use of Hydrogen Peroxide as an Indicator	Water	2,023	cc-by	10,684	Citation: Rahman, M.; Asaeda, T.; Abeynayaka, H.D.L.; Fukahori, K. An Assessment of the Effects of Light Intensities and Temperature Changes on Cyanobacteria’s Oxidative Stress via the Use of Hydrogen Peroxide as an Indicator. Water 2023, 15, 2429. https://doi.org/10.3390/w15132429 Keywords: Phormidium ambiguum; Pseudanabaena foetida; hydrogen peroxide; temperatures; light intensities; protein content; chlorophyll-a Article An Assessment of the Effects of Light Intensities and Temperature Changes on Cyanobacteria’s Oxidative Stress via the Use of Hydrogen Peroxide as an Indicator Mizanur Rahman 1,* , Takashi Asaeda 1,2,3,, Helayaye Damitha Lakmali Abeynayaka 1 and Kiyotaka F 1 Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan; hdlakmali@yahoo.com (H.D.L.A.); fukahori@mail.saitama-u.ac.jp (K.F.) 2 Hydro Technology Institute, Shimo-meguro, Tokyo 153-0064, Japan 3 Research and Development Center, Nippon Koei, Tsukuba 300-1259, Japan Correspondence: masudbiochem2012@gmail.com (M.R.); asaedat@gmail.com (T.A.); Tel.: +81-70-8480-3949 (M.R.); +81-90-8043-2268 (T.A.) 1 Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan; hdlakmali@yahoo.com (H.D.L.A.); fukahori@mail.saitama-u.ac.jp (K.F.) 2 Hydro Technology Institute, Shimo-meguro, Tokyo 153-0064, Japan 3 Research and Development Center, Nippon Koei, Tsukuba 300-1259, Japan * Correspondence: masudbiochem2012@gmail.com (M.R.); asaedat@gmail.com (T.A.); Tel.: +81-70-8480-3949 (M.R.); +81-90-8043-2268 (T.A.) * Correspondence: masudbiochem2012@gmail.com (M.R.); asaedat@gmail.com (T.A.); Tel.: +81-70-8480-3949 (M.R.); +81-90-8043-2268 (T.A.) Abstract: Humans and other organisms are adversely affected by cyanobacterial blooming. This study aims to investigate the long-term effects of light intensities and different temperatures on Phormidium ambiguum and Pseudanabaena foetida. Enough P. ambiguum and P. foetida cells were acclimated for 24 days at 30 ◦C, 20 ◦C, and 10 ◦C in separate incubators. The starting day sample was collected after 24 days of acclimatization, and a second sample was collected seven days later at light intensities of 10, 30, 50, 200, and 600 µmol m−2 s−1 for each temperature. The optical density (OD730), hydrogen peroxide (H2O2) concentration, protein content, chlorophyll-a (Chl-a) concentration, and catalase (CAT) activity were measured. Light intensity changes soon after collection resulted in nearly identical starting day samples at each individual temperature. The H2O2 concentration and algal biomass increased until a light intensity of 200 µmol m−2 s−1 was reached and decreased afterward in each temperature for both species after seven days. In association with an increasing H2O2 concentration, the Chl-a concentration decreased after 50 µmol m−2s−1 of light intensity, affecting the protein content. The algal biomass was signiﬁcantly lower at 10 ◦C compared to 30 ◦C. The CAT activity increased proportionately with the H2O2 concentration and algal biomass. Therefore, water bodies in the ﬁeld can be illuminated with long-term high light intensities in different temperatures to reduce algal biomass. water water water 1. Introduction Academic Editor: Genuario Belmonte There are frequent outbreaks of cyanobacteria blooms in eutrophic lakes and reser- voirs which can cause severe environmental and economic problems [1,2]. Cyanobacteria produce toxins that have adverse effects on aquatic organisms and human health [3]. Cyanobacteria species can cause allergies, skin irritation, and respiratory problems [4–6]. Climate change and nutrient availability have contributed to the spread of cyanobacteria. The production of cyanotoxins by cyanobacteria can cause instability in water bodies [7,8]. Cyanobacteria produce toxic secondary metabolites that harm ecosystems [9] and volatile organic compounds that deteriorate water quality by increasing unpleasant odors [10]. Light intensities and temperatures greatly affect cyanobacterial habitat preferences and growth proliferation conditions [11,12]. Received: 10 June 2023 Revised: 27 June 2023 Accepted: 28 June 2023 Published: 30 June 2023 Received: 10 June 2023 Revised: 27 June 2023 Accepted: 28 June 2023 Published: 30 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cyanobacteria are photoautotrophs in nature. The presence of light directly affects the growth of cyanobacteria, and even moderate light changes are stressful [13]. The ability of cyanobacteria to respond to varying light intensities provides insights into photosynthe- sis and biotechnology applications [14]. The physiological metabolism of cyanobacteria https://www.mdpi.com/journal/water Water 2023, 15, 2429. https://doi.org/10.3390/w15132429 Water 2023, 15, 2429 2 of 16 depends on light intensity, so the effects of different light intensities must be investi- gated [12,15]. Due to extreme light conditions (very low or very high), photoinhibition can occur [16]. In photoinhibition, the electron transport in photosystem II is reversibly reduced to protect the photosynthesis apparatus [17] and other photoprotective mech- anisms, macroalgae, and phytoplankton [18–20]. Changes in chlorophyll ﬂuorescence characteristics in response to light result from photoinhibition [21]. Reactive oxygen species (ROS) cause photoinhibition. In the presence of excessive oxidative stress, ROS will become imbalanced in comparison to the antioxidant’s capacity to scavenge them [22,23]. The electron transport process, as well as the metabolism of substances and energy, create ROS as byproducts within chloroplasts, mitochondria, perox- isomes, and the cytoplasm [24,25]. Among ROS, H2O2 is the most common. Superoxide radicals are also converted into H2O2 by superoxide dismutase, which is then detoxiﬁed into water by antioxidants [26,27]. ROS accumulation harms cyanobacterial cells as it can destroy the cytoplasmic membrane, Chl-a concentration, photosynthetic apparatus, protein content, and DNA [28,29]. Phormidium ambiguum Gomont and Pseudanabaena foetida Niiyama, Tuji et Ichise are two cyanobacterial species that have been linked to water quality and safety problems around the world [30,31]. Eutrophication and global warming have led to increases in their worldwide occurrences [32,33]. Eutrophication is an alarming problem in developing countries with limited ﬁnancial resources for waste treatment [34]. P. ambiguum is a ﬁla- mentous benthic cyanobacterial species [35]. Benthic cyanobacteria are capable of forming bioﬁlms in water bodies. As cyanobacteria form mats or clusters close to banks, animals consume high levels of toxins, often resulting in their death [36]. As high concentrations of these toxins can be found in ﬂoating mats, they can also pose a health risk to humans via ingestion and direct skin exposure. Benthic cyanobacteria have also been found to contain hepatotoxins [37,38]. P. 2.2. Experimental Procedure We transferred sufﬁcient amounts of P. ambiguum and P. foetida into three incubators at 30 ◦C (±0.3 ◦C), 20 ◦C (±0.3 ◦C), and 10 ◦C (±0.3 ◦C) for 24 days at a light intensity of 10–20 µmol m−2 s−1 for P. ambiguum and a light intensity of 20–30 µmol m−2 s−1 for P. foetida. The effects of light exposure on P. ambiguum and P. foetida at each temperature were examined by introducing white, ﬂuorescent light at intensities of 10, 30, 50, 200, and 600 µmol m−2 s−1. In order to maintain 12 h of light and 12 h of darkness, an automatic timer was set (REVEX PT7, Kawaguchi city, Saitama, Japan). A quantum sensor (Apogee, MQ-200, Logan, UT, USA) was used to measure the light intensities, which were then uniformly adjusted in the medium. The culture ﬂasks were gently shaken three times per day for homogeneous exposure to light. Samples were taken twice for analysis. The ﬁrst samples were taken at 12:00 noon on the 24th day of acclimatization, considered the starting day. Following the ﬁrst sampling, a second sampling was conducted after an interval of seven days at the same time of day. The starting day collection samples were identical because the preliminary light intensities were changed soon after the collection. Using 1.5 mL tubes, 1 mL of P. ambiguum and P. foetida cells were collected separately. The cells were centrifuged at 10,000 rotations per minute (rpm) for 10 min at 4 ◦C. The supernatant was removed and kept at −80 ◦C until biochemical analysis. The buffer (speciﬁed for different bioassays) cannot break down hard cyanobacterial cell walls. To homogenize the cyanobacterial cells, ﬁve to eight 3 mm beads (Bio Medical Science Inc., Tokyo, Japan) were added to the buffer, and the mixture was vigorously shaken. The mixture was centrifuged at 10,000 rpm for 10 min at 4 ◦C, and the supernatant was used as an extract for further analysis. Three replicates of each culture group were conducted. 2.3. Estimation of Total Soluble Protein Content The total soluble protein concentrations were measured following Abeynayaka’s [57] method with minor changes. The cyanobacterial cells were homogenized using 500 mM of NaOH solution. The supernatant was collected after centrifugation at 4 ◦C for 15 min at 10,000 rpm. Then, 51 µL of supernatant extract was added to 969 µL of Coomassie Bradford protein assay for each sample. The mixture was kept at room temperature for 10 min, and the absorbance was recorded at 595 nm using a UVmini−1240. A series of known albumin concentration standard curves were used to measure the known protein concentrations. 2.1. P. ambiguum and P. foetida Cell Cultures Strains of P. ambiguum (NIES-2119) and P. foetida (NIES-512) were provided by the Na- tional Institute for Environmental Studies, Japan. Throughout the experiment, an autoclaved BG 11 medium [56] was provided to culture P. ambiguum and P. foetida. The strains were cultured at 30 ◦C (±0.3 ◦C) with a white, fluorescent lamp that provided a 12 h:12 h cycle of light and darkness in an incubator (KMH-259, AS ONE Corporation, Osaka, Japan). The light intensity in the incubator ranged between 10 and 20 µmol m−2 s−1 for P. ambiguum and between 20 and 30 µmol m−2 s−1 for P. foetida. When the cultures were incubated under light, they were manually shaken three times a day. They were subcultured until enough P. ambiguum and P. foetida were produced [57,58]. foetida is a non-blooming odorous compound containing cyanobac- terial species. P. foetida produces 2-methylisoborneol (2-MIB), which causes operational issues with water supplies [39]. Both cyanobacterial species are known to release harmful cytotoxins and grow in tropical and subtropical waters [35,40]. During the growth of cyanobacteria, higher temperatures promote metabolic accel- eration. At the same time, at the ecosystem level, they contribute to the formation of thermal stratiﬁcation, which promotes biomass accumulation at the water’s surface [41]. P. ambiguum and P. foetida grow best at 25 ◦C to 33 ◦C [42–44]. Subtropical and tropical regions can experience cyanobacterial blooms all year round due to their ability to prolifer- ate at high temperatures [45,46]. Aside from the higher temperatures of their preferential growth conditions, little is known about how ﬂuctuating temperatures affect the growth of cyanobacteria. y Considering the environmental factors that inﬂuence cyanobacterial growth and metabolism is essential to solving real-life problems associated with cyanobacteria. The growth of cyanobacteria is dependent on water temperature, light intensity, the availability of nitrogen (N) and phosphorus (P), salinity, and turbidity [47–49]. Light intensity and tem- perature are the major factors that affect the growth of cyanobacteria [47]. It is important for freshwater resource management to understand how light intensity and temperature affect changes in the growth of cyanobacteria. Cyanobacterial blooms have been eradicated using a variety of chemical and physical methods globally [50,51]. However, it is discouraging to apply chemical methods to reduce cyanobacteria due to limitations in their application and their peripheral impact on the environment [52]. There is a huge possibility of using abiotic stresses like light intensities and temperatures instead of chemical methods [53–55]. Therefore, this study aims to (1) ﬁnd the oxidative stress threshold level of cyanobacteria, using H2O2 as an indicator, and (2) determine the long-term effects of different light intensities at different temperatures. Water 2023, 15, 2429 3 of 16 2.6. H2O2 Concentration Measurement Assay The modiﬁed ferrous oxidation xylenol orange (commonly known as eFOX) assay was adapted to quantify the H2O2 concentration [62,63]. First, 100 µL of supernatant was mixed with 1 mL of the assay solution, which contained 0.250 mM of ferrous ammonium sulfate, 0.1 mM of sorbitol, 0.1 mM of xylenol orange, and 25 mM of H2SO4 (sulfuric acid), and 1% ethanol was added to increase the sensitivity of the reaction with H2O2 by 50% (Fujiﬁlm wako pure chemical corporation, Osaka, Japan). The reaction mixtures were incubated for a period of 15 min at room temperature. Spectrophotometric measurements were carried out at 560 nm (UVmini-1240, Shimadzu, Japan). A H2O2 standard curve was developed via dilution solutions of commercially available 9.8 M H2O2 (30%) (w/w) [55,64]. Trials were conducted to conﬁrm the H2O2 concentrations inside and outside of the cells. The cyanobacterial cells were separated with ﬁlter paper (0.22 µm pore size). The separated water was analyzed to quantify the H2O2 concentration. No H2O2 was detected from the separated water. It was conﬁrmed that the H2O2 concentration obtained via the methodology described above indicated the sum of the H2O2 contained by the cells and the intercellular H2O2. 2.4. Chlorophyll-a Concentration Measurements The Chl-a concentrations were quantiﬁed using the method described in [59,60]. First, 1.5 mL of 90% ethanol was added to the unfrozen cyanobacteria cells. The mixture was homogenized with 3 mm beads via vertexing and kept at room temperature (25 ± 2 ◦C) for 24 h in darkness by wrapping it with aluminum foil. Each sample was centrifuged at 12,000 rpm, and the supernatant was measured with a UVmini−1240 at absorption wave- lengths of 750 nm, 665 nm, and 649 nm. The 750 nm reading was obtained to conﬁrm whether the extract interfered with organic matters or not. Each value lay between −0.003 Water 2023, 15, 2429 4 of 16 and 0.003, indicating the purity of the extract. The formula for calculating the Chl-a content is as follows: and 0.003, indicating the purity of the extract. The formula for calculating the Chl-a content is as follows: Chl ( / L) (13 95 A 6 88 A ) 1 5 Chl-a (µg/mL) = (13.95 ∗A665 −6.88 ∗A649) ∗1.5 2.8. Statistical Analysis A one-way analysis of variance (ANOVA), followed by Tukey’s test, was performed to check the statistical signiﬁcance of the variations among different PAR groups. To determine how the light intensities and temperatures interacted, we used a two-way ANOVA. Pearson’s correlation analysis was conducted to evaluate the correlations among parameters. IBM SPSS Statistics (Version 28.0 IBM Corporation, Chicago, IL, USA) software was used to execute the statistical analyses. 2.7. CAT Assay The CAT activity was measured spectrophotometrically at room temperature by monitoring the decrease in absorbance at 240 nm resulting from the decomposition of H2O2. The CAT activity was measured via the method of Aebi [65]. The enzyme was extracted using 3 mm beads with 1 mL of potassium phosphate buffer (0.05 M, pH 7.0) containing 0.1 mM of EDTA. The reaction mixture contained 15 µL of 0.75 M H2O2, 920 µL of potassium phosphate buffer, and 65 µL of enzyme extract. Measurements were obtained every 10 s for 3 min, and calculations were conducted using the 39.4 mM/cm extinction coefﬁcient. 2.5. Identiﬁcation of Cell Growth OD730 measurements were carried out with the help of a UV-Vis spectrophotometer (UVmini-1240, Shimadzu, Japan) in order to quantify the growth of the cyanobacteria. The OD730 was measured using a previously proposed methodology [61] with a minor modiﬁcation. An amount of 1 mL of cyanobacterial cells was homogenized with 3 mm beads (Bio Medical Science Inc., Tokyo, Japan) and measured at a wavelength of 730 nm. 3. Results OD730, H2O2 concentration, protein concentration, Chl-a content, and CAT activity were monitored in P. ambiguum and P. foetida at 30 ◦C, 20 ◦C, and 10 ◦C based on each light intensity for the starting day and after the seven-day period. Due to changes in the preliminary light intensities soon after the samples were collected, the starting day collection for each temperature did not show much ﬂuctuation. Over the seven days of exposure, the H2O2 concentration steadily increased with increases in the light inten- sity until a light intensity of 200 µmol m−2 s−1, while it declined at a light intensity of 600 µmol m−2 s−1 at each temperature in both species (Figures 1b and 2b). In both species, Water 2023, 15, 2429 5 of 16 and pro h the OD730 and protein concentration, which had been lower at the starting day at 20 ◦C, increased up to the 30 ◦C level in seven days (Figure 1a,c and Figure 2a,c). The OD730 values were higher in P. ambiguum than P. foetida at each temperature after seven days of exposure. For example, at 30 ◦C, the OD730 value for P. ambiguum was ~2, while the OD730 value for P. foetida was ~0.6. Over the seven days of treatment, the protein concentrations of P. ambiguum and P. foetida decreased as the temperature decreased from 30 ◦C to 10 ◦C. The Chl-a concentrations of P. ambiguum and P. foetida under different light intensities at each temperature are shown in Figures 1d and 2d. The Chl-a concentration substantially decreased as the light intensity increased from 50 µmol m−2 s−1 to 200 µmol m−2 s−1 (p < 0.001 for both species) and thereafter at 30 ◦C, 20 ◦C, and 10 ◦C in both species over seven days of light exposure (Figures 1d and 2d). After seven days, P. ambiguum had higher Chl-a contents than P. foetida at 30 ◦C, 20 ◦C, and 10 ◦C. The antioxidant activity of CAT increased until a light intensity of 200 µmol m−2 s−1 was reached in response to oxida- tive damage due to the H2O2 concentration and decreased with higher light intensities at 30 ◦C, 20 ◦C, and 10 ◦C in both species (Figures 1e and 2e). In Figure 3a,b, OD730 is pre- sented with respect to the protein and Chl-a contents, respectively. 3. Results There is a signiﬁcantly high proportionate correlation observed between OD730 and protein for all temperatures (at 30 ◦C for P. ambiguum r = 0.991, p < 0.001 and at 30 ◦C for P. foetida r = 0.952, p < 0.001; at 20 ◦C for P. ambiguum r = 0.978, p < 0.001 and at 20 ◦C for P. foetida r = 0.990, p < 0.001; at 10 ◦C for P. ambiguum r = 0.829, p < 0.001 and at 10 ◦C for P. foetida r = 0.715, p < 0.001) after the seven days of exposure. On the starting day, both Chl-a and OD730 were signiﬁ- cantly proportionate with respect to protein concentration, whereas after seven days, the Chl-a content was signiﬁcantly smaller compared to the starting level (p < 0.001 for both species). For both species, the concentration of H2O2 per biomass was expressed by the change in H2O2/OD730 after seven days, which is presented in Figure 4. In every case, the H2O2/OD730 increased following the light intensity until 200 µmol m−2 s−1, while it declined at a light intensity of 600 µmol m−2 s−1. The H2O2/OD730 values were lower at 10 ◦C compared to 30 ◦C in both species. There was a positive correlation observed between H2O2/OD730 and different light intensities at temperatures ranging from 30 ◦C to 10 ◦C in P. ambiguum (r = 0.390, p = 0.176) and P. foetida (r = 0.282, p = 0.307). y g g biguum than P. foetida at each temperature after seven days of exposure. For example, a 30 °C, the OD730 value for P. ambiguum was ~2, while the OD730 value for P. foetida was ~0.6 Over the seven days of treatment, the protein concentrations of P. ambiguum and P. foetid decreased as the temperature decreased from 30 °C to 10 °C. The Chl-a concentrations o P. ambiguum and P. foetida under diﬀerent light intensities at each temperature are show n Figures 1d and 2d. The Chl-a concentration substantially decreased as the light intensit ncreased from 50 µmol m−2 s−1 to 200 µmol m−2 s−1 (p < 0.001 for both species) and thereafte at 30 °C, 20 °C, and 10 °C in both species over seven days of light exposure (Figures 1 and 2d). After seven days, P. ambiguum had higher Chl-a contents than P. foetida at 30 °C 20 °C, and 10 °C. 3. Results Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), pro tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard devia tions. Figure 1. Light intensities at 30 ◦C, 20 ◦C, and 10 ◦C affected OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard deviations. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), pro- tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard devia- tions. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), pro tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-da treatment, whereas blank quadrate indicates starting day. The error bars indicate standard devia tions. Figure 1. Light intensities at 30 ◦C, 20 ◦C, and 10 ◦C affected OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard deviations. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), pro- tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard devia- tions. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), p tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven- treatment, whereas blank quadrate indicates starting day. The error bars indicate standard de tions. Figure 1. Light intensities at 30 ◦C, 20 ◦C, and 10 ◦C affected OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicate seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard deviations. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), p tein concentration (c), Chl-a content (d), and CAT activity (e). 3. Results The antioxidant activity of CAT increased until a light intensity of 20 µmol m−2 s−1 was reached in response to oxidative damage due to the H2O2 concentratio and decreased with higher light intensities at 30 °C, 20 °C, and 10 °C in both species (Fig ures 1e and 2e). In Figure 3a,b, OD730 is presented with respect to the protein and Chl- contents, respectively. There is a signiﬁcantly high proportionate correlation observed be ween OD730 and protein for all temperatures (at 30 °C for P. ambiguum r = 0.991, p < 0.00 and at 30 °C for P. foetida r = 0.952, p < 0.001; at 20 °C for P. ambiguum r = 0.978, p < 0.00 and at 20 °C for P. foetida r = 0.990, p < 0.001; at 10 °C for P. ambiguum r = 0.829, p < 0.00 and at 10 °C for P. foetida r = 0.715, p < 0.001) after the seven days of exposure. On th starting day, both Chl-a and OD730 were signiﬁcantly proportionate with respect to protein concentration, whereas after seven days, the Chl-a content was signiﬁcantly smaller com pared to the starting level (p < 0.001 for both species). For both species, the concentratio of H2O2 per biomass was expressed by the change in H2O2/OD730 after seven days, whic s presented in Figure 4. In every case, the H2O2/OD730 increased following the light inten sity until 200 µmol m−2 s−1, while it declined at a light intensity of 600 µmol m−2 s−1. Th H2O2/OD730 values were lower at 10 °C compared to 30 °C in both species. There was positive correlation observed between H2O2/OD730 and diﬀerent light intensities at tem peratures ranging from 30 °C to 10 °C in P. ambiguum (r = 0.390, p = 0.176) and P. foetida ( = 0.282, p = 0.307). Figure 1. Cont. Figure 1 Cont Figure 1. Cont. ater 2023, 15, 2429 6 of 1 2023, 15, x FOR PEER REVIEW 6 o Figure 1. Cont. Water 2023, 15, 2429 ater 2023, 15, x FOR PE 6 of 16 6 of 1 Figure 1. Cont. 7 of 16 7 of 1 7 of 16 7 of 1 Water 2023, 15, 2429 ater 2023, 15, x FOR P Figure 1. 3. Results Solid quadrate indicates seven-d treatment, whereas blank quadrate indicates starting day. The error bars indicate standard dev tions. Figure 2. Cont. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), pr tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-da treatment, whereas blank quadrate indicates starting day. The error bars indicate standard devi tions. Figure 1. Light intensities at 30 ◦C, 20 ◦C, and 10 ◦C affected OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard deviations. Figure 1. Light intensities at 30 °C, 20 °C, and 10 °C aﬀected OD730 (a), H2O2 concentration (b), pro tein concentration (c), Chl-a content (d), and CAT activity (e). Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard devia tions. Figure 2. Cont. Figure 2. Cont. 8 of 16 8 of 1 Water 2023, 15, 2429 ter 2023, 15, x FOR PE Figure 2. Changes in OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content and CAT activity (e) with respect to light intensities at the start and after seven days of P. foetid 30 °C, 20 °C, and 10 °C. Solid quadrate indicates seven-day treatment, whereas blank quadrate dicates starting day. The error bars indicate standard deviation. Figure 2. Changes in OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d and CAT activity (e) with respect to light intensities at the start and after seven days of P. foetida a 30 ◦C, 20 ◦C, and 10 ◦C. Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard deviation. igure 2. Changes in OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d nd CAT activity (e) with respect to light intensities at the start and after seven days of P. foetida a 0 °C, 20 °C, and 10 °C. Solid quadrate indicates seven-day treatment, whereas blank quadrate in icates starting day. The error bars indicate standard deviation. Figure 2. Changes in OD730 (a), H2O2 concentration (b), protein concentration (c), Chl-a content (d), and CAT activity (e) with respect to light intensities at the start and after seven days of P. foetida at 30 ◦C, 20 ◦C, and 10 ◦C. 4. Discussion 4.1. Eﬀects of Light Intensities on the Response of H2O2 4.1. Effects of Light Intensities on the Response of H2O2 4. Discussion 4.1. Eﬀects of Light Intensities on the Response of H2O2 4.1. Effects of Light Intensities on the Response of H2O2 i u io 4.1. Eﬀects of Light Intensities on the Response of H2O2 4.1. Effects of Light Intensities on the Response of H2O2 ﬀ f g p f Excess levels of ROS can be generated through abiotic stress [66–68]. The accumula- tion of ROS in each cyanobacteria species under high light (higher than 200 µmol m−2 s−1) and alterations in temperature may result in oxidative damage, which may be one reason for the inhibition of cell growth (Figures 1a and 2a). Cells exposed to high levels of envi- ronmental stress produce and accumulate H2O2. As cyanobacteria are exposed to various abiotic stresses in natural water, they are more likely to undergo oxidative stress, produc- ing H2O2 in the process, which may deteriorate cyanobacterial biomass by producing hy- droxyl radicals. The production of H2O2 may not necessarily be cumulative in abiotic stresses [69,70]. The H2O2 concentration was enhanced until a light intensity of 200 µmol m−2 s−1 and decreased afterward at each temperature (Figures 1b and 2b). The trend was more prominent at 30 °C in the seven days of exposure. In the present study, photoinhi- bition was seen even with a light intensity of 200 µmol m−2 s−1, whereas a light intensity of 1000 µmol m−2 s−1 has been reported in ﬁeld observations [71,72]. In ﬁeld monitoring, light intensity in water decreases relatively quickly with depth, despite surface colonies receiv- ing high-intensity solar radiation. As a result, the colony of cyanobacteria does not receive strong solar radiation directly, especially P. ambiguum and P. foetida, since they stay in relatively deep water (~2 m) [73]. In addition, they avoid the highest level of solar radia- tion during the day by migrating to deeper zones [74,75], where they are likely to avoid the high level of solar radiation and oxidative stress before the recovery of homeostasis d Excess levels of ROS can be generated through abiotic stress [66–68]. The accumulation of ROS in each cyanobacteria species under high light (higher than 200 µmol m−2 s−1) and alterations in temperature may result in oxidative damage, which may be one reason for the inhibition of cell growth (Figures 1a and 2a). Cells exposed to high levels of environmental stress produce and accumulate H2O2. 4. Discussion 4.1. Eﬀects of Light Intensities on the Response of H2O2 4.1. Effects of Light Intensities on the Response of H2O2 As cyanobacteria are exposed to various abiotic stresses in natural water, they are more likely to undergo oxidative stress, producing H2O2 in the process, which may deteriorate cyanobacterial biomass by producing hydroxyl radicals. The production of H2O2 may not necessarily be cumulative in abiotic stresses [69,70]. The H2O2 concentration was enhanced until a light intensity of 200 µmol m−2 s−1 and decreased afterward at each temperature (Figures 1b and 2b). The trend was more prominent at 30 ◦C in the seven days of exposure. In the present study, photoinhibition was seen even with a light intensity of 200 µmol m−2 s−1, whereas a light intensity of 1000 µmol m−2 s−1 has been reported in ﬁeld observations [71,72]. In ﬁeld monitoring, light intensity in water decreases relatively quickly with depth, despite surface colonies receiving high-intensity solar radiation. As a result, the colony of cyanobacteria does not receive strong solar radiation directly, especially P. ambiguum and P. foetida, since they stay in relatively deep water (~2 m) [73]. In addition, they avoid the highest level of solar radiation during the day by migrating to deeper zones [74,75], where they are likely to avoid the high level of solar radiation and oxidative stress before the recovery of homeostasis via increases in antioxidant activities [74]. via increases in antioxidant activities [74]. In the past, laboratory incubations under diﬀerent H2O2 concentrations provided the lethal H2O2 dosage for cyanobacteria [76–78], which implies that cyanobacterial biomass is degraded at higher H2O2 concentrations. The lethal H2O2 dosage for the suppression of cyanobacteria in vitro under diﬀerent H2O2 concentrations ranges from 1 to 1000 µmol L−1 [76,79,80]. This study suggests that if the H2O2/OD730 exceeded ~2 to 6 µmol/L (depending on temperatures) (Figure 4) after seven days of treatment, it signiﬁcantly declined after- ward, even against higher temperatures or light intensities. The high H2O2 concentration In the past, laboratory incubations under different H2O2 concentrations provided the lethal H2O2 dosage for cyanobacteria [76–78], which implies that cyanobacterial biomass is degraded at higher H2O2 concentrations. The lethal H2O2 dosage for the suppression of cyanobacteria in vitro under different H2O2 concentrations ranges from 1 to 1000 µmol L−1 [76,79,80]. This study suggests that if the H2O2/OD730 exceeded ~2 to 6 µmol/L (depending on temperatures) (Figure 4) after seven days of treatment, it signiﬁ- cantly declined afterward, even against higher temperatures or light intensities. 3. Results Solid quadrate indicates seven-day treatment, whereas blank quadrate indicates starting day. The error bars indicate standard deviation. Water 2023, 15, 2429 W 2023 15 FOR 9 of 16 9 f 16 2023, 15, x FOR PEER REVIEW 9 of Figure 3. The relationship between protein and OD730, as well as the relationship between prote and Chl-a in P. ambiguum (a) and P. foetida (b) at 30 °C, 20 °C, and 10 °C. Figure 3. The relationship between protein and OD730, as well as the relationship between prote and Chl-a in P. ambiguum (a) and P. foetida (b) at 30 ◦C, 20 ◦C, and 10 ◦C. Figure 3. The relationship between protein and OD730, as well as the relationship between protein and Chl-a in P. ambiguum (a) and P. foetida (b) at 30 °C, 20 °C, and 10 °C. Figure 3. The relationship between protein and OD730, as well as the relationship between protein and Chl-a in P. ambiguum (a) and P. foetida (b) at 30 ◦C, 20 ◦C, and 10 ◦C. Figure 3. The relationship between protein and OD730, as well as the relationship between protein and Chl-a in P. ambiguum (a) and P. foetida (b) at 30 °C, 20 °C, and 10 °C. Figure 3. The relationship between protein and OD730, as well as the relationship between protein and Chl-a in P. ambiguum (a) and P. foetida (b) at 30 ◦C, 20 ◦C, and 10 ◦C. 10 of 16 10 of 16 10 of 16 10 of 16 Water 2023, 15, 2429 Water 2023, 15, x FOR Figure 4. Changes in H2O2/OD730 after seven days of exposure to diﬀerent light intensities in P. am- biguum and P. foetida at 30 °C, 20 °C, and 10 °C. Figure 4. Changes in H2O2/OD730 after seven days of exposure to different light intensities in P. ambiguum and P. foetida at 30 ◦C, 20 ◦C, and 10 ◦C. Figure 4. Changes in H2O2/OD730 after seven days of exposure to diﬀerent light intensities in P. am- biguum and P. foetida at 30 °C, 20 °C, and 10 °C. Figure 4. Changes in H2O2/OD730 after seven days of exposure to different light intensities in P. ambiguum and P. foetida at 30 ◦C, 20 ◦C, and 10 ◦C. 4. Discussion 4.1. Eﬀects of Light Intensities on the Response of H2O2 4.1. Effects of Light Intensities on the Response of H2O2 The high H2O2 concentration deteriorates the physiological condition without generating further Water 2023, 15, 2429 11 of 16 11 of 16 H2O2 [55,81]. In contrast, the Chl-a concentration was affected by even lower levels of light intensities (after 50 µmol m−2 s−1). A low H2O2 concentration is generated with sufﬁciently low PAR (50 µmol m−2 s−1), and the Chl-a concentration ﬂuctuates positively with the PAR (data are not shown). This may be due to an adaptation to increase productiv- ity. Chl-a concentration increases due to the breakdown of photosynthesis components, such as photosystems I and II [82] and phycobilisomes [16]. In contrast, a high Chl-a concentration increases the photosynthesis rate, generating more H2O2 and deteriorating the photosynthesis apparatus. Thus, an increased H2O2 concentration negatively alters the Chl-a concentration under highly stressful conditions [55]. The H2O2 concentration also becomes a useful indicator for demonstrating the effects of environmental stress on other plants and their productivities [55]. In the present study, we used the protein content as an indicator of biomass rather than the Chl-a content for both cyanobacterial species [83]. One-third to one-half of the biomass of cyanobacteria cells is protein [84]. There was a high correlation between OD730 and protein content (Figure 3a,b), indicating that the protein content is a proper biomass reference. Compared with the OD730 values and protein contents, the Chl-a contents were affected by lower light intensities. A suitable parameter should be selected to identify the cyanobacterial biomass. H2O2/OD730 could be a suitable parameter for evaluating the H2O2 amount in a cell. In the ﬁeld treatment for artiﬁcially inducing H2O2, the light intensity is the primary component in increasing the H2O2 concentration in nature and accumulating it in a cell. The required amount of H2O2 to be induced should be identiﬁed using a suitable factor, such as H2O2/OD730, for the suppression of algal blooms. The seasonal dynamics of phytoplankton are largely determined by temperature [85]. When temperatures are relatively high, cyanobacteria grow the fastest [86]. Depending on the species, they respond differently to low temperatures. In the present study, the growth of P. ambiguum was signiﬁcantly decreased by decreasing the temperature from 30 ◦C to 10 ◦C (p < 0.001 for both species), similar to other studies [26,87,88] (Figures 1a and 2a). Decreasing temperatures (from 30 ◦C to 10 ◦C) also signiﬁcantly affected the H2O2, Chl-a, and protein concentrations. 4.2. Antioxidant Activity in Response to Oxidative Stress The tissue H2O2 concentration at a particular time is determined as the balance be- tween the generation rate and the scavenging by antioxidant activities. This concentration works as a signal to activate antioxidant behavior. Antioxidant activity increases in response to oxidative stress to prevent cell damage [89]. Oxidative stress and antioxidative enzymes are triggered by abiotic stress [26,90]. In the present study, high light intensities (200 to 600 µmol m−2 s−1) decreased the H2O2 concentration, and low light intensities (10 to 200 µmol m−2 s−1) increased it. CAT activity proportionally increased with H2O2 concen- tration until 200 µmol m−2 s−1 at each temperature and decreased afterward, following the reduction in H2O2 concentration with high light intensities (Figures 1e and 2e). 4. Discussion 4.1. Eﬀects of Light Intensities on the Response of H2O2 4.1. Effects of Light Intensities on the Response of H2O2 The amount of H2O2 in a cell (H2O2/OD730) was also lower at 10 ◦C compared to 30 ◦C (Figure 4). These results indicate that P. ambiguum and P. foetida maintain higher metabolisms with higher temperatures, bringing about high growth rates with high temperatures. 4.3. Management of Cyanobacterial Blooms Long-term exposure to light intensities at different temperatures is highly appreciated as a non-chemical approach to controlling P. ambiguum and P. foetida. This result opposes the hypothesis that low-light exposure suppresses cyanobacterial growth. According to Visser et al. [73], artiﬁcial water mixing in lakes and reservoirs is only sometimes efﬁcient. During artiﬁcial mixing, oxygen levels increase in the water, the temperatures of the deep layers are increased, and the temperature on the surface is lowered. For instance, de- stratiﬁcation (the development of vertical mixing) decreases the surface temperature from 28.9 ◦C to 26.4 ◦C, whereas deep-water aeration increases the temperature from 8 ◦C to 23.7 ◦C [91]. The mixing occurs deep enough to limit light availability, and the mixing Water 2023, 15, 2429 12 of 16 12 of 16 devices are well distributed horizontally over the lake. A reduction in light exposure will lead to decreases in the growth of cyanobacteria and algae. Among the drawbacks of artiﬁ- cial mixing are the cost of installation and operation and the energy required during the entire growing season. Using intermittent mixing might be beneﬁcial in reducing energy costs and reducing the ﬂotation velocities of cyanobacteria. The availability of light under water can inﬂuence cyanobacterial growth composition [92]. When the wind is mixed with the unsustainable vertical heterogeneity of an algal biomass, light cannot enter the deeper zone of water. On the other hand, under calm conditions (a stable water column, vertical migration, and a sustained surface biomass maximum) in a lake, light may enter the deeper zone [92]. For example, in comparison with the euphotic depth, the Secchi depth is roughly half. Water transparency is measured via the Secchi depth, where the Secchi depth increases with increasing transparency. A lake’s bottom is illuminated when the Secchi depth is roughly half its euphotic depth [92]. Phytoplankton may experience light limitations when the Secchi depth/total euphotic depth ratio is below 0.5. It is important to consider both species’ low-light and high-light vulnerabilities when introducing the system. A destratiﬁcation process must be designed beforehand based on the intensity and distribution of light and temperature [93,94]. Other practical methods can be intro- duced to control cyanobacterial species, such as exposing deep water to high intensities of light without raising the temperature. There should be a variety of methods developed to illuminate the water column with a greater light intensity than what is tolerated by these species. References Zhang, C.; Chen, J.; Ding, A.; Hou, Y.; Li, Z.; Niu, X.; Su, Y.; Xu, E. Laws The 2007 water crisis in Wux origin. J. Hazard. Mater. 2010, 182, 130–135. [CrossRef] g 11. Cao, H.-S.; Tao, Y.; Kong, F.-X.; Yang, Z. Relationship between Temperature and Cyanobacterial Recruitment from Sediments in Laboratory and Field Studies. J. Freshw. Ecol. 2008, 23, 405–412. [CrossRef] y J [ ] 12. Asaeda, T.; Rahman, M.; Abeynayaka, H.D.L. Hydrogen peroxide can be a plausible biomarker in cyanobacterial bloom treatment. Sci. Rep. 2022, 12, 1–11. [CrossRef] p 13. Welkie, D.; Rubin, B.E.; Diamond, S.; Hood, R.D.; Savage, D.F.; Golden, S.S. A Hard Day’s Night: Cyanobacteria in Diel Cycles. Trends Microbiol. 2019, 27, 231–242. [CrossRef] [PubMed] 14. Flombaum, P.; Gallegos, J.L.; Gordillo, R.A.; Rincón, J.; Zabala, L.L.; Jiao, N.; Karl, D.M.; Li, W.K.W.; Lomas, M.W.; Veneziano, D.; et al. Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus. Proc. Natl. Acad. Sci. USA 2013, 110, 9824–9829. [CrossRef] 15. Barnuevo, A.; Asaeda, T. Integrating the ecophysiology and biochemical stress indicators into the paradigm of mangrove ecology and a rehabilitation blueprint. PLoS ONE 2018, 13, e0202227. [CrossRef] aeda, T. Integrating the ecophysiology and biochemical stress indicators into the paradigm of mangrove ecol ion blueprint. PLoS ONE 2018, 13, e0202227. [CrossRef] 16. Walsh, K.; Jones, G.J.; Dunstan, R.H. Effect of irradiance on fatty acid, carotenoid, total protein composition and growth of Microcystis aeruginosa. Phytochemistry 1997, 44, 817–824. [CrossRef] 17. Hanelt, D.P. Photoinhibition of photosynthesis in marine macroalgae. Sci. Mar. 1996, 60, 243–248. 18. Hortonn, P.; Ruban, A. Molecular design of the photosystem II light-harvesting antenna: Photosynthesis and photoprotection. J. Exp. Bot. 2005, 56, 365–373. [CrossRef] 19. Kirilovsky, D. Photoprotection in cyanobacteria: The orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism. Photosynth. Res. 2007, 93, 7–16. [CrossRef] 20. Zhang, Y.; Hu, M.; Zhong, L.; Wiedensohler, A.; Liu, S.; Andreae, M.; Wang, W.; Fan, S. Regional Integrated Experiments on Air Quality over Pearl River Delta 2004 (PRIDE-PRD2004): Overview. Atmos. Environ. 2008, 42, 6157–6173. [CrossRef] 21. Jones, R.J.; Hoegh-Guldberg, O. Diurnal changes in the photochemical efﬁciency of the symbiotic dinoﬂa corals: Photoprotection, photoinactivation and the relationship to coral bleaching. Plant Cell Environ. 2 21. Jones, R.J.; Hoegh-Guldberg, O. Diurnal changes in the photochemical efﬁciency of the symbiotic dinoﬂagellates (Dinophyceae) of corals: Photoprotection, photoinactivation and the relationship to coral bleaching. Plant Cell Environ. 2001, 24, 89–99. [CrossRef] 22. 5. Conclusions Cyanobacterial species exhibit photoinhibition even after seven days of exposure to a light intensity of 200 µmolm−2s−1 at 30 ◦C, 20 ◦C, and 10 ◦C. High light intensities (higher than 200 µmol m−2 s−1) signiﬁcantly affect H2O2 concentrations, protein concentrations, and Chl-a contents at each temperature. CAT activity increased until a light intensity of 200 µmolm−2s−1 in response to the H2O2 concentration and the ability of the algal biomass to scavenge oxidative stress, and it decreased afterward with higher light intensities. The algal biomass decreased from 30 ◦C to 10 ◦C due to the effect of temperature. The opportunity of developing control mechanisms based on high light intensities that result in slower growth could be explored, as could the further development of existing methods that rely on low light intensities, which could possibly result in reductions in growth rates. Water bodies could be effectively controlled via illumination with a greater light intensity than the intensities tolerated by these species. Author Contributions: Conceptualization, T.A. and M.R.; methodology, T.A.; software, M.R.; valida- tion, T.A., H.D.L.A., K.F. and M.R.; formal analysis, M.R. and H.D.L.A.; investigation, M.R.; resources, T.A.; data curation, M.R.; writing—original draft preparation, M.R.; writing—review and editing, T.A. and K.F.; visualization, T.A. and M.R.; supervision, T.A. and K.F.; project administration, T.A.; funding acquisition, T.A. All authors have read and agreed to the published version of the manuscript. Funding: The Grant-in-Aid for Scientiﬁc Research (B) (19H02245), and (C) (20K04714) provided ﬁnancial support for this work, and the Japanese Society for the Promotion of Science (JSPS) Fund for Joint International Research (18KK0116) also provided support. Data Availability Statement: The authors state that we will make all data available to anyone who requests the data without undue preservation. This manuscript contains information supporting this study’s ﬁndings in the Materials and Methods section in the form of a preprint at “Research square” with the identiﬁer (https://doi.org/10.21203/rs.3.rs-2326349/v1) (accessed on 11 June 2023); (https://doi.org/10.21203/rs.3.rs-2276741/v1) (accessed on 11 June 2023); and (https://doi.org/10.2 1203/rs.3.rs-2232330/v1) (accessed on 11 June 2023). The authors declare that previous DOIs will be updated with the new DOI upon acceptance. Conﬂicts of Interest: The authors declare no conﬂict of interest. 13 of 16 13 of 16 Water 2023, 15, 2429 References 1. Dokulil, M.T.; Teubner, K. Cyanobacterial dominance in lakes. Hydrobiologia 2000, 438, 1–12. [CrossRef 2. Chen, Y.; Weng, Y.; Zhou, M.; Meng, Y.; Liu, J.; Yang, L.; Zuo, Z. Linalool-and α-terpineol-induced programmed cell death in Chlamydomona sreinhardtii. Ecotoxicol. Environ. Saf. 2019, 167, 435–440. [CrossRef] y f 3. Carmichael, W.W. 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https://openalex.org/W4380685774	https://repositorio.ul.pt/bitstream/10451/58350/1/rsos.230525%20%281%29.pdf	English	null	Intraspecific variation for host immune activation by the spider mite <i>Tetranychus evansi</i>	Royal Society open science	2,023	cc-by	12,493	Author for correspondence: Author for correspondence: Jéssica Teodoro-Paulo e-mail: jessicapaulo94@gmail.com Intraspecific variation for host immune activation by the spider mite Tetranychus evansi royalsocietypublishing.org/journal/rsos Research Cite this article: Teodoro-Paulo J, Alba JM, Charlesworth S, Kant MR, Magalhães S, Duncan AB. 2023 Intraspecific variation for host immune activation by the spider mite Tetranychus evansi. R. Soc. Open Sci. 10: 230525. https://doi.org/10.1098/rsos.230525 1cE3c—Centre for Ecology, Evolution and Environmental Changes & CHANGE—Global Change and Sustainability Institute, Faculty of Sciences, University of Lisbon, Lisbon, Portugal 2Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands 3Institut des Sciences de l’Évolution, University of Montpellier, CNRS, IRD, Montpellier, France JT-P, 0000-0003-2933-9940; ABD, 0000-0002-6499-2913 Many parasites can interfere with their host’s defences to maximize their fitness. Here, we investigated if there is heritable variation in the spider mite Tetranychus evansi for traits associated with how they interact with their host plant. We also determined if this variation correlates with mite fecundity. Tetranychus evansi can interfere with jasmonate (JA) defences which are the main determinant of anti-herbivore immunity in plants. We investigated (i) variation in fecundity in the presence and absence of JA defences, making use of a wild-type tomato cultivar and a JA-deficient mutant (defenseless-1), and (ii) variation in the induction of JA defences, in four T. evansi field populations and 59 inbred lines created from an outbred population originating from controlled crosses of the four field populations. We observed a strong positive genetic correlation between fecundity in the presence (on wild-type) and the absence of JA defences (on defenseless-1). However, fecundity did not correlate with the magnitude of induced JA defences in wild-type plants. Our results suggest that the performance of the specialist T. evansi is not related to their ability to manipulate plant defences, either because all lines can adequately reduce levels of defences, or because they are resistant to them. Jéssica Teodoro-Paulo1,2,3, Juan M. Alba2, Steven Charlesworth1, Merijn R. Kant2, Sara Magalhães1 and Alison B. Duncan3 1. Introduction Antagonistic interactions between organisms, such as between parasites and their hosts, are abundant in nature. Hosts have evolved numerous traits relating to resistance or avoidance to minimize the negative effects on fitness caused by parasites Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c. 6673594. © 2023 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 Interference of host defences is a strategy that has evolved in diverse plant parasites, including viruses [9], nematodes [10], lepidopteran larvae [11], mites [12–14] and aphids [15]; and animal parasites, such as parasitoid wasps [16], Plasmodium [17] and HIV [18]. Immune interference is often associated with the production of molecules by the parasite that alter host–cell structure and function (in plants [19]; in animals [20]) referred to as ‘effectors’. Many effectors have been identified and some of these have been characterized in detail (reviewed in [21]), but for many the mode of action, genetic variation and their costs are unclear. Theory predicts that their production may incur direct physiological costs for the parasite [22]. There may also be ecological costs due to parasite-mediated changes in host immune traits, as a disarmed host may be more suitable for competing parasites (e.g. helminths [23]; reviewed for spider mites [24]; reviewed in [25]; theoretically shown in [26]). These physiological and ecological costs may be responsible for the maintenance of genetic diversity for immune interference of host defences [27], although evidence for this is still scarce [9,16,28]. os R. Soc. Open Sci. 10: 230525 Plant immunity largely depends on the upstream action of plant hormones, with salicylic acid (SA) and jasmonic acid (JA) being the central players in mediating defences such as the production of toxic secondary metabolites and proteins that interfere with herbivore digestion and development [29–31]. The SA pathway is mainly involved in the response against biotrophic pathogens and phloem-feeding herbivores, while the JA pathway is involved in the defence against necrotrophic pathogens and chewing or cell-content-feeding herbivores [31]. Several herbivores have been shown to interfere with these defence pathways and this has been linked to increases in arthropod fitness (e.g. weight, fecundity or survival; lepidopteran larvae [11]; white flies [32]; aphids [33,34]; spider mites [12,13,35]). In some cases, interference occurred after the initial onset of induced defences [13,36]. There is evidence that plant defence interference (e.g. spider mites [37]; aphids [38]) and effector production by herbivores (spider mites [39,40]; Lepidoptera [41]) are plastic traits that can be influenced by environmental cues such as competition [37], light–dark cycles [42] or temperature [41]. This plasticity may serve to limit physiological or ecological costs associated with immune interference. For instance, the arrival of competitors has been shown to modulate immune interference in spider mites, by stronger suppression of local defences in plant leaves [37]. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 In addition, mites were also shown to shield their feeding site from competitors via physical barriers (through webbing [43], or by using leaf hairs as a refuge [44]), and to mitigate competitive population growth via reproductive interference [45]. This may act to buffer competitor-driven selection against immune interference [24]. Several studies have reported immune interference (often referred to as defence suppression) in tetranychid spider mites [12,13,39,40,43]. Knegt et al. [46] observed levels of immune interference to differ among T. evansi populations collected on different continents. In this study, we measure intraspecific variation in immune interference at the population and genotype level, and how it relates to fitness differences. We used four field populations collected in Portugal and 59 inbred lines generated from an outbred population, created from controlled crosses of all four field populations [44], thus capturing the genetic variation present in them. First, we compared the fecundity of the four field populations and inbred lines on a common tomato variety (referred to as wild-type, WT) and on a mutant tomato unable to activate the JA pathway (defenseless-1, def-1) [47–49] to assess how they cope with JA defences. We then assessed the magnitude of induced defences in the WT for 19 lines using marker genes involved in the mite-induced JA and SA pathways. We observed that fecundity across the four field populations differed marginally, with no difference in the expression of the tomato defence genes. Yet we did find variation in fecundity and their effect on the expression of defence genes across the lines, with some lines inducing immune responses. This variation in fecundity has a genetic basis but did not correlate with their effect on induced tomato defences. [1–4]. This, in turn, selects for parasites that can overcome, resist or interfere with host resistance [5–7], which can lead to the maintenance of genetic variation in host and parasite populations [8]. [1–4]. This, in turn, selects for parasites that can overcome, resist or interfere with host resistance [5–7], which can lead to the maintenance of genetic variation in host and parasite populations [8]. 2 2.2. Tetranychus evansi field and outbred populations The collection of the four field populations and the generation of the outbred mite population are described in detail in [44]. In summary, at four locations in Portugal in 2017 T. evansi mites were collected from field-grown tomato plants. We refer to these four mite populations as VIT, 6M1, QG and PBS. All four populations had the same ITS haplotype [44]. To remove Wolbachia, these populations were heat shocked at 33°C for 42 days and then used to create an outbred laboratory population, with a maximal level of genetic diversity, by performing controlled crosses while avoiding over-representation of genotypes from a given population [44]. Field and outbred populations were maintained on detached tomato leaves (28 days old, cv. Moneymaker) with the petioles submerged in water. All the females used in the experiment were obtained from cohorts of 100 mated females maintained on tomato cv. Moneymaker leaves. ed from https://royalsocietypublishing.org/ on 26 June 2023 2.1. The study system Tetranychus evansi is an arrhenotokous mite species, feeding mostly on Solanaceae plants [50]. In the laboratory, this species has a life cycle of approximately 13 days (egg to adult) at 25°C [51], with all life-stages residing on the host plant. We performed the experiments on tomato plants (Solanum lycopersicum L.) cv. Castlemart (WT) and the mutant defenseless-1 in the Castlemart genetic background (def-1) [48]. def-1 is unable to mount JA-related defences. All plants were grown in a climatic chamber (photoperiod of 16 : 8 h, 25:18°C, day:night, 50–60% relative humidity). Infestations with T. evansi occurred when plants were 28 days old, with four fully expanded leaves. All mite strains, prior to and for the experiments, were maintained in a temperature-controlled room (photoperiod of 16 : 8 h, 23.5 ± 2°C, 70% RH). climatic chamber (photoperiod of 16 : 8 h, 25:18°C, day:night, 50–60% relative humidity). Infestations with T. evansi occurred when plants were 28 days old, with four fully expanded leaves. All mite strains, prior to and for the experiments, were maintained in a temperature-controlled room (photoperiod of 16 : 8 h, 23.5 ± 2°C, 70% RH). 3 2.4. Benchmark mite strains For all our experiments, T. urticae Santpoort-2 (‘KMB’ in [35]) and T. evansi Baker & Pritchard Viçosa-1 [43] were used as benchmark controls for the induction and suppression of plant defences, respectively. These lines are referred to as the ‘inducer’ and ‘suppressor’ benchmarks as they have been shown in previous experiments to induce and suppress JA defences in tomato, respectively (e.g. [12,13,43]). The T. urticae strain was maintained on detached bean leaves (10 days old, cv. Speedy), and the T. evansi Viçosa-1 population on detached tomato leaves (28 days old, cv. Castlemart). Note that it was not possible to use a strain of T. evansi as a control for immune induction as this trait has not been found in this species. 2.3. Tetranychus evansi inbred lines Downloaded from https://royalsocietypublishing.org/ on 2 The generation of the 59 inbred mite lines, each originating from the outbred population, is described in detail in [44]. Brother–sister mating was imposed for each line for 15 generations resulting in 59 lines giving an expected level of 93.6% homozygosity [44]. All lines were maintained on detached tomato leaves (28 days old, cv. Moneymaker) placed on water-saturated cotton wool in 10 cm diameter Petri dishes. All the females used in the experiment were obtained from cohorts of 60 mated females maintained on tomato cv. Moneymaker leaves. Downloaded from https://royalsocietypublis royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 4 We measured the expression of salivary effector 84 (Te84 and Tu84 in [14]) in the different spider mite populations and of genes implicated in JA defences (Proteinase Inhibitor IIc (WIPI-IIc) and Proteinase Inhibitor IIf (WIPI-IIf) [13]) in WT plants infested with mites. The mite Ribosomal protein 49 (RP49) and the tomato Actin were used as housekeeping reference genes for spider mites and tomato plants, respectively (see electronic supplementary material, table S1, for primer sequences). For this, we used the leaflets from WT plants from the performance assay described above. In parallel, we also infested plants with the T. urticae inducer benchmark strain and the T. evansi immune suppressor benchmark strain in the same way as described above to have a quantitative reference for induction and suppression [13]. The part of the leaflet with glue and lanolin was discarded and the rest of the leaflet (plant and mite material) was collected, flash-frozen in liquid nitrogen and stored at −80°C for RNA extraction. For each mite population (field and outbred), we included 5–11 replicates. Uninfested plants (i.e. without mites) were used as a control. os R. Soc. Open Sci. 10: 230525 oaded from https://royalsocietypublishing.org/ on 26 June 2023 Total RNA from the sampled WT leaves was isolated using a protocol adapted from Verwoerd et al. [53]. Our protocol differs from it in that: (i) we used phenol at room temperature and (ii) the 5 min sample incubation step was completed at room temperature. Next, 2 µg of RNA was DNAse-treated with Ambion Turbo DNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was synthesized with RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific). Next, 1 µl of 10× diluted cDNA was used as a template for a 20 µl quantitative reserve-transcriptase polymerase chain reaction (RT-qPCR) using the SsoFast EvaGreenSupermix (Bio-Rad, Hercules, CA, USA) and the CFX96 Real-Time system (Bio-Rad). Gene expression was normalized using the ΔCt method [13] and, for graphical representation, scaled to the value with the lowest normalized average expression per gene. 2.5.3. Impact of infestation with the field and outbred populations on Tetranychus urticae Santpoort-2 and host immune responses Immune interference can be investigated by comparing the fecundity of a mite strain negatively affected by JA defences (T. urticae inducer Santpoort-2) on plants previously exposed to spider mites that suppress defences, with their fecundity on clean plants (i.e. not having had their defences suppressed) [35]. We used this method to assess the immune interference of the four field populations and the outbred population. We first infested WT plants with females from the field or outbred population for four days and then removed adults and eggs. Next, cleaned leaflets were re-infested with three T. urticae inducer mated females (15 ± 1 days old). Female survival and the number of eggs of the T. urticae inducer strain were recorded 48 h later (6 days after primary infestation) and mean fecundity per surviving female calculated as described above. We also assessed whether immune interference of the field and outbred populations persists following secondary infestation by measuring gene expression on day 6 using the same assay as described above. There were six replicates (n = 6) for each pre-infestation treatment divided across seven experimental blocks. Plants pre-infested with the T. urticae inducer and T. evansi suppressor strains were used as benchmarks for induction and suppression. Uninfested plants were used as a control for the basal level of plant defences. Note that although this was set up in parallel to the experiment measuring gene expression 4 dpi (days post infestation) described above, two additional replicates were performed. 5.1. Fecundity of Tetranychus evansi field and outbred populations on WT and def-1 tomato plants First, we assessed fecundity in the four field populations and the outbred population in the presence (WT) or absence of functional JA-mediated defences (def-1). To this end, 25 mated females (15 ± 1 days old) from each of the T. evansi populations were placed on one non-terminal leaflet of one fully expanded leaf of WT or def-1 tomato plants (day 0). Mite dispersal was prevented by isolating the adaxial surface of this leaflet with a 1 : 1 mix of entomological glue (Tanglefoot, The Scotts Company LLC, OH, USA), and lanolin (Sigma-Aldrich, St Louis, MO, USA), distributed around the adaxial edge of the leaflet. For each population and replicate, individual plants were used. The number of surviving females and their eggs were assessed four days after infestation. With these two measures, we calculated fecundity per female assuming linear mortality [52] by using [total eggs]/[(alive females + total females)/2] and using these numbers as the average per female. This equation accounts for differential female mortality, which is measured at the end of the assay and thus enables a more accurate representation of per capita fecundity. For each mite population, we included 8–11 replicates (i.e. one leaflet per plant). Assays for WT and def-1 plants were performed separately, and done in three or four temporal experimental blocks, respectively. 2.5.2. Expression of salivary effectors and tomato defences after infestation with the field and outbred populations 2.5.2. Expression of salivary effectors and tomato defences after infestation with the field and outbred populations 4 2.7. Statistical analysis All statistical analyses were performed with the software R (version 4.2.2) [54]. All models for gene expression were repeated including or excluding the benchmark controls for immune induction (T. urticae inducer), immune suppression (T. evansi suppressor) and uninfested plants. This was mostly to ensure that induction of immune defences occurred as in previous experiments [13,46], but also enabled us to test for differences among T. evansi populations or inbred lines, i.e. in models excluding benchmark controls. We fitted two independent generalized linear mixed models (GLMMs) (i.e. one for each host plant type) with a normal error structure (lmer, lme4 package [55]) to investigate whether fecundity (4 dpi) varied among the field and outbred populations on WT and def-1. The models included population (T. evansi Outbred, VIT, 6M1, QG and PBS) as a fixed explanatory variable. To analyse the transcript accumulation of effector 84, we fitted a GLMM with a gamma distribution and a log link function (glmmTMB package [56]). The model included population (T. urticae inducer, T. evansi suppressor, Outbred, VIT, 6M1, QG and PBS) as a fixed explanatory variable. To investigate whether transcript accumulation changed in tomato plants infested with field or outbred populations (4 dpi) and if the levels changed when subsequently infested with the T. urticae inducer population (2 days after re-infestation), GLMMs with a gamma distribution and a log link function (glmmTMB package [56]) were fitted for each gene separately (WIPI-IIc, WIPI-IIf). These models included population, time of gene expression (4 dpi or 2 days after re-infestation) and their interaction as fixed explanatory variables. glmmTMB models were used instead of glmer to improve model convergence. To evaluate if the fecundity of T. urticae inducer changed following previous infestation with the T. evansi field and outbred populations, we fitted a GLMM assuming a Gamma distribution and a log-link function (lme4 package [55]) since normality was not met (Shapiro–Wilk test: p = 0.008), and variances were not homogeneous. In these models, the pre-infestation population (T. evansi Outbred, VIT, 6M1, QG, PBS, and the inducer and manipulator benchmark populations) was included as a fixed explanatory variable. A GLMM assuming a Gamma distribution and a log-link function (lme4 package [55]) was used to investigate whether there was variation in fecundity of the inbred lines on the different host plant types since normality was not met, but here variances were homogeneous. Inbred line, host plant (i.e. 2.6.2. Expression of tomato defences in response to infestation by Tetranychus evansi inbred lines ublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 The expression of marker genes implicated in JA defences (WIPI-IIc, WIPI-IIf) was measured to assess variation in the defensive response of WT plants for 19 of the inbred lines (n = 5–7). The 19 inbred lines that were chosen present the range of fecundity levels observed across the WT and def-1 plants (electronic supplementary material, figure S1). Induction of defences was also measured for a subset of the benchmark control populations (i.e. inducer and suppressor populations, n = 4) and uninfested plants (n = 4). os R. Soc. Open Sci. 10: 230525 Genetic variation in Tetranychus evansi inbred lines for fecundity on WT and def-1 tomato plants We assessed variation in fecundity between lines on the different plant types: in the presence (WT) and absence (def-1) of functioning JA immune defences following a similar experimental set-up described for the populations, with some modifications. These were: (i) 12 mated females (13 ± 1 days old) were placed on each leaflet, (ii) leaves were detached from plants with the petiole in water, and (iii) infestations were only for 2 days. Mean fecundity per surviving female was calculated as described above. Owing to a large number of inbred lines, we randomly tested subsets of inbred lines (incomplete block design). In total, we assessed mite performance across thirty-five temporal blocks over a year. Each block included twelve inbred lines placed on both WT and immunocompromised plants (WT: n = 4–7; def-1: n = 4–7). In each block, there were also two to three replicates of each benchmark control population, i.e. T. urticae inducer (WT: n = 75; def-1: n = 72) and T. evansi suppressor (WT: n = 76; def-1: n = 67) populations also on both plant types, in addition to clean plants (WT: n = 27; def-1: n = 25). 5 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 societypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 We investigated whether the fecundity of the inbred lines on WT plants and on def-1 plants correlated with normalized gene expression for the defence genes WIPI-IIc and WIPI-IIf using Pearson correlations [58]. Normalized gene expression was log-transformed to improve the normality of data. For all analyses, block was included in models as a random variable. For each model, when significant differences were found, multiple comparisons were performed using estimated marginal means (emmeans, emmeans package [59]) and the p-values corrected using the false discovery rate method (α = 0.05) [60]. rsos R. Soc. Open Sci. 10: 230525 ed from https://royalsocietypublishing.org/ on 26 June 2023 3.1. Fecundity, expression of salivary effector 84 and plant defences after infestation with Tetranychus evansi field and outbred populations There was variation in levels of fecundity among field and outbred populations on WT ( population: x2 4 ¼ 12:204, p = 0.016; figure 1a) and on def-1 ( population: x2 4 ¼ 15:928, p = 0.003; figure 1b) plants. On WT, PBS had the highest fecundity, while 6M1 had the lowest. On def-1, VIT had the highest fecundity and differed significantly from PBS and QG. Fecundity of the outbred population did not differ from any of the field populations on either plant type. Downloaded from https://royalsocietypublis There was a significant effect of population on the expression of salivary effector 84 (x2 6 ¼ 116:290, p < 0.001; figure 1c). This was mainly explained by low levels of expression of this effector for the T. urticae inducer benchmark control, with all T. evansi populations having similar levels of expression. There was no difference in transcript accumulation levels among T. evansi populations. We observed differences between populations for WIPI-IIc (x2 7 ¼ 64:097, p < 0.001; figure 1d) and WIPI-IIf (x2 7 ¼ 53:344, p < 0.001; figure 1e), in analyses including the benchmarks, with both JA marker genes being induced by T. urticae inducer. Expression levels of JA marker genes in plants infested with field or outbred populations were similar to defence levels found in plants infested with the T. evansi suppressor control, and sometimes to levels observed for uninfested plants (i.e. all populations for WIPI-IIc and VIT and 6M1 for WIPI-IIf). 3.2. Impact of infestation with the field and outbred populations on a JA-susceptible Tetranychus urticae population Fecundity (2 days after re-infestation) of the JA-susceptible T. urticae inducer population was not affected by pre-infestation with any of the field, outbred populations, or benchmark controls ( population: x2 7 ¼ 7:7907, p = 0.3514; figure 2a). However, the expression of JA marker genes changed following secondary infestation depending on the pre-infestation treatment ( population × time of gene expression analysis; WIPI-IIc: x2 7 ¼ 24:516, p = 0.001; WIPI-IIf: x2 7 ¼ 56:020, p < 0.001; figure 2b,c). The expression of these genes only increased in previously uninfested plants and plants pre-infested with the inducer benchmark control population. By contrast, plants pre-infested with each of the T. evansi populations had no increase in expression, with levels remaining the same or being lower than at 4 dpi. 2.7. Statistical analysis WT or def- 1) and their interaction were included in the model as fixed explanatory variables. A similar model was repeated for the 19 inbred lines selected to investigate variation in the induction of plant defences. We calculated broad-sense heritability (H2) [57] for the fecundity of the inbred lines on WT and def-1 by performing separate generalized mixed linear models for each host type with a Gamma distribution and a log link function (lme4 package [55]) with inbred line included in the model as a random explanatory variable. From the summary of the models, we extracted the variance for inbred line, block and residual variance of the model and calculated H2 as follows: varðinbred lineÞ= ðvarðinbred lineÞ þ varðblockÞ þ varðresidualsÞÞ. To determine the significance of H2, we compared models, using ANOVA, including and excluding the inbred line random factor. To analyse if there is variation in the induction of plant defences after infestation with the inbred lines, GLMMs with a gamma distribution and a log link function (glmmTMB package [56]) were fitted for each gene separately (WIPI-IIc or WIPI-IIf). Inbred line was included in the model as a fixed explanatory variable. glmmTMB models were used instead of glmer to improve the convergence of the models. To test how JA defences influence T. evansi fecundity, a genetic correlation between fecundity on WT and def-1 plants was performed using a generalized mixed linear model assuming a Gamma distribution and a log link function (lme4 package [55]) with host plant as a fixed variable and inbred line nested within plant (0 + host plant \| inbred line). From the summary of the model, we extracted the correlation obtained in the random effects section. To determine the significance of the genetic correlation, we compared models with and without the correlation using an ANOVA. 6 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences 10: 230525 7 (c) b a a a a a a 200 100 0 I S O VIT infesting populations normalized expression effector 84 6M1 QG PBS y yp g g j 7 30 (a) (b) (d) (e) (c) 25 20 fecundity on WT 15 10 5 0 I S O VIT ab bc bc bc bc bc d b bc bc cd bcd bcd a a b c ab ab ab ab b a b b b a a a a a a a infesting populations 6M1 QG PBS 12 600 26 000 6000 4000 2000 0 12 000 6000 normalized expression WIPI-IIc normalized expression WIPI-IIf 4000 2000 0 C I S infesting populations O VIT 6M1 QG PBS C I S infesting populations O VIT 6M1 QG PBS 30 200 100 0 I S O VIT infesting populations normalized expression effector 84 6M1 QG PBS 25 20 fecundity on def-1 15 10 5 0 I S O VIT infesting populations 6M1 QG PBS royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 7 (b) ab ab a b b 30 25 20 fecundity on def-1 15 10 5 0 I S O VIT infesting populations 6M1 QG PBS 30 (a) 25 20 fecundity on WT 15 10 5 0 I S O VIT ab ab ab b a infesting populations 6M1 QG PBS 7 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 7 infesting populations (d) normalized expression WIPI-IIc infesting populations infesting populations Figure 1. Phenotypic characterization of the field populations and the outbred population. Mean fecundity (± s.e.) of each population on (a) WT and (b) def-1 plants at 4 dpi, and the mean normalized gene expression (± s.e.) of (c) salivary effector 84, and JA-related genes (d) WIPI-IIc and (e) WIPI-IIf at 4 dpi. The black circle denotes the mean and each grey circle a replicate. Different lowercase letters indicate statistical differences among populations according to multicomparison analysis performed using estimated marginal means. The benchmark control treatments are denoted as ‘I’ for the T. urticae inducer control, ‘S’ for the T. evansi suppressor control, and ‘C’ for uninfested plants. 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences (a) 12 8 4 0 C I S O VIT 6M1 QG PBS infesting populations fecundity of susceptible population 2000 1500 1000 500 0 C a a bc cd cd d bcd b I S O VIT 6M1 QG PBS infesting populations normalized expression WIPI-IIc 4000 3000 2000 1000 0 a a b b b b b b C I S O VIT 6M1 QG PBS infesting populations normalized expression WIPI-IIf (b) (c) Figure 2. Effect of the prior infestation with T. evansi populations on a JA-susceptible and inducer T. urticae population. Mean (a) fecundity (± s.e.) 2 days after re-infestation and mean normalized gene expression (± s.e.) of JA-related genes (b) WIPI-IIc and (c) WIPI-IIf 2 days after re-infestation. Each grey circle represents a replicate and the black circle the mean. Different lowercase letters indicate statistical differences between populations according to multicomparison analysis performed using estimated marginal means. The benchmark control treatments are denoted as ‘I’ for the T. urticae inducer mite population, ‘S’ for the T. evansi suppressor population control, and ‘C’ for uninfested plants. (a) 12 8 4 0 C I S O VIT 6M1 QG PBS infesting populations fecundity of susceptible population ( 4000 3000 2000 1000 0 a a b b b b b b C I S O VIT 6M1 QG PBS infesting populations normalized expression WIPI-IIf (c) infesting populations infesting populations infesting populations ffect of the prior infestation with T. evansi populations on a JA-susceptible and inducer T. urticae population. Mean (a) Figure 2. Effect of the prior infestation with T. evansi populations on a JA-susceptible and inducer T. urticae population. Mean (a) fecundity (± s.e.) 2 days after re-infestation and mean normalized gene expression (± s.e.) of JA-related genes (b) WIPI-IIc and (c) WIPI-IIf 2 days after re-infestation. Each grey circle represents a replicate and the black circle the mean. Different lowercase letters indicate statistical differences between populations according to multicomparison analysis performed using estimated marginal means The benchmark control treatments are denoted as ‘I’ for the T urticae inducer mite population ‘S’ for the T evansi Figure 2. Effect of the prior infestation with T. evansi populations on a JA-susceptible and inducer T. urticae po Figure 2. Effect of the prior infestation with T. evansi populations on a JA-susceptible and inducer T. urticae population. 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences There were significant differences among inbred lines for fecundity (inbred line: x2 58 ¼ 156:611, p < 0.001; electronic supplementary material, figure S1a) with overall higher fecundity on the immunocompromised plants than on WT (host plants: x2 1 ¼ 5:371, p = 0.021; electronic supplementary material, figure S1a). The interaction between inbred line and host plant was not significant (inbred line × host plants: x2 58 ¼ 42:549, p = 0.936). The genetic component for this observed phenotypic variation in fecundity was similar on both WT (H2= 0.094, x2 1 ¼ 75:435, p < 0.001) and def-1 30 (a) (b) (d) (e) (c) 25 20 fecundity on WT 15 10 5 0 I S O VIT ab bc bc bc bc bc d b bc bc cd bcd bcd a a b c ab ab ab ab b a b b b a a a a a a a infesting populations 6M1 QG PBS 12 600 26 000 6000 4000 2000 0 12 000 6000 normalized expression WIPI-IIc normalized expression WIPI-IIf 4000 2000 0 C I S infesting populations O VIT 6M1 QG PBS C I S infesting populations O VIT 6M1 QG PBS 30 200 100 0 I S O VIT infesting populations normalized expression effector 84 6M1 QG PBS 25 20 fecundity on def-1 15 10 5 0 I S O VIT infesting populations 6M1 QG PBS Figure 1. Phenotypic characterization of the field populations and the outbred population. Mean fecundity (± s.e.) of each population on (a) WT and (b) def-1 plants at 4 dpi, and the mean normalized gene expression (± s.e.) of (c) salivary effector 84, and JA-related genes (d) WIPI-IIc and (e) WIPI-IIf at 4 dpi. The black circle denotes the mean and each grey circle a replicate. Different lowercase letters indicate statistical differences among populations according to multicomparison analysis performed using estimated marginal means. The benchmark control treatments are denoted as ‘I’ for the T. urticae inducer control, ‘S’ for the T. evansi suppressor control, and ‘C’ for uninfested plants. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences Mean (a) fecundity (± s.e.) 2 days after re-infestation and mean normalized gene expression (± s.e.) of JA-related genes (b) WIPI-IIc and (c) WIPI-IIf 2 days after re-infestation. Each grey circle represents a replicate and the black circle the mean. Different lowercase letters indicate statistical differences between populations according to multicomparison analysis performed using estimated marginal means. The benchmark control treatments are denoted as ‘I’ for the T. urticae inducer mite population, ‘S’ for the T. evansi suppressor population control, and ‘C’ for uninfested plants. (H2= 0.1217, x2 1 ¼ 136:490, p < 0.001) plants. There was a strong positive genetic correlation for fecundity in the presence and absence of JA defences across inbred lines (rg = 0.72, p < 0.001; figure 3a). g On the subset of 19 inbred lines selected to study the expression of JA plant defences (highlighted in black in electronic supplementary material, figure S1a), fecundity varied among inbred lines but did not (a) (b) (c) 15 10 5 0 800 normalized expression WIPI-IIc 400 200 0 600 normalized expression WIPI-IIf 400 200 0 C S 11 28 60 59 37 56 inbred lines 12 42 48 32 O I 61 16 15 54 1 8 30 39 38 C I 12 39 32 16 54 15 inbred lines 59 S O 11 61 28 56 60 37 48 8 38 0 1000 2000 3000 4000 30 42 1 0 5 10 I S rg = 0.72 p < 0.001 fecundity on def-1 15 fecundity on WT Figure 3. Phenotypic variation of the 59 inbred lines. (a) Genetic correlation between mean fecundity (± s.e.) on WT and def-1. Black points correspond to the 19 inbred lines selected to study the induction of defences. The black line represents the correlation between traits, excluding benchmark controls, and the grey shadow the 95% confidence intervals. Mean normalized gene expression (± s.e.) of JA-related genes (b) WIPI-IIc and (c) WIPI-IIf on WT plants. The 19 inbred lines selected to measure the response of the JA marker genes after infestation are highlighted in black in (a). Panel (c) has two graphs, to account for the high variation in normalized gene expression of WIPI-IIf among lines. Each grey circle represents one replicate. The benchmark control treatments are denoted as ‘I’ for the benchmark inducer population, ‘S’ for the benchmark suppressor population, ‘O’ for the outbred population, and ‘C’ for uninfested plants. 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 8 (a) 15 10 5 0 I S rg = 0.72 p < 0.001 fecundity on WT 8 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 8 I (c) 600 normalized expression WIPI-IIf 400 200 0 C I 12 39 32 16 54 15 inbred lines 59 S O 11 61 28 56 60 37 48 8 38 0 1000 2000 3000 4000 30 42 1 5 10 fecundity on def-1 15 pen Sci. 10: 230525 5 15 5 10 fecundity on def-1 10 0 (b) 800 normalized expression WIPI-IIc 400 200 0 C S 11 28 60 59 37 56 inbred lines 12 42 48 32 O I 61 16 15 54 1 8 30 39 38 0 5 f normalized inbred lines Phenotypic variation of the 59 inbred lines. (a) Genetic correlation between mean fecundity (± s.e.) on WT and def-1. Figure 3. Phenotypic variation of the 59 inbred lines. (a) Genetic correlation between mean fecundity (± s.e.) on WT and def-1. Black points correspond to the 19 inbred lines selected to study the induction of defences. The black line represents the correlation between traits, excluding benchmark controls, and the grey shadow the 95% confidence intervals. Mean normalized gene expression (± s.e.) of JA-related genes (b) WIPI-IIc and (c) WIPI-IIf on WT plants. The 19 inbred lines selected to measure the response of the JA marker genes after infestation are highlighted in black in (a). Panel (c) has two graphs, to account for the high variation in normalized gene expression of WIPI-IIf among lines. Each grey circle represents one replicate. The benchmark control treatments are denoted as ‘I’ for the benchmark inducer population, ‘S’ for the benchmark suppressor population, ‘O’ for the outbred population, and ‘C’ for uninfested plants. differ on WT or def-1 plants (inbred line: x2 18 ¼ 45:952, p < 0.001; host plants: x2 1 ¼ 3:232, p = 0.072; inbred line × host plants: x2 18 ¼ 18:235, p = 0.440). There were differences in levels of expression for WIPI-IIc (x2 22 ¼ 78:470, p < 0.001; figure 3b) and WIPI-IIf (WIPI-IIf: x2 22 ¼ 98:584, p < 0.001; figure 3c), with the T. urticae inducer inducing the highest and the T. evansi suppressor showing lower levels of induced defences. Post hoc comparisons (electronic supplementary material, table S2) revealed that most of the T. 1. The fecundity of Tetranychus evansi does not correlate with levels of immune interfe JA defences reduced the fecundity of the inbred lines of T. evansi as, overall, fecundity was higher on def-1 than on WT plants. Indeed, plants with a functioning immune response are a more hostile environment for several herbivores, as has been shown for mites and caterpillars [13,48,49,61]. Similarly, plants pre- infested by T. evansi have generally been found to increase the fecundity of both T. evansi and T. urticae [12,13,36,39] although this effect can depend on the timing of the co-infestation [62]. Taken together with our results, it seems that immunocompromised plants, either artificially or via herbivore immune interference mechanisms, confer obvious benefits to arthropod herbivores feeding on them, including T. evansi (as shown here with lines laying, on average, 1.07-fold more eggs per female on immunocompromised plants). However, despite genetic variation for the propensity of the T. evansi inbred lines to induce immune responses, we did not find a correlation between the degree of immune interference and fecundity. Fecundity is often used as a proxy for performance, is heritable, and can be influenced by the environment [63–65]. In a sister species, T. urticae, fecundity has been observed to have variable levels of narrow-sense heritability (h2= 0.72 [63]; h2= 0.11 [64]; h2= 0.05 [65]). Although these studies revealed genetic and environmental correlations among fecundity and other life-history traits (e.g. development time and juvenile mortality), fecundity was found to be independent of longevity [66] and web production [63]. Similarly, our results suggest that fecundity is independent of the magnitude of JA defences, as heritability was similar on WT and immunocompromised plants and fecundity on WT and def-1 across lines was positively correlated. It is possible that absolute expression of two marker genes at an arbitrary time point does not adequately reflect the complexity and dynamics of the relevant downstream induced-defence response of tomato plants. Another recent study found that T. urticae mites that suppress defensive bubble formation in honeysuckle did not have higher fecundity than inducer mites [67]. In addition, defence induction may affect other traits that were not measured here. It may be that variation in the ability of T. evansi to interfere with immune defences is correlated with other life-history traits (longevity, lifetime reproductive performance, juvenile survival, etc.). As such, how other life-history traits covary with immune induction, in longer-term experiments, should be considered in future. It is generally found that T. 3.4. Influence of JA defences on Tetranychus evansi fecundity There was no phenotypic correlation between the fecundity of the 19 inbred lines on WT plants and the normalized gene expression for either of the genes implicated in JA defences after mite infestation (WIPI- IIc: r = −0.079, t107= −0.821, p = 0.414; WIPI-IIf: r = 0.077, t107 = 0.811, p = 0.419; electronic supplementary material, figure S2a,b). The same was observed for the correlation between the fecundity of the 19 inbred lines on def-1 and the normalized gene expression for either of the genes (WIPI-IIc: r = −0.052, t17 = −0.214, p = 0.833; WIPI-IIf: r = −0.093, t17 = −0.386, p = 0.704; electronic supplementary material, figure S3a,b). royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences Influence of JA defences on Tetranychus evansi fecundity 4. Discussion We investigated intraspecific variation, in different field populations and inbred lines derived from these populations, for the ability to interfere with host immune responses and how this relates to fecundity. We found no differences among four field populations in the gene expression of effector 84 and the immune interference of JA defences, despite differences in fecundity among populations on WT and def-1 plants. We did though find genetic variation among the inbred lines for oviposition on WT plants and variation in induction of gene expression for both marker genes implicated in the JA pathway (WIPI-IIc and WIPI- IIf). We also found that overall, the different lines had higher fecundity on immunocompromised host plants, indicating that the active JA pathway in WT plants negatively affects T. evansi mites. However, higher levels of fecundity in the inbred lines did not correlate with lower levels of immune defences (i.e. higher levels of immune interference). os R. Soc. Open Sci. 10: 230525 ed from https://royalsocietypublishing.org/ on 26 June 2023 3.3. Genetic variation in Tetranychus evansi inbred lines for fecundity and expression of plant defences evansi inbred lines (17/19) had similar levels of WIPI-IIc expression to the suppressor control, with 7/19 lines also not differing from uninfested plants. For WIPI-IIf, levels of induction for most inbred lines did not differ from either of the benchmark controls (15/19), with 18/19 T. evansi inbred lines not differing from the T. urticae inducer benchmark. However, only three inbred lines (lines 1, 8 and 42) had higher levels of expression than the T. evansi suppressor benchmark. In analyses excluding the benchmark controls, there were also differences among lines for the expression of each gene (WIPI-IIc: x2 18 ¼ 32:119, p = 0.021; WIPI-IIf: x2 18 ¼ 54:515, p < 0.001). 3.4. Influence of JA defences on Tetranychus evansi fecundity There was no phenotypic correlation between the fecundity of the 19 inbred lines on WT plants and the normalized gene expression for either of the genes implicated in JA defences after mite infestation (WIPI- IIc: r = −0.079, t107= −0.821, p = 0.414; WIPI-IIf: r = 0.077, t107 = 0.811, p = 0.419; electronic supplementary material, figure S2a,b). The same was observed for the correlation between the fecundity of the 19 inbred lines on def-1 and the normalized gene expression for either of the genes (WIPI-IIc: r = −0.052, t17 = −0.214, p = 0.833; WIPI-IIf: r = −0.093, t17 = −0.386, p = 0.704; electronic supplementary material, figure S3a,b). 4. Discussion We investigated intraspecific variation, in different field populations and inbred lines derived from these populations, for the ability to interfere with host immune responses and how this relates to fecundity. We found no differences among four field populations in the gene expression of effector 84 and the immune interference of JA defences, despite differences in fecundity among populations on WT and def-1 plants. We did though find genetic variation among the inbred lines for oviposition on WT plants and variation in induction of gene expression for both marker genes implicated in the JA pathway (WIPI-IIc and WIPI- IIf). We also found that overall, the different lines had higher fecundity on immunocompromised host plants, indicating that the active JA pathway in WT plants negatively affects T. evansi mites. However, higher levels of fecundity in the inbred lines did not correlate with lower levels of immune defences (i.e. higher levels of immune interference). royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 9 9 3.4. 4.3. Immune interference in Tetranychus evansi populations and consequences for heterospecifics 4.3. Immune interference in Tetranychus evansi populations and consequences for heterospecifics Not only the magnitude of defence activation but also the expression levels of effector 84 were similar across the T. evansi field populations. This was despite variation in levels of fecundity among the field populations on both WT and def-1 plants. effector 84 is a salivary effector protein that is involved in the suppression of the JA and SA pathways in plants [14,37]. To date, no study has addressed variability in the expression of this effector within the Tetranychus genus, as most studies addressing effector genes mainly focus on the mechanism and mode of action of such molecules (reviewed in [21]). The absence of significant variation in the expression of this effector may be explained by the fact that all these populations belong to the same haplotype (ITS lineage I [44]). It is possible that another haplotype (ITS lineage II) may suppress plant defences more strongly [46]. However, Knegt et al. [46] also observed little variability in traits related to immune interference across populations. Nevertheless, it would be interesting to assay these populations for variation in the expression of effector 84 and see how it relates to the expression of plant defence genes and T. evansi life-history traits. More studies investigating the dynamics in effector gene expression and linking this to the expression of immune genes in host plants could improve our understanding of the mechanisms of coevolution between plants and their parasites. We did find that infestation with the T. evansi field and outbred populations prevented plants from mounting an effective immune response against the JA defence susceptible T. urticae [13]. In plants pre- infested with mites from these T. evansi populations, levels of induction for all defence genes were maintained at the same or lower levels after subsequent infestation with the T. urticae inducer population. This lasting effect of defence interference could result from a latency period required for the host plant to re-establish its normal defensive status in response to a secondary infection. Moreover, in several co-infection studies when infections are sequential, host-mediated facilitation by a suppressor parasite has been shown to increase fitness-related traits in a second parasite [6,12,13,36,39]. This facilitation may in turn promote competition with herbivores co-habiting the same host (e.g. spider mites [12,43,72]; beetles [73]). 4.3. Immune interference in Tetranychus evansi populations and consequences for heterospecifics Investigating how the lasting effect of defence interference persists through time and its benefits for con- and heterospecific individuals would increase our understanding of how species may evolve in communities. In the experiments presented here, however, despite defences being maintained at low levels, we did not observe facilitation of the JA-susceptible T. urticae population in terms of fecundity. Previous studies showed that the oviposition of this heterospecific competitor of T. evansi is higher on suppressed and uninfested plants, compared to plants with induced defences [12,13,35,39]. As previously reported, the outcome of facilitation experiments may be variable and strongly depend on the timing of the infestation and the number of mites used [24,36,37,74]. 2. Genetic variation for immune interference among lines, but not among population There were differences in immune interference among the different inbred lines, but the four field populations did not differ in the extent to which they upregulated tomato defences. It is most probable that this is because the different populations contain similar levels of genetic variation that was captured across the different lines. This is supported by the observation that the variation in defence gene induction by the outbred population overlaps with variation across inbred lines. Thus, in our lines, we probably fixed the genetic variation present within the field populations. s R. Soc. Open Sci. 10: 230525 ed from https://royalsocietypublishing.org/ on 26 June 2023 1. The fecundity of Tetranychus evansi does not correlate with levels of immune interfe evansi can suppress tomato immune responses, with corresponding beneficial effects on fecundity, juvenile survival and development rate when compared to T. urticae [12,13,36,44,68]. However, previous studies that identified a link between defence induction and herbivore performance included comparisons among either T. urticae lines [35] or Tetranychus species [13]. It is generally found that T. evansi can suppress tomato immune responses, with corresponding beneficial effects on fecundity, juvenile survival and development rate when compared to T. urticae [12,13,36,44,68]. However, previous studies that identified a link between defence induction and herbivore performance included comparisons among either T. urticae lines [35] or Tetranychus species [13]. It is possible that some of the inbred lines with higher fecundity may be more resistant to tomato defences than other lines. Plant defence resistance has been demonstrated in T. urticae whereby some [12,13,36,44,68]. However, previous studies that identified a link between defence induction and herbivore performance included comparisons among either T. urticae lines [35] or Tetranychus species [13]. It is possible that some of the inbred lines with higher fecundity may be more resistant to tomato defences than other lines. Plant defence resistance has been demonstrated in T. urticae whereby some It is possible that some of the inbred lines with higher fecundity may be more resistant to tomato defences than other lines. Plant defence resistance has been demonstrated in T. urticae whereby some lines induce host immune genes but maintain high levels of fecundity [35,69]. If this is the case, resistance to plant defences may be via detoxification mechanisms (e.g. metabolite modification, degradations and/or secretion) [66,69–71]. One study found T. urticae to have increased expression of genes involved in detoxification following only five generations on tomato (a novel stressful environment after bean host plants), with correlated beneficial effects in other toxic environments such as the presence of pesticides [69]. 10 4.5. Immune interference as a public good Immune interference could be seen as a public good, if it benefits other parasites sharing the same host [6,12,13,36,39]. This could lead to the emergence of cheaters/exploiters of the same or different species, which do not suppress defences but have higher fitness when it occurs, since they pay no energetic costs associated with immune interference. A theoretical study revealed that it is possible for two strains with extreme immune interference strategies (i.e. zero and maximum) to coexist in a population [26]. This could produce antagonistic coevolutionary dynamics between parasite strains that can interfere with their host immune system, that strive to monopolize the suppressed environment, and cheaters that aim to reap the benefits [83]. Cheaters that benefit from, but do not contribute to, immune interference have been identified in Pseudomonas aeruginosa and Yersinia pestis [84]. The emergence of cheaters exploiting public goods has also been identified in vitro in the bacterium Pseudomonas flourescens [85,86]. Selection for suppression and cheating would be possible in T. evansi considering the potential for genetic variation of this trait. However, support for this hypothesis in this system would require identifying the (benefits) and costs of immune interference either energetically (i.e. via the production of interfering molecules, such as salivary effectors), or the presence of competitors exploiting and benefitting from the resource more than suppressor lines. It would be interesting to establish the relationship or co-occurrence between the fecundity of putative cheaters and their proximity to suppressors in natural populations. The fact that suppression enables overcoming host immune responses and can be beneficial for competitor parasites of the same or different species, means this trait may coevolve in response to both the host and other parasites [7,25]. These may be important factors responsible for the maintenance of genetic variation in this trait. For instance, selection for immune interference may depend on the host environment, and the frequency at which parasites encounter hosts upon which immune interference is effective, or in co-infections with other parasites that exploit the manipulated host environment. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 11 oaded from https://royalsocietypublishing.org/ on 26 June 2023 R. Soc. Open Sci. 10: 230525 Spider mites also show plasticity in other traits related to immune interference [37]. An ecological cost for parasites may be the presence of competitors in the within-host environment that benefit from, but do not contribute to, immune interference [24,26,82]. In the presence of competitors, T. evansi mites can increase web density [43], local levels of immune interference and their oviposition [37], showing plasticity in traits facilitating their monopolization of the immunosuppressed environment. This indicates that trait plasticity may help mitigate ecological costs associated with immune interference. 4.4. Plasticity of immune interference We found no evidence for metabolic costs of immune interference as lines showing higher levels of immune interference did not have lower levels of fecundity on def-1 plants (electronic supplementary material, figure S3). This type of trade-off has been investigated in other parasites, with varying results. Immune interference by a parasitoid was lost following selection on a diversity of hosts, possibly due to being ineffective, and/or costly to maintain on some host species [27]. Another study found metabolic detoxification to trade-off with increased population growth rate in the Sitobion avanae aphid [75]. By contrast, no trade-off was found between the growth rate of Depressaria pastinacella or Heliothis zea caterpillars and the production of detoxification enzymes [76,77]. Also, there was no apparent cost for Schistocephalus solidus manipulating the behaviour of its intermediate host (to increase predation by its definitive host [78]). Another study, with T. evansi, showed the maintenance of suppression after evolution on immunosuppressed (def-1) plants for 60 generations suggesting marginal costs associated with this trait in this species [79]. Low costs for the maintenance of immune interference may be explained through trait plasticity. Indeed, responses to induced plant defences are phenotypically plastic for some herbivores, only being expressed on plants when needed [49,70,80]. For T. evansi this seems to be the case, as the effector 84 was found to be highly plastic in certain conditions (e.g. the presence of heterospecific competitors [37]; light–dark cycles [42]; developmental stages [81]). royalsocietypublishing.org/journal/rsos R 1 11 References Lond. B 271, 1611–1615. (doi:10.1098/rspb. 2004.2762) Lond. B 271, 1611–1615. 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(doi:10.1111/j.1558-5646. 1949.tb00004.x) 26. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 12 The authors would like to thank Sara Magalhães’s team at the University of Lisbon, especially Cátia Eira and Lucie de Sousa, for the help in the creation and maintenance of the populations and lines used in this study and for fruitful discussions throughout the project. Also, we would like to thank Giacomo Zilio for helpful discussion. Finally, we would like to thank João Valeriano-Santos for his help in creating large functions as a way to prepare the datasets for analysis. This is ISEM contribution number ISEM 2023-095. s R. Soc. Open Sci. 10: 230525 Acknowledgements. The authors would like to thank Sara Magalhães’s team at the University of Lisbon, especially Cátia Eira and Lucie de Sousa, for the help in the creation and maintenance of the populations and lines used in this study and for fruitful discussions throughout the project. Also, we would like to thank Giacomo Zilio for helpful discussion. Finally, we would like to thank João Valeriano-Santos for his help in creating large functions as a way to prepare the datasets for analysis. This is ISEM contribution number ISEM 2023-095. d from https://royalsocietypublishing.org/ on 26 June 2023 Downloaded from https://royalsocietypublishing.org/ on 2 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230525 12 Data accessibility. Data are available in an open-access data repository: https://doi.org/10.5061/dryad.hmgqnk9mx [87]. The data are provided in electronic supplementary material [88]. The data are provided in electronic supplementary material [88]. Authors’ contributions. J.T.-P.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, visualization, writing—original draft, writing—review and editing; J.M.A.: supervision, writing— review and editing; S.C.: investigation; M.R.K.: funding acquisition, supervision, validation, writing—review and editing; S.M.: conceptualization, funding acquisition, resources, writing—review and editing; A.B.D.: funding acquisition, supervision, writing—original draft, writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. Conflict of interest declaration. We declare we have no competing interests. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. Conflict of interest declaration. We declare we have no competing interests. Funding. Funds were provided by an ERC consolidator grant (COMPCON, GA 725419) to S.M.; the Dutch Research Council (ALWOP.283) to M.R.K.; and FCT Doctoral grant (SFRH/BD/136416/2018) to J.T.-P. Acknowledgements. The authors would like to thank Sara Magalhães’s team at the University of Lisbon, especially Cátia Eira and Lucie de Sousa, for the help in the creation and maintenance of the populations and lines used in this study and for fruitful discussions throughout the project. Also, we would like to thank Giacomo Zilio for helpful discussion. Finally, we would like to thank João Valeriano-Santos for his help in creating large functions as a way to prepare the datasets for analysis. This is ISEM contribution number ISEM 2023-095. p g Funding. Funds were provided by an ERC consolidator grant (COMPCON, GA 725419) to S.M.; the Dutch Research Council (ALWOP.283) to M.R.K.; and FCT Doctoral grant (SFRH/BD/136416/2018) to J.T.-P. Acknowledgements. The authors would like to thank Sara Magalhães’s team at the University of Lisbon, especially Cátia Eira and Lucie de Sousa for the help in the creation and maintenance of the populations and lines used in this study p g Funding. Funds were provided by an ERC consolidator grant (COMPCON, GA 725419) to S.M.; the Dutch Research Council (ALWOP.283) to M.R.K.; and FCT Doctoral grant (SFRH/BD/136416/2018) to J.T.-P. ownloaded from https://royalsocietypublishing.org/ on 26 June 2023 Council (ALWOP.283) to M.R.K.; and FCT Doctoral grant (SFRH/BD/136416/2018) to J.T.-P. Acknowledgements. 5. Conclusion and perspectives Our results show genetic variation for fecundity within a T. evansi population, but this does not correlate with variation in levels of induction of immune defences. This might be because intraspecific variation for immune interference might be linked to other T. evansi life-history traits that, for example, are more targeted by host immune defences. 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https://openalex.org/W2606360535	https://repository.arizona.edu/bitstream/10150/626103/1/biophys-04-00284.pdf	English	null	Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony-forming cells	AIMS biophysics	2,017	cc-by	6,069	Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony-forming cells Item Type Article Authors Ahern, Megan A.; Black, Claudine P.; Seedorf, Gregory J.; Baker, Christopher D.; Shepherd, Douglas P. Citation Megan A. Ahern, Claudine P. Black, Gregory J. Seedorf, Christopher D. Baker, Douglas P. Shepherd. Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony- forming cells[J]. AIMS Biophysics, 2017, 4(2): 284-297. DOI 10.3934/biophy.2017.2.284 Publisher AMER INST MATHEMATICAL SCIENCES-AIMS Journal AIMS Biophysics Rights © 2017 Douglas P. Shepherd, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License. Download date 24/10/2024 04:04:47 Item License https://creativecommons.org/licenses/by/4.0/ Version Final published version Link to Item http://hdl.handle.net/10150/626103 Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony-forming cells Item Type Article Authors Ahern, Megan A.; Black, Claudine P.; Seedorf, Gregory J.; Baker, Christopher D.; Shepherd, Douglas P. Citation Megan A. Ahern, Claudine P. Black, Gregory J. Seedorf, Christopher D. Baker, Douglas P. Shepherd. Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony- forming cells[J]. AIMS Biophysics, 2017, 4(2): 284-297. DOI 10.3934/biophy.2017.2.284 Publisher AMER INST MATHEMATICAL SCIENCES-AIMS Journal AIMS Biophysics Rights © 2017 Douglas P. Shepherd, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License. Download date 24/10/2024 04:04:47 Item License https://creativecommons.org/licenses/by/4.0/ Version Final published version Link to Item http://hdl.handle.net/10150/626103 Megan A. Ahern 1,2,†, Claudine P. Black 2, Gregory J. Seedorf 2, Christopher D. Baker 2 and Douglas P. Shepherd 1,2,* 1 Department of Physics, University of Colorado Denver, Denver, CO 80212, USA 1 Department of Physics, University of Colorado Denver, Denver, CO 80212, USA 2 Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado Denver Anschutz Medical Campus, Aurora CO 80045, USA 2 Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado Denver Anschutz Medical Campus, Aurora CO 80045, USA † Current address: Department of Physiology, School of Medicine, University of Arizona, Tucson, USA † Current address: Department of Physiology, School of Medicine, University of Arizona, Tucson, USA AIMS Biophysics, 4(2): 284-297. DOI: 10.3934/biophy.2017.2.284 Received: 09 January 2017 Accepted: 10 April 2017 Published: 19 April 2017 http://www.aimspress.com/journal/biophysics Hyperoxia impairs pro-angiogenic RNA production in preterm endothelial colony-forming cells Megan A. Ahern 1,2,†, Claudine P. Black 2, Gregory J. Seedorf 2, Christopher D. Baker 2 and Douglas P. Shepherd 1,2,* AIMS Biophysics, 4(2): 284-297. DOI: 10.3934/biophy.2017.2.284 Received: 09 January 2017 Accepted: 10 April 2017 Published: 19 April 2017 1. Introduction Developmentally appropriate genetic signaling depends on internal feedback networks responding to external signals. Recent works have demonstrated that single-cell measurements are key to understanding how these networks respond to external factors because single-cell behavior is rarely normally distributed about a mean value [1,2]. This is partially due to RNA bursting, during which RNA are stochastically produced in the nucleus and after a series of steps arrive in the cytoplasm for translation [1,2]. This spatial separation provides information on the timing of events, even for populations of fixed cells. For example, Neuert et al. measured the single-cell RNA expression of thousands of budding yeasts in response to osmotic shock for multiple genes. Using this dense dataset, they created in silico models that predicted how the internal regulatory networks would respond to new external conditions or genetic perturbations to the network itself [3]. This insight would not be possible using standard ensemble methods because the variability in spatial distribution of RNA and copy number variation in RNA would be lost due to population averaging [1–4]. This set of techniques provides a powerful tool to image fixed cell populations and infer the temporal sequence of events within a signaling network. Vascular development is partially driven by highly proliferative endothelial progenitor cells (EPC). Numerous assays have been utilized to enumerate and isolate EPC by flow cytometry and primary cell culture [5,6,7]. Although conflicting definitions exist in the literature, Critser and Yoder concluded in 2010 that late outgrowth endothelial colony-forming cells (ECFC), which demonstrate the ability for self-renewal and neo-angiogenesis, both in vitro and in animal models, can uniquely be described as progenitors of endothelial cells [6,8]. A key feature of ECFC is tube formation when plated on Matrigel or cross-linked collagen, allowing for the study of in vitro angiogenesis without the need for complicated growth matrices. We previously demonstrated that ECFC are reduced in prematurely born infants that go on to develop bronchopulmonary dysplasia (BPD) and infants born to mothers with preeclampsia [9,10,11]. For prematurely born infants the introduction to room air after birth leads to an increase in environmental oxygen at a developmentally abnormal time. One potential regulator of ECFC environmental response to hyperoxia is Thymosin 4 (T4). Cytosolic T4 protein mediates the response of endothelial cells to changes in oxygen environment by modulating the stability of Hypoxia Inducible Factor 1α (HIF-1α). HIF-1α is historically associated with cellular response to hypoxia, not hyperoxia. * Correspondence: Email: douglas.shepherd@ucdenver.edu; Tel: +303-556-6380. * Correspondence: Email: douglas.shepherd@ucdenver.edu; Tel: +303-556-6380. Abstract: Disruptions in the response of endothelial progenitor cells to changes in oxygen environment may present a possible mechanism behind multiple pediatric pulmonary disease models, such as bronchopulmonary dysplasia. Using high-throughput fixed single-cell protein and RNA imaging, we have created “stop-motion” movies of Thymosin 4 (T4) and Hypoxia Inducible Factor 1α (HIF-1α) protein expression and vascular endothelial growth factor (vegf) and endothelial nitric oxide synthase (eNOS) mRNA in human umbilical cord-derived endothelial colony-forming cells (ECFC). ECFC were grown in vitro under both room air and hyperoxia (50% O2). We find elevated basal T4 protein expression in ECFC derived from prematurely born infants versus full term infants. T4 is a potent growth hormone that additionally acts as an actin sequestration protein and regulates the stability of HIF-1α. This basal level increase of T4 is associated with lower HIF- 1α nuclear localization in preterm versus term ECFC upon exposure to hyperoxia. We find altered expression in the pro-angiogenic genes vegf and eNOS, two genes that HIF-1α acts as a transcription factor for. This provides a potential link between a developmentally regulated protein and previously observed impaired function of preterm ECFC in response to hyperoxia. 285 Keywords: single-molecule; single-cell; fluorescence microscopy; endothelial colony-forming cells; endothelial cell biology Keywords: single-molecule; single-cell; fluorescence microscopy; endothelial colony-forming cells; endothelial cell biology 1. Introduction Recent work has demonstrated a role for HIF-1α in signaling preservation across all changes in oxygen environment, particularly in endothelial cells [12–16]. One key feature of this signaling network is a self-modulating feedback mechanism for T4. Jo et al. demonstrated altered HIF-1α nuclear localization as a function of increasing T4 expression in endothelial cells [12]. Based on these results, we applied high- throughput imaging to determine if changes in the spatial localization of T4 and HIF-1α in ECFC Volume 4, Issue 2, 284-297. AIMS Biophysics 286 are associated with changes in RNA copy number of vascular endothelial growth factor (vegf) and endothelial nitric oxide synthase (eNOS) at the single-cell level. vegf and eNOS are potent pro-proliferation and pro-angiogenesis genes that are associated with many developmental diseases in prematurely born infants [9,17–20]. Our laboratory previously showed that ECFC derived from the umbilical cord blood of prematurely born infants (pt-ECFC) demonstrated markedly different proliferation in vitro as compared to ECFC derived from the umbilical cord blood of full term birth infants (t-ECFC) [9,10,11]. Based on these results and others, we hypothesized that exposure to hyperoxia introduces a specific environmental signal that blunts pro-proliferation and pro-angiogenesis genetic signaling networks in a sub-population of pt-ECFC. Specifically, we hypothesized that elevated basal cytosolic T4 protein concentration in a subpopulation of pt-ECFC is associated with diminished HIF-1α nuclear localization and diminished early-time (within the first 60 minutes) vegf and eNOS RNA production in response to hyperoxia as compared to t-ECFC. To test this hypothesis, we utilized high-throughput single-cell imaging of protein and RNA expression to quantify pt-ECFC and t-ECFC grown in vitro room air and hyperoxia. We quantified T4 and HIF-1α protein expression using immunofluorescence as well as vegf and eNOS RNA expression using single-molecule fluorescence in-situ hybridization (smFISH [21,22]). Because these techniques require cell fixation we were unable to track single-cell temporal correlations. We instead built statistical spatial measures of how independent populations of pt-ECC or t-ECFC respond to either room air or hyperoxia treatment by measuring cell populations across multiple time points and multiple environmental conditions. This allowed us to infer potential casual relationships through statistical analysis. We found that pt-ECFC have elevated basal cytosolic T4 as compared to t-ECFC. This was statistically associated with lower HIF-1α nuclear localization. During the first hour of in vitro hyperoxia growth, distinct single-cell populations emerged in pt-ECFC that are not present in t- ECFC. 1. Introduction Based on these findings we speculate that this pathway may directly control the pro- angiogenic response of ECFC and propose a set of future experiments. AIMS Biophysics 2.2. ECFC in vitro culture ECFC were removed from cryo-suspension and studied at passage 3–5 in complete EGM-2 media. Individual #1 cover slips (Corning, 2845-25) were cleaned in ethanol followed by 1 × PBS. ECFC were plated onto coverslips at low density and cultured in either room air with 5% CO2 or 50% O2 with 5% CO2. At 0, 15, 30 or 60 minutes the EGM-2 medium was replaced with 3.7% paraformaldehyde (Sigma, 47608) for 15 minutes, followed by 1 × PBS (Sigma, P5493). All steps were performed within the incubator to ensure a near constant oxygen environment. Following completion of all coverslips, the PBS in each well was exchanged for 70% ethanol (Sigma, 277649). 2.3. High-throughput single-molecule, single-cell imaging Automated imaging was performed using a custom-built microscope, consisting of an Olympus IX71 microscope base, X-Y translation stage (Mad City Labs Microstage), objective piezo (Mad City Labs F200S), oil-immersion 100 × NA1.3 objective (Olympus UPLSAPO 100XO), multi-color LED light source (Lumencor Spectra-X), LED light source specific filter set (Semrock LED- DA/FI/TR/Cy5-4X-A), and a sCMOS camera (Hamamatsu C11400-22CU). Automation and acquisition were performed using MicroManager v1.4 running on a Windows 7 64-bit laptop [24]. Analysis was performed on a computing cluster with 48 cores, 128 gigabytes of RAM, 2 NVidia TITAN GPUs, and hot swappable storage (Titanus Computers). Custom analysis software was used to analyze all data in both ImageJ [19] and MATLAB (Mathworks). Fifty image areas, containing at minimum two cells per area, were identified using the automated stage and objective piezo. At each image area, a simple contrast based autofocus routine implemented in MicroManager was used to correct mechanical or thermal drift. Forty axial image planes were acquired at 250 nm spacing, centered around the algorithm selected best focus point. At each focal plane, four independent images were captured, one each for DAPI, Alexa 488, Alexa 561, and Alexa 647 fluorescent labels. 2.1. Cord blood collection and ECFC isolation Cord blood (CB) collection and endothelial colony forming cells (ECFC) were prepared as previously described [23]. CB samples were obtained following informed consent from term (gestational age greater than 37 weeks) and preterm (gestational age less than 36 weeks) infants at the University of Colorado Anschutz Inpatient Pavilion. The Colorado Multiple Institutional Review Board approved all protocols. All CB was processed within 24 hours of collection. Mononuclear cells (MNCs) were plated on cell culture plates coated with type 1 rat-tail collagen (BD Biosciences; San Jose, CA) at a density of 5 × 106 cells/cm2. Complete EGM-2 medium (Lonza, CC-3612; with 10% fetal bovine serum) was changed daily for seven days. After seven days, media were changed three times per week and ECFC colonies were enumerated on day 14 using light microscopy. To ensure that ECFC were not migrating or being removed during feeding, the removed Volume 4, Issue 2, 284-297. 287 media from 6 cell wells was re-plated on collagen-coated plates and observed for 14 days. Cells were then collected into cryogenic tubes and frozen under liquid nitrogen until future studies were performed. media from 6 cell wells was re-plated on collagen-coated plates and observed for 14 days. Cells were then collected into cryogenic tubes and frozen under liquid nitrogen until future studies were performed. 2.5.3. The basic workflow of protein detection and quantification was: 1. image filtering and 2. corrected total cell fluorescence (CTCF) calculation. In-house MATLAB and ImageJ software were utilized to median filter and then calculate the CTCF value for every cell. The background for CTCF was independently determined for each image. 2.5.2. Cell and nuclei identification Custom in-house MATLAB software was utilized to filter and identify individual cells in every image prior to protein analysis [4,26,27]. The user was presented a maximum intensity projection image of the nuclei and actin cytoskeleton fluorescent labels. Each cell was manually circled and recorded until all cells in an image are identified. A watershed algorithm was used to automatically detect the nucleus within each identified cell. Overlapping cells are not included in further data analysis to avoid over-counting. Cell and nuclei outlines are saved as text-files for use later in the analysis. 2.4. T4 and HIF-1α fluorescent labeling All fluorescent labeling steps were adapted from previous work by our group and others [20,21,22]. Following overnight permeabilization in 70% ethanol at 4 °C, coverslips were washed in 2 mL of 1 × PBS at room temperature followed by incubation in blocking buffer (Thermo- Fisher, 37515). Coverslips were incubated in primary antibody (1:100 dilution in 1 × PBS) for T4 (Abcam, ab14335) and HIF-1α (Abcam, ab51608) overnight, washed in 2 mL of 1 × PBS, incubated in secondary antibody (1:100 dilution in 1 × PBS; Abcam, ab175471; Abcam, ab150115) at 37 °C, and washed in 2 mL of 1 × PBS with 1 drop of pre-mixed DAPI (Life Technologies, R37606). Volume 4, Issue 2, 284-297. AIMS Biophysics 288 The following steps were performed in low light to prevent bleaching. Coverslips were hybridized with 20 μL of GLOX and Phalloidin-Alexa488 (Life Technologies, A22287) for 30 minutes at room temperature and then washed with GLOX buffer for 15 minutes at room temperature. Coverslips were plated on standard microscope slides with 8 μL GLOX plus enzyme (imaging buffer) and nail polish was applied around the coverslip to seal. 2.5. T4 and HIF-1α image analysis 2.5.1. GPU-based de-convolution 2.5.1. GPU-based de-convolution Image de-convolution was performed in ImageJ, utilizing software provided by the Butte Group [25], prior to any further image processing steps. 2.5.2. Cell and nuclei identification 2.6.1. Single-molecule fluorescence in-situ hybridization All hybridization steps were adapted from previous work by our group and others [4,21,22]. Following the overnight permeabilization, coverslips were washed in 2mL of 10% wash buffer for 30 minutes at 37 °C. Coverslips were incubated with 20 μL of vegf and eNOS probe solution (1:1:1000 in 10% hybridization buffer) for 8 hours at 37 °C. Both vegf and eNOS probe sets were designed using both in-house and commercially available probe designing technologies (Biosearch Technologies). Following hybridization, coverslips were washed with 10% wash buffer for 30 minutes at 37 °C and then incubated in 10% wash buffer with 1 drop pre-mixed DAPI solution (Life AIMS Biophysics AIMS Biophysics Volume 4, Issue 2, 284-297. 289 Technologies, R37606) in each well for 30 minutes at 37 °C. Coverslips were then washed with GLOX buffer for 15 minutes at room temperature. Technologies, R37606) in each well for 30 minutes at 37 °C. Coverslips were then washed with GLOX buffer for 15 minutes at room temperature. 2.6.2. Hybridization buffers 10% wash buffer: 5 mL deionized formamide (Ambion, AM9342), 5 mL 20 × SSC (Ambion, AM9763), 40 mL nuclease-free water (Ambion, AM9932). Hybridization buffer: 1 mL deionized formamide (Ambion, AM9342), 10 mg E. coli tRNA (Roche, 10109541001), 1 mL 20× SSC (Ambion, AM9763), 40 μL ultrapure BSA (Ambion, AM2618), 1 g dextran sulfate (Sigma, D8906- 50G), 100 μL of 200mM vanadyl-ribonucleoside complex (Sigma, 94742-1ML), nuclease-free water (Ambion, AM9932) to 10mL total solution. GLOX buffer: 100 μL 1M Tris (Ambion, AM9855G), 1mL 20× SSC (Ambion, AM9763), 400 μL of 10% Glucose, 8.5mL nuclease-free water (Ambion, AM9932). 2.6.4. Imaging buffer GLOX plus enzyme: 1 μL 3.7 mg/mL Glucose Oxidase (Sigma, G7141), 1 μL Catalase (Sigma, C30), 100 μL GLOX buffer. 2.7. vegf and eNOS image analysis 2.7. vegf and eNOS image analysis 2.7. vegf and eNOS image analysis 2.7.1. GPU-based de-convolution 2.6.3. Imaging preparation The following steps were performed in low light to prevent bleaching. Coverslips were hybridized with 20 μL of GLOX and Phalloidin-Alexa488 (Life Technologies, A22287) for 30 minutes at room temperature and then washed with GLOX buffer for 15 minutes at room temperature. Coverslips were plated on standard microscope slides with 8 μL GLOX plus enzyme (imaging buffer) and nail polish was applied around the coverslip to seal. 2.7.3. RNA detection and quantification The basic workflow of RNA detection and quantification was: (1) image filtering, (2) spot pre- detection, (3) spot fitting, (4) non-specific versus specific determination, (5) spot assignment to individual cells, (6) batch processing. FISH-QUANT was utilized to identify, fit, and quantify individual RNA labeled using smFISH. The details of FISH-QUANT are described in a prior publication [26]. Background fluorescence is typically due to FISH probes that were either not washed out or non-specifically bound during the labeling procedure [4]. FISH-QUANT utilizes a three-dimensional Gaussian to fit each possible spot and the user must discriminate background from specific binding by manual thresholding of several extracted fit parameters (e.g. lateral fit uncertainty, axial fit uncertainty, amplitude of Gaussian). 2.8. Statistical analysis of imaging results Single-cell protein CTCF and RNA distributions were loaded in MATLAB for statistical analysis. Before further analysis, all measurements were divided by the maximum intensity projection of the measurement’s corresponding cell compartment area (nucleus, cytoplasm, or total cell). Area normalized marginal distributions were tested for independence using the two sample Kolmogorov Smirnov (K-S) test implemented in the Statistics Toolbox for MATLAB (null hypothesis rejection at α = 0.05). Area normalized marginal distribution independence were tested at each measured time for a given protein or RNA species for all cell types and growth conditions (population response). Additionally, we tested within each protein or RNA species for a specific cell type and growth condition against measure time points (temporal response). Area normalized multivariate joint distributions were tested for independence using the non-parametric and distribution free multivariate distribution distance tests, implemented in the highdim package for MATLAB (null hypothesis rejection at α = 0.05) [28]. This allowed for direct comparison of the non-Gaussian distributions common in single-cell gene expression imaging experiments using a non- parametric test. 2.7.1. GPU-based de-convolution Image de-convolution was performed in ImageJ, utilizing software provided by the Butte Group [25], prior to any further image processing steps. 2.7.2. Cell and nuclei identification FISH-QUANT and custom in-house MATLAB software were utilized to filter and identify individual cells in every image prior to RNA analysis [4,26,27] The user was presented a maximum intensity projection image of the nuclei and actin cytoskeleton fluorescent labels. Each cell was manually circled and recorded until all cells in an image are identified. A watershed algorithm was used to automatically detect the nucleus within each identified cell. Overlapping cells were not AIMS Biophysics Volume 4, Issue 2, 284-297. 290 included in further data analysis to avoid over-counting. Cell and nuclei outlines are saved as text- files for use later in the analysis. included in further data analysis to avoid over-counting. Cell and nuclei outlines are saved as text- files for use later in the analysis. AIMS Biophysics 3.1. Cytosolic basal T4 expression in ECFC To begin, we tested if pt-ECFC T4 expression was elevated in multiple primary ECFC cultures. To determine T4 expression, we thawed and prepared individual populations of ECFC from four term and four preterm patients. After three to five passages, we fixed each population and labeled for T4 using immunofluorescence. We assessed protein content using corrected total cell fluorescence (CTCF) for the nucleus, cytoplasm, and total cell. We normalized CTCF values by the maximum projection area of the feature of interest to limit copy number variability due to cell size. Volume 4, Issue 2, 284-297. AIMS Biophysics 291 Using T4 expression from at least 200 cells per patient we find that cytosolic T4 protein expression is elevated across pt-ECFC (n = 4 patients) as compared to t-ECFC (n = 4 patients) at basal conditions (Figure 1). We found that total cell and nuclear T4 expression are not statistically different. Using T4 expression from at least 200 cells per patient we find that cytosolic T4 protein expression is elevated across pt-ECFC (n = 4 patients) as compared to t-ECFC (n = 4 patients) at basal conditions (Figure 1). We found that total cell and nuclear T4 expression are not statistically different. Figure 1. Area normalized T4 CTCF values. t-ECFC (200 cells from each patient, 4 patients) had lower expression than pt-ECFC (200 cells from each patient, 4 patients) after thawing and passage (p = 1.1e-11 using two-sample K-S test). Figure 1. Area normalized T4 CTCF values. t-ECFC (200 cells from each patient, 4 patients) had lower expression than pt-ECFC (200 cells from each patient, 4 patients) after thawing and passage (p = 1.1e-11 using two-sample K-S test). 3.2. T4 protein expression and HIF-1α protein nuclear localization After establishing that pt-ECFC have elevated T4, we moved on to test if pt-ECFC display diminished HIF-1α nuclear localization. While nuclear localization is not a perfect indicator of transcription factor activity, it is a requirement for a transcription factor to be in the nucleus to activate a gene network. To determine protein localization, we cultured a mixture population of the four patients used in Figure 1 in room air or hyperoxia (50% O2). At 0, 15, and 30 minutes of growth we fixed and labeled for both T4 and HIF-1α using immunofluorescence. We assessed protein content in the nucleus, cytoplasm, and total cell using CTCF. We normalized CTCF values by the maximum projection area of the feature of interest to limit copy number variability due to cell size. We found that HIF-1α nuclear localization was diminished for pt-ECFC grown in vitro room air or hyperoxia (top 2 rows; blue line; Figure 2A). In contrast, we found that HIF-1 nuclear localization was immediate and sustained for t-ECFC grown in vitro room air and hyperoxia (bottom 2 rows; blue line; Figure 2A). We found that there was minimal difference in T4 protein localization for pt-ECFC grown in vitro room air or hyperoxia versus t-ECFC grown in vitro room air or hyperoxia respectively (top 2 rows versus bottom 2 rows; Figure 2B). This suggests that it was the initial increased concentration in T4, or other factors not measured here, that lead to the observed reduction in nuclear HIF-1α localization for pt-ECFC grown in vitro hyperoxia [12]. Volume 4, Issue 2, 284-297. AIMS Biophysics 292 Figure 2. (A) HIF-1α and (B) T4 protein expression. Marginal probability distributions for nuclear (blue) and cytosolic (red) protein expression for pt-ECFC (top 2 rows) and t- ECFC grown (bottom 2 rows) in vitro room air (RA) and hyperoxia (HO) for 0, 15, and 30 minutes for HIF-1α and T4. Nuclear localization of HIF-1α was blunted in pt-ECFC as compared to t-ECFC in hyperoxia (p < 0.01 for all time points, K-S test). Additionally, nuclear and cytoplasmic expression for HIF-1α and T4 was different between in vitro room air and hyperoxia for both pt-ECFC and t-ECFC (p < 0.01 for all time points, K-S test). (C) Representative maximum projection image of pt-ECFC grown in vitro hyperoxia (nucleus—magenta; actin—blue; HIF-1α—green; T4— red). Figure 2. (A) HIF-1α and (B) T4 protein expression. 3.2. T4 protein expression and HIF-1α protein nuclear localization Marginal probability distributions for nuclear (blue) and cytosolic (red) protein expression for pt-ECFC (top 2 rows) and t- ECFC grown (bottom 2 rows) in vitro room air (RA) and hyperoxia (HO) for 0, 15, and 30 minutes for HIF-1α and T4. Nuclear localization of HIF-1α was blunted in pt-ECFC as compared to t-ECFC in hyperoxia (p < 0.01 for all time points, K-S test). Additionally, nuclear and cytoplasmic expression for HIF-1α and T4 was different between in vitro room air and hyperoxia for both pt-ECFC and t-ECFC (p < 0.01 for all time points, K-S test). (C) Representative maximum projection image of pt-ECFC grown in vitro hyperoxia (nucleus—magenta; actin—blue; HIF-1α—green; T4— red). 3.3. vegf and eNOS RNA expression One consequence of reduced HIF-1α nuclear localization was a potential reduction in vegf and eNOS RNA production. We utilized the same experimental design as above but instead labeled for RNA transcripts at each time point. We found the pt-ECFC do not begin production of vegf or eNOS RNA in either room air or hyperoxia until 60 minutes (top 2 rows; blue line; Figure 3A and B). In contrast, we found t-ECFC began and sustained vegf and eNOS RNA production almost immediately (bottom 2 rows; blue line; Figure 3A and 3B). This production resulted in increased mature RNA in the cytoplasm by 60 minutes (bottom 2 rows; red line; Figure 3A and 3B). Volume 4, Issue 2, 284-297. AIMS Biophysics 293 Figure 3. (A) vegf and (B) eNOS RNA expression. Marginal probability distributions for nuclear (blue) and cytosolic (red) single-cell RNA expression for pt-ECFC and t-ECFC grown in vitro room air and hyperoxia for 0, 15, 30, and 60 minutes for vegf and eNOS. Nuclear vegf RNA production began almost immediately in t-ECFC as compared to pt- ECFC for both in vitro room air and hyperoxia (p < 0.01, K-S test). This production resulted in increased cytosolic vegf RNA at 60 minutes in t-ECFC as compared to pt- ECFC (p < 0.01, K-S test). A similar result was observed for eNOS, however with sustained nuclear expression in t-ECFC through 60 minutes for both in vitro room air and hyperoxia (p < 0.01 except for 60-minute time point, K-S test). The exception was at 60 minutes, where pt-ECFC and t-ECFC eNOS expression were statistically indistinguishable (p > 0.05, K-S test). (C) Representative maximum projection image of t-ECFC grown in vitro hyperoxia (nucleus—magenta; actin—blue; vegf—green; eNOS— red). Figure 3. (A) vegf and (B) eNOS RNA expression. Marginal probability distributions for nuclear (blue) and cytosolic (red) single-cell RNA expression for pt-ECFC and t-ECFC grown in vitro room air and hyperoxia for 0, 15, 30, and 60 minutes for vegf and eNOS. Nuclear vegf RNA production began almost immediately in t-ECFC as compared to pt- ECFC for both in vitro room air and hyperoxia (p < 0.01, K-S test). This production resulted in increased cytosolic vegf RNA at 60 minutes in t-ECFC as compared to pt- ECFC (p < 0.01, K-S test). 3.3. vegf and eNOS RNA expression A similar result was observed for eNOS, however with sustained nuclear expression in t-ECFC through 60 minutes for both in vitro room air and hyperoxia (p < 0.01 except for 60-minute time point, K-S test). The exception was at 60 minutes, where pt-ECFC and t-ECFC eNOS expression were statistically indistinguishable (p > 0.05, K-S test). (C) Representative maximum projection image of t-ECFC grown in vitro hyperoxia (nucleus—magenta; actin—blue; vegf—green; eNOS— red). AIMS Biophysics 3.4. Protein or RNA expression as a function of nucleus to cytoplasm area ratio Based on our above findings that there were differences in T4, HIF-1α, vegf, and eNOS marginal distributions both between pt-ECFC and t-ECFC as well as in vitro room air and hyperoxia growth, we utilized our single-cell dataset to construct multi-dimensional distributions. Based on AIMS Biophysics Volume 4, Issue 2, 284-297. 294 previous findings that the ratio of nuclear area to cytoplasmic area (N:C ratio) may be a marker of proliferating ECFC [29], we plotted the N:C ratio versus T4 CTCF and HIF-1α CTCF (Figure 4A) and N:C ratio versus vegf RNA transcripts and eNOS RNA transcripts (Figure 4B). Unlike the marginal distributions presented in Figures 2–3, we utilized the total amount of protein or RNA in each cell to simplify the analysis. This analysis provides a visualization of how the four different cell populations evolved over time, fully leveraging the information within this single-cell dataset. pt-ECFC grown in vitro HO (green) demonstrated different behavior from all other populations for both protein (Figure 4A) and RNA (Figure 4B) (p < 0.01, multi-dimensional distance test). A large heterogeneity in both cell size and protein or RNA expression was also apparent using this analysis, supporting the idea that ensemble measurements may be averaging over several different single cell behaviors, masking these differences. Figure 4. Multivariate (A) protein (x-axis: T4 CTCF normalized by cell area; y-axis: HIF-1α CTCF normalized by cell area; and z-axis: nuclear to cytoplasm area ratio) and (B) RNA (x-axis: vegf RNA transcripts normalized by cell area; y-axis: eNOS RNA transcripts normalized by cell area; and z-axis: nuclear to cytoplasm area ratio) single- cell distributions at each time point for all conditions. Figure 4. Multivariate (A) protein (x-axis: T4 CTCF normalized by cell area; y-axis: HIF-1α CTCF normalized by cell area; and z-axis: nuclear to cytoplasm area ratio) and (B) RNA (x-axis: vegf RNA transcripts normalized by cell area; y-axis: eNOS RNA transcripts normalized by cell area; and z-axis: nuclear to cytoplasm area ratio) single- cell distributions at each time point for all conditions. 5. Conclusion In this study, we attempted to address our hypothesis that elevated basal cytosolic T4 protein concentration in a subpopulation of pt-ECFC diminishes early-time (within the first 60 minutes) vegf and eNOS RNA production in response to hyperoxia as compared to t-ECFC. Using single-cell imaging, we found statistical evidence that elevated cytosolic T4 in pt-ECFC were associated with diminished HIF-1α nuclear localization and diminished vegf and eNOS RNA copy number. Our data and analyses, combined with previous studies, suggest that T4 may play a key role in determining ECFC vegf and eNOS response to changes in oxygen environment. Our data additionally suggest a role for HIF-1α, further expanding the scope of this key transcription factor to the ECFC hyperoxia response. 4. Discussion Our analyses suggested that previous observations of blunted proliferation of pt-ECFC in hyperoxia [9,23] may be due to developmentally appropriate levels of pro-growth proteins, such as T4. These levels were elevated as compared to t-ECFC, most likely due to internal and external factors driving ECFC proliferation. Paradoxically, the release of T4 into the cytosol in response to hyperoxia reduced HIF-1α stability in this high-T4 sub-population [12]. That in turn lead to lowered vegf and eNOS RNA production and the emergence of a less progenitor-like population. The AIMS Biophysics Volume 4, Issue 2, 284-297. 295 overall results of this network motif matched previous ensemble proliferation measurements, but the details are only observable using single-cell methodologies. overall results of this network motif matched previous ensemble proliferation measurements, but the details are only observable using single-cell methodologies. Because these data were “stop-motion” movies of distinct cell populations, we cannot confirm that those cells with elevated T4 were the same cells that emerged with reduced HIF-1α nuclear localization and subsequent lower vegf and eNOS RNA expression. Future experiments that rely on live-cell fluorescent reporters of T4 and HIF-1α protein followed by smFISH measurement of vegf and eNOS RNA may provide key insight into the temporal evolution of these heterogeneous cell populations. Conflict of Interest All authors declare no conflicts of interest. Acknowledgements M.A.A. and D.P.S acknowledge startup funding from the University of Colorado Denver College of Liberal Arts and Sciences. G.J.S. acknowledges funding from the National Institute of Health (NHLBI HL68702). C.D.B. acknowledges funding from the National Institutes of Health (K23 HL121090-01A1). AIMS Biophysics References 1. Munsky B, Fox Z, Neuert G (2015) Integrating single-molecule experiments and discrete stochastic models to understand heterogeneous gene transcription dynamics. Methods 85: 12–21. 2. Munsky B, Neuert G, Oudenaarden AV (2012) Using gene expression noise to understand gene regulation. Science 336: 183–187. 3. Neuert G, Munsky B, Tan RZ, et al. (2013) Systematic identification of signal-activated stochastic gene regulation. Science 339: 584–587. 3. Neuert G, Munsky B, Tan RZ, et al. (2013) Systematic identification of signal-activated stochastic gene regulation. Science 339: 584–587. 4. Shepherd DP, Li N, Micheva-Viteva SN, et al. 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Nat Biotechnol 32: 1151–1157. © 2017 Douglas P. Shepherd, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) Volume 4, Issue 2, 284-297. AIMS Biophysics
https://openalex.org/W1998189375	https://link.springer.com/content/pdf/10.1007/s11926-014-0493-9.pdf	English	null	Physicochemical and Biomechanical Stimuli in Cell-Based Articular Cartilage Repair	Current rheumatology reports	2,015	cc-by	10,356	Curr Rheumatol Rep (2015) 17: 22 DOI 10.1007/s11926-014-0493-9 Curr Rheumatol Rep (2015) 17: 22 DOI 10.1007/s11926-014-0493-9 OSTEOARTHRITIS (MB GOLDRING, SECTION EDITOR) Physicochemical and Biomechanical Stimuli in Cell-Based Articular Cartilage Repair Holger Jahr & Csaba Matta & Ali Mobasheri Published online: 2 April 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Published online: 2 April 2015 Cartilage bioengineering . Mesenchymal stem cell . Intracellular signaling pathways Abstract Articular cartilage is a unique load-bearing connec- tive tissue with a low intrinsic capacity for repair and regen- eration. Its avascularity makes it relatively hypoxic and its unique extracellular matrix is enriched with cations, which increases the interstitial fluid osmolarity. Several physico- chemical and biomechanical stimuli are reported to influence chondrocyte metabolism and may be utilized for regenerative medical approaches. In this review article, we summarize the most relevant stimuli and describe how ion channels may contribute to cartilage homeostasis, with special emphasis on intracellular signaling pathways. We specifically focus on the role of calcium signaling as an essential mechanotransduction component and highlight the role of phosphatase signaling in this context. Abbreviations ACI Autologous chondrocyte implantation ALP Alkaline phosphatase CCI Characterized chondrocyte implantation Cn Calcineurin CREB cAMP response element binding protein CsA Cyclosporine A ECM Extracellular matrix ERK Extracellular signal-regulated kinase FAK Focal adhesion kinase FCD Fixed charge density FTE Functional tissue engineering GAG Glycosaminoglycan HP Hydrostatic pressure MAPK Mitogen-activated protein kinase Abbreviations ACI Autologous chondrocyte implantation ALP Alkaline phosphatase CCI Characterized chondrocyte implantation Cn Calcineurin CREB cAMP response element binding protein CsA Cyclosporine A ECM Extracellular matrix ERK Extracellular signal-regulated kinase FAK Focal adhesion kinase FCD Fixed charge density FTE Functional tissue engineering GAG Glycosaminoglycan HP Hydrostatic pressure MAPK Mitogen-activated protein kinase Keywords Articularcartilage .Cartilagerepair .Regenerative medicine . Autologous chondrocyte implantation . Keywords Articularcartilage .Cartilagerepair .Regenerative medicine . Autologous chondrocyte implantation . Chondrocyte metabolism . Functional tissue engineering . Chondrocyte metabolism . Functional tissue engineering . This article is part of the Topical Collection on Osteoarthritis H. Jahr Department of Orthopaedic Surgery, University Hospital RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany H. Jahr: C. Matta: A. Mobasheri (*) The D-BOARD European Consortium for Biomarker Discovery, Surrey, UK e-mail: a.mobasheri@surrey.ac.uk A. Mobasheri e-mail: ali.mobasheri.manuscripts@gmail.com URL: http://www.d-board.eu/dboard/index.aspx C. Matta: A. Mobasheri Department of Veterinary Preclinical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Duke of Kent Building, Guildford, Surrey GU2 7XH, UK C. Matta Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen 4032, Hungary C. Physicochemical and Biomechanical Stimuli in Cell-Based Articular Cartilage Repair Matta Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen 4032, Hungary C. Matta Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen 4032, Hungary A. Mobasheri Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, Arthritis Research UK Pain Centre, Medical Research Council and Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK A. Mobasheri Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, Arthritis Research UK Pain Centre, Medical Research Council and Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK A. Mobasheri Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd Medical Research Center (KFMRC), King AbdulAziz University, Jeddah 21589, Kingdom of Saudi Arabia A. Mobasheri Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd Medical Research Center (KFMRC), King AbdulAziz University, Jeddah 21589, Kingdom of Saudi Arabia MSC Mesenchymal stem cell NFAT Nuclear factor of activated T lymphocytes OA Osteoarthritis OP Osmotic pressure PEMF Pulsed electromagnetic field PG Proteoglycan PKA Protein kinase A PKC Protein kinase C RVD Regulatory volume decrease TRPV Transient receptor potential vanilloid channel FF Fluid flow 22 Page 2 of 12 22 Page 2 of 12 22 Page 2 of 12 Curr Rheumatol Rep (2015) 17: 22 MSC Mesenchymal stem cell NFAT Nuclear factor of activated T lymphocytes OA Osteoarthritis OP Osmotic pressure PEMF Pulsed electromagnetic field PG Proteoglycan PKA Protein kinase A PKC Protein kinase C RVD Regulatory volume decrease TRPV Transient receptor potential vanilloid channel FF Fluid flow 22 Page 2 of 12 signaling apparatus that convert various environmental forces (e.g., deformation, shear stress and fluid flow, hydrostatic pressure (HP), and extracellular ionic milieu (i.e., OP) as well as magnetic and electric forces), into cellular responses. We provide a brief overview of how these forces might be exploited to facilitate cartilage regeneration, with special em- phasis on intracellular signaling, which is often understudied in the context of cartilage bioengineering. Introduction The unique biomechanical properties of articular cartilage are attributed to the structure, composition, and organization of its extracellular matrix (ECM) macromolecules. The cartilage ECM is mainly composed of a collagen fiber network (type II collagen with type IX and XI) and large aggregating pro- teoglycans (PGs) entrapped within. The fixed negative charges on the glycosaminoglycan (GAG) side chains of PGs attract counteracting ions, which drive the movement of large amounts of osmotically obliged water into the matrix [1]. The high osmotic milieu and the tissue swelling maintain the hydrostatic pressure and viscoelastic properties of cartilage. The highly sulfated GAG side chains of PGs, through attracting mobile cations, are responsible for the characteristi- cally high negative fixed-charge density (FCD) [2]. Intertwined collagens and PGs combine to create the tissue rigidity by entrapping solutes and water, giving cartilage its unique biomechanical properties, to withstand large compres- sive and shear forces without failing [3]. Articular cartilage absorbs stresses generated during joint loading and contrib- utes to joint lubrication [1, 4]. An intact collagen network restricts swelling and, in combination with sulfated GAGs, determines the osmotic pressure (OP) of the extracellular fluid around chondrocytes, which ranges from 350 to 480 mOsm in healthy cartilage [5]. Cartilage Pathologies Traumatic local damage [13–15], usually in younger patients, and whole joint erosion, as in osteoarthritis (OA) [16] in the elderly, are challenging areas of regenerative orthopedics [17]. To date, there is no successful targeted therapy that would halt or even reverse OA progression; current management includ- ing inflammatory medications, total joint replacements, or an- algesics only allow palliative treatment [18•]. There is a press- ing need for targeted treatment options, ideally at the early, asymptomatic stages of the disease. The earliest signs of ar- ticular cartilage degeneration during OA are net depletion of PGs [10, 19–21], subsequent loss of the collagen network [22], and diminished intrinsic compressive stiffness, affecting chondrocyte deformation, metabolic activities, and electrome- chanical events within cartilage under body load [10, 23]. Severity-dependent catabolic events during the course of OA reduce extracellular osmolarity, resulting in reduced viscoelas- tic tissue properties, corresponding biomechanical inferiority [5, 24], and eventually increased deformation of cartilage un- der mechanical load. Elevated levels of inflammatory media- tors that promote matrix degradation may also accompany these changes. Electrical Stimulation Electrical Stimulation Mechanical stimulation is an important regulator of chon- drocyte metabolism that is required for maintaining normal cartilage matrix properties [54, 55] and a well-established cue for improving mechanical properties of tissue- engineered cartilage [27], as reviewed by Vunjak-Novakovic et al. [56] and Lee et al. [57]. A plethora of bioreactors have been developed in which mechanical forces are applied via compression, HP, shear, multimodal compression and shear, vibration, bi-axial tension, and friction [18•]. While static loading of tissue-engineered cartilage constructs, in general, results in suppression of ECM biosynthesis, intermittent dy- namic loading is usually beneficial and increases the biosyn- thetic activity of chondrocytes. The cellular response to me- chanical stimuli always depends on magnitude, frequency, and duration of the stimulus, as well the relative timing of the loading, the culture period, and the subpopulation of chondrocytes. Importantly, the balanced activities of catabolic and anabolic factors may be needed to stimulate native-like ECM synthesis [58, 59]. Mechanoelectrical transduction phenomena occurring natural- ly within the cartilage due to the FCD have prompted the development of experimental electrical stimulation protocols for therapeutic cartilage repair [35, 36]. Therapeutic devices involving electrical stimulation are increasingly entering the clinical market [37], despite rather discouraging early results [38, 39]. More recently, Brighton et al. observed anabolic effects [40] that may hold potential to treat osteoarthritic le- sions [18•]. Our current appreciation of underlying molecular mechanisms, however, is rudimentary. Stimulating Functional Cartilage Tissue Engineering Stimulating Functional Cartilage Tissue Engineering Regenerative Approaches Autologous chondrocyte implantation (ACI) [25] surpasses existing procedures for treating focal defects, but is unable to fully restore functional hyaline cartilage ad integrum. A potentially better procedure for structurally repairing symp- tomatic cartilage defects in the knee is characterized chondro- cyte implantation (CCI) which has a more favorable outcome compared to microfracturing [26]. The electrochemical properties of articular cartilage arise from the flow of “free” electrolytes (e.g., Na+, K+, Ca2+) pass- ing the relatively “fixed” FCD (e.g., SO4 2−) distributed along the PGs [6] resulting in electrokinetic phenomena and charge- dependent osmotic swelling pressures (i.e., Donnan osmotic pressure) [7–9]. The FCD permits tissue hydration, charged species transport, and other electrochemical responses [10]. Mow et al. postulated that the ECM is a mechanical signal transducer, receiving loading as input to generate an output of multiple biophysical signals [11]. A major challenge is the complex zonal structure of carti- lage tissue, which is important for its load-bearing properties [27–29]. The sparse available data indicate that mechanical properties significantly vary between articular cartilage zones [30–32]. The lack of mechanical homogeneity may be impor- tant for mechanosensation, signal transduction, and chondro- cyte phenotypic stability. Interestingly, reports on physiologically relevant values of tensile or shear forces in natural cartilage or in tissue- engineered constructs are sparse [12], as is the knowledge about the molecular identity of the sensory components and Another major challenge of present tissue engineering strategies for cartilage repair is the limited integration of the Page 3 of 12 22 Curr Rheumatol Rep (2015) 17: 22 22 Mechanical Stimulation constructs into the surrounding host tissue [33], often resulting in local cell death at the defect margins [34]. The goal, there- fore, is to create tissue functionality prior to implantation by enhancing the rate and quality of tissue growth through creat- ing in vivo-like conditions in vitro. Various environmental stimuli for promoting cartilage regeneration are discussed in the following sections. In comparison to the rather limited evidence for effects of mag- netic and electrical stimulation, a vast body of studies have described the consequences of mechanical stimulation on artic- ular cartilage or chondrocytes in tissue engineering strategies [18•]. We review mainly cellular responses of chondrocytes and aspects closely related to osmotic stress, such as compression- induced changes in HP and OP, as both are essential for stim- ulating chondrocyte physiology and useful for manipulating chondrocyte metabolism and phenotype [53]. Regenerative Approaches In comparison to the rather limited evidence for effects of mag- netic and electrical stimulation, a vast body of studies have described the consequences of mechanical stimulation on artic- ular cartilage or chondrocytes in tissue engineering strategies [18•]. We review mainly cellular responses of chondrocytes and aspects closely related to osmotic stress, such as compression- induced changes in HP and OP, as both are essential for stim- ulating chondrocyte physiology and useful for manipulating chondrocyte metabolism and phenotype [53]. Magnetic Stimulation By manipulating substrate elasticity and adhesion density 22 Page 4 of 12 22 Page 4 of 12 Curr Rheumatol Rep (2015) 17: 22 [68], stiffness may affect proliferation and RGD adhesion site density during cellular differentiation. While the banding pe- riodicity of collagen fibers in the ECM is 67 nm [69] and the RGD epitopes of fibronectin fibers are ≥73 nm [70, 71], cells are clearly sensitive to changes in interparticle spacing of about 1 nm over a cell length [63], [68], stiffness may affect proliferation and RGD adhesion site density during cellular differentiation. While the banding pe- riodicity of collagen fibers in the ECM is 67 nm [69] and the RGD epitopes of fibronectin fibers are ≥73 nm [70, 71], cells are clearly sensitive to changes in interparticle spacing of about 1 nm over a cell length [63], chondrocytes can be technically challenging [53], while OP is robustly defined as chemical. Unlike OP, tonicity is influ- enced only by solutes that cannot cross the cell membrane. Although chemical loading (i.e., OP) and mechanical loading (i.e., HP) may not be exactly equivalent [85], the combination of HP and OP produce gene expression profiles different from those with OP alone, each stimulus by itself often results in similar effects such as the stimulation of sulfated GAG syn- thesis [53]. That MSC fate can be re-directed, even after weeks, by switching the biophysical microenvironment [72••] holds promises for several cartilage-related tissue engineering appli- cations. In contrast to generally unfavorable static compres- sion, static pre-stretching of biomaterials may beneficially al- ter (stem) cell behavior [73] through regulation of epigenetic events [74]. A meshwork of intermediate filaments and lamins physically links chromatin to the cytoskeleton-mediated ex- tracellular signal reception [75, 76]. Mechanical forces arising from matrix rigidity and nanotopography can physically affect the structural organization of the nucleus [77], possibly direct- ly altering gene expression and mechanical properties [63]. [ ] As OP is a state quantity, it changes during compressive joint loading and off-loading; with zone-dependent concentra- tions of sulfated GAGs causing OP gradients in articular car- tilage [86]. In each zone, chondrocytes are subject to different HPs and OPs due to weight bearing and joint loading [53, 32]. Magnetic Stimulation Applying HPs from 0 to 0.5 MPa at 0.5 Hz and OPs from 300 to 450 mOsm can upregulate anabolic and catabolic molecules in all three major zones in a descending order of magnitude from the surface to the deep zone. Interestingly, HP off- loading maintains anabolic messenger RNAs (mRNAs) and reduces catabolic mRNAs, while high OP retains mainly cat- abolic mRNAs [53]. Superficial zone-derived cells are most sensitive to changes in HP or OP [53], which may explain discrepancies between chondrocytes isolated from “normal” and OA cartilage. The effects of OP on viscoelastic and physical properties of chondrocytes are well described [87]. Tonicity enhancer binding protein (TonEBP, also known as nuclear factor of activated T cells (NFAT)5) stimulates multiple cellular pathways for adaptation to osmotic stress [88, 89] and organic osmolyte-dependent and independent pathways [90]. Physiological and path- ophysiological stimuli such as cytokines, growth factors, receptor and integrin activation, contractile agonists, ions, and reactive oxygen species have been implicated in the positive regulation of TonEBP expression and activity in diverse cell types [91]. In combination with mechanical stimulation, incorporation of chemical groups such as sulfates may improve chondrocyte proliferation while inhibiting hypertrophic differentiation [78, 79]. By using intrinsic biomaterial cues to stimulate migration, cell-seeded scaffolds appear promising for cartilage repair. Similar to MSCs, chondrocytes respond to HP, fluid flow (FF) and the accompanying shear stress, substrate strain and stiffness or topography, and electromagnetic fields [80]. Fully synthetic hydrogels can provide independent control over physical and adhesive properties [81] for use in cartilage re- generative medicine [82]. Magnetic Stimulation Since its FDA approval in 1979, pulsed electromagnetic field (PEMF) therapy has been widely used in orthopedics to treat poorly healing fractures [41, 42•]. Although the biology of how PEMF stimulates bone formation is only partially under- stood [43] and may originate from stimulating progenitor cell differentiation [44], its clinical use has provided a rationale for applying (P)EMF in musculoskeletal tissue engineering [45, 46]. Intrinsic Mechanical Stimulation and Substrate Properties Cyclic compression is required for chondrogenesis [60], while its impact on the intrinsic material properties of cartilage is an underappreciated aspect in tissue engineering. Using surface topography, stiffness, or patterns to induce mesenchymal stem cell (MSC) proliferation or differentiation [61] holds a lot of potential for enhancing musculoskeletal regeneration [62•]. ] Data from randomized controlled trials now suggest that PEMF improves clinical scores and function even in pa- tients with knee OA [47]. While (low-frequency) PEMF therapy barely influences the biosynthetic activity of hu- man OA chondrocytes in vitro [48], it increases PG release in alginate culture [49]. PEMF increases anti-inflammatory effects in the human costal chondrocyte cell line T/C-28a2 [50], and, like IGF-1, it augments chondroprotective anabolic activities such as PG synthesis in human OA cartilage explants, possibly by counteracting the effects of IL-1β in early stages of OA [51]. A study in ovariectomized rats, aiming at simu- lating postmenopausal osteoarthritis, reported an interest- ing systemic effect of PEMF therapy on estrogen metab- olism that reduced apoptosis and matrix metalloproteinase (MMP)13 expression in knee joint cartilage [52]. Howev- er, since current evidence for PEMF as a disease- modifying OA therapy is still weak, further studies are needed to elucidate its molecular basis. Our understanding of how cells sense the stiffness of ECM or biomimetic substrates is rudimentary. Emerging mecha- nisms of biophysically induced signals include focal adhe- sions and cytoskeletal or Rho GTPase functions [63, 64]. Lo- cal matrix stiffness can determine cell development, differen- tiation, and regeneration through adhesion complexes [64] with the actin–myosin cytoskeleton generating intrinsic con- tractile forces by “sensing” substrate properties via pre- stretching through actin stress fibers; linking integrin trans- membrane receptors to ECM in mechanosensation enables primary cells to alter their function in response to exogenous forces [65] or oxygen tension and local cell density [66]. Ini- tial attempts suggest the feasibility of creating 3D stiffness gradients in hydrogels [67] to re-differentiate chondrocytes. Osmotic Stimulation Human OA chondrocytes have a more positive membrane potential (i.e., −26±4 mV) than healthy cells and show reduced [Ca2+]o independent protein kinase C (PKC)α-mediated hyperpolarization upon hyperosmotic stimulation [105]. A precisely set balance between the activities of protein kinases and phosphoprotein phosphatases is crucial to regulating chondrogenesis and maintaining the chondro- cyte phenotype. All of the major protein kinase families, including protein kinase A (PKA), PKC, mitogen- activated protein kinase (MAPK), and CaMK, as well as all major protein phosphatases (PP1, PP2A, and PP2B) play fundamental roles in molecular regulation in chondrocytes [112]. These signaling pathways even- tually converge on targets that are involved in defining the chondrocyte phenotype, and they regulate cell shape, proliferation, differentiation, and gene expression (via transcriptional regulators such as Sox9, cAMP response element binding protein (CREB), and NFAT; see details below). Osmotic loading is known to modulate chondrocyte height, width, and volume in situ, and OP may modulate cell shape in accordance with the primary collagen fibril direction [106], as well as altering nuclear size and shape [107]. Interestingly, osmotic sensitivity of nuclear shape and volume appeared to be independent of the actin cyto- skeleton. While compression (and thus increased OP) re- duces the ECM, cellular, nuclear, rER, and mitochondrial volumes, the Golgi apparatus seems relatively resistant to intraorganelle water loss [108]. This may, at least partial- ly, explain some of the observed posttranscriptional ef- fects of OP [97]. The Ca2+-dependent serine/threonine phosphoprotein phosphatase calcineurin (Cn; also known as PP2B) has been identified as a potential target to improve the chondrocyte phenotype. The Cn inhibitor FK506 (also known as Tacroli- mus) increases the expression of chondrogenic markers during in vitro expansion in hypoosmotic culture medium [113]. Iso- lation and expansion of adult human articular chondrocytes in culture medium of physiologic osmolarity (i.e., 380 mOsm) improves chondrogenic marker gene expression and ECM production through NFAT5 [92]. Interestingly, FK506 within the range of 0.1 and 1000 ng/mL increased not only COL2A1 but also COL10A1 expression, while in human OA cells FK506 suppressed the osmolarity-induced COL10A1 expres- sion [93]. Generally, similar anabolic and anti-hypertrophic effects were observed in ex vivo cartilage explant cultures and non-OA chondrocytes. Similar data were reported with alternative Cn inhibitors (i.e., cyclosporine A, CsA) in human cells [114] and in the murine AT805-derived chondrogenic ATDC5 cell line [115], where FK506 increased PG content in a dose-dependent manner without elevating alkaline phos- phatase (ALP) activity. Osmotic Stimulation Chondrocytes in cartilage represent cells under pressures of different natures, like deformation, hydrostatic pressure, ex- tracellular ion composition (i.e., OP), and streaming potential (i.e., FF) [54]. Further, the concept is generally accepted that matrix turnover by chondrocytes is influenced by changes to the intracellular composition (e.g., cell volume, pH, and ionic content). The pericellular microenvironment functions in situ to mediate the chondrocyte (or chondron) responses to phys- icochemical changes associated with joint loading [83]. Dur- ing compression-induced changes in OP, the pericellular ma- trix exerts important functions through amplifying cell vol- ume changes [84]; such findings argue in favor of using chondrons, rather than isolated chondrocytes, for osmo- induced cartilage tissue engineering. Under standard FCS-containing expansion culture conditions, proliferation of human chondrocytes seems to be unhampered up to physiological osmolarity levels (i.e., ∼350–400 mOsm) [92, 93]. Proteomic analysis of serum-free expanded chondrocytes has confirmed a cut- off threshold of about 350 mOsm, above which cell cycle progression and proliferation appears compromised [94, 95]. Molecular Aspects The response to osmotic loading seems to depend on the nature of the osmotic stimulation and the chon- drocyte phenotype, which is related to passage number and pathological state [96]. Osmotic loading differentially regulates SOX9 and COL2A1 mRNA stability posttranscriptionally [97]. In nucleus pulposus cells, NFAT5 [98], together with intracel- lular Ca2+ [99] and MEK/extracellular signal-regulated kinase (ERK) signaling [100], control cell function, survival, and sul- fated GAG synthesis [101, 102]. Hyperosmotic stress induces About 15 years ago [11], Mow et al. described the some- times counter-intuitive effects of flow-induced compression of the ECM and hypothesized that this friction-drag effect is likely of major importance for fluid flow through the ECM. Changes in HP and OP are essential for chondrocyte physiol- ogy and useful for manipulating their metabolic function and phenotype [53]. Applying controlled HP to cartilage or Curr Rheumatol Rep (2015) 17: 22 Page 5 of 12 22 Regulation of Phosphatases by Chemo- and Biomechanics Calcineurin as a Potential Target Molecule volume changes and Ca2+ transients in chondrocytes by trans- membrane ion channels, phospholipids, and G-protein coupled pathways [103]. Pritchard et al. [104] found that IL-1α alters the normal volumetric and Ca2+ signaling response of porcine chondrocytes to OP through mechanisms involving F-actin remodeling and Rho GTPases. Osmotic Stimulation Clinical Relevance Chondrocyte shrinkage by raised hyperosmotic pressure (≥480 mOsm) may protect cells. While most cell-based chondral repair strategies aim at re-differentiation of routinely expanded, dedifferentiated chondrocytes, van der Windt et al. showed that dedif- ferentiation can be delayed by harvesting and expanding cells under elevated (i.e., physiological, 380 mOsm) os- molarity [92]. Interestingly, combining physiological os- molarity with inhibition of calcineurin activity can in- crease the expression of anabolic genes and suppress catabolic genes, as well as hypertrophic markers, in hu- man OA and “normal” chondrocytes [93] and may be a promising strategy for improving cell-based chondral defect repair. The clinical potential of applying osmolar- ity to improve the chondrocyte phenotype is hard to predict from present in vitro data, given the depth zone dependence of osmotic responses [109] and the current clinical practice of harvesting chondrocytes irrespective of their original zonal location. The exact mechanism underlying the effects of Cn inhibi- tion under different osmolarities is not yet understood, but Cn is known to induce FGF18, which can suppress noggin and facilitate BMP-related chondrogenesis-like effects [116]. This pathway may involve, among others, NFAT4-mediated induc- tion of BMP2 [117]. FK506, but not CsA, induces ATDC5 differentiation [118], suggesting that FK506 promotes chondrogenic differentiation, at least partly, by Cn- independent signaling routes. Since FK506 has been proven effective and safe as an anti-rheumatoid arthritis drug [119, 120], this approach may improve cell-based chondral repair strategies by interfering with adverse inflammatory or im- mune cell-mediated effects. The effects of OP, to a certain extent, also depend on the culture model: in alginate, higher proliferation rates, with diminished sulfated GAG production, were found at 280 mOsm [110]. Of note, the pHi is also osmolarity- dependent and its contribution to sulfated GAG produc- tion remains speculative. Finite element modeling showed that charged tissues (or synthetic matrices) always support larger loads than un- charged tissues. This load support derives from three sources: intrinsic matrix stiffness, HP, and OP [111]. 22 Page 6 of 12 Curr Rheumatol Rep (2015) 17: 22 regulators of NFAT activity in chondrocytes have been iden- tified to date. Not surprisingly, pharmacological inhibition of GSK-3β signaling increases cartilage degeneration in rats [132], while FK506 in the same species protects the collage- nous ECM of articular cartilage against osteoarthritic wear- and-tear erosion [133]. Cn–NFAT Signaling in Cartilage Pathologies Cn regulates the activity of NFAT family members in a spe- cific and Ca2+/calmodulin-dependent manner [121]. For a de- tailed overview of this vertebrate-specific phosphatase in chondrocyte physiology, the reader is referred elsewhere [112]. NFATs have arisen from an ancient precursor with a Rel domain, and Cn–NFAT signaling may be an essential pro- cess during vertebrate development [122]. After the original study by Glimcher’s group had shown that all four NFATc1–4 proteins are expressed in the cartilage [123], Greenblatt and colleagues recently expanded the earlier studies of Ranger et al. and Wang et al. [123, 124] by demon- strating essential functions of NFATs (i.e., NFATc1 and NFATc2) in articular cartilage homeostasis [125]. NFATs have the potential to link many extracellular signals to the nuclear transcriptional machinery [126]. Overall, in vivo and in vitro data from mice and humans strongly suggest a dynamic control of NFATc2 expression in articular cartilage and a crucial role of NFAT family members in cartilage homeostasis and joint health. NFATs might be involved in distinguishing articular from growth plate chondrocytes, the origins of which are still not understood completely [136]. Novel small molecular compounds with higher specificities may make NFATs potential therapeutic targets for cartilage regenerative medicine and anti- osteoarthritic treatment regimes. Greenblatt’s cartilage-specific NFATc1 and NFATc2 dou- ble mutant mice showed accelerated cartilage degeneration and expression of OA markers, such as increased expression of genes encoding proteases involved in ECM degradation such as MMP13, ADAMTS-5, and hypertrophic chondrocyte markers, including COL10A1, and reduced expression of Sox9 and PRG4, encoding lubricin. Intriguingly, NFATc1 pro- tein expression is restricted to the superficial zone of articular cartilage, and its mRNA expression is reduced around carti- lage lesions in human osteoarthritic patients [125]. A number of earlier in vitro studies suggest that NFATsignaling may also induce catabolic genes such as ADAMTS4 and 9 in chondrogenic cells [127, 128], which are findings contradic- tory to the protective roles observed in vivo. While NFATc3 seems less important for cartilage homeostasis, it may still be relevant in chondrogenesis [117]. Most notably, multiple path- ways co-regulate the subcellular localization of the four Ca2+- dependent NFAT proteins (NFATc1–4). In contrast, osmotic stress, rather than Ca2+ signaling primarily regulates the more distantly related fifth family member NFAT5, as discussed above. Osmotic Stimulation At present, it is not clear whether these effects are due to altered NFAT activity or other pathways affected by the inhibitors of Cn (CsA or FK506) or GSK (GIN). Earlier studies suggest a link between osmolarity- induced signaling pathways such as MAPK or Ca2+ signaling and Cn–NFAT signaling, which may be integrated through NFAT5. NFAT activity and expression in chondrocytes in vitro seems also to be dependent on both Notch and Wnt5a signaling, at least in growth plate chondrocytes [134, 135], and its relevance for articular chondrocytes, especially in vivo, remains to be shown. Cn–NFAT Signaling in Cartilage Pathologies The Role of PKA and PP2A in Mechanical Stimulation and the Chondrocyte Phenotype A main function of articular cartilage is to absorb shock during joint movements. Chondrocytes are sensitive to mechanical load, one of the most physiological stimuli that trigger the activation of key signaling molecules. Although appropriate mechanical stimuli are essential for limb development [137], differentiation of MSCs [138], and cartilage regeneration dur- ing OA [139], mechanotransduction pathways in differentiat- ing or mature chondrocytes are still incompletely understood [140•]. A recent study showed that lentiviral shRNA-mediated Nfatc2 knockdown in articular chondrocytes in vitro largely matches the in vivo phenotype and also upregulates pro- inflammatory cytokines [129]. In tracheal cartilage, CaV3.2 T-type Ca2+ channels may be involved in Cn–NFAT-depen- dent modulation of Sox9 expression [130••]. A previous study using other Cn inhibitors such as CsA had already suggested the participation of Ca2+ channels [131]. Although mechanosensitive ion channels, primary cil- ia, and the actin cytoskeleton have all been implicated as mechanosensors in chondrocytes, downstream path- ways are even less well characterized. Of the major signaling pathways, integrins and focal adhesion kinases (FAKs), the ERK, and the PI 3-kinase/Akt pathways have been reported [141]. It is of note that activation of the cAMP–PKA–CREB axis following mechanical stimuli has been documented in different models [142]. PP2A also plays a regulatory role in p38 MAPK acti- vation during cyclic strain [143]. Oscillating mechanical load promotes chondrogenesis and stimulates cartilage ECM production in chicken limb bud-derived micromass cultures, and the observed effects can be Pharmacological inhibition of Cn by FK506 promotes chondrogenic marker expression in dedifferentiated human adult chondrocytes, probably through upregulation of TGFβ1 [113]. NFAT activity seems tightly regulated by upstream sig- naling pathways: both activators (Cn) and inhibitors (e.g., GSK-3) can link a large number of mechanical and biochem- ical stimuli to this protein family, but few extracellular Curr Rheumatol Rep (2015) 17: 22 Page 7 of 12 22 Page 7 of 12 22 attributed to the activation of PKA/CREB–Sox9 signal- ing and concurrent inhibition of the PP2A pathway [144]. Here, we propose that increased PKA activity results in enhanced Sox9 and CREB phosphorylation and nuclear translocation; these in turn facilitate chondrogenic differentiation and ECM matrix produc- tion. Given that PP2A is a negative regulator of chon- drogenesis and balances the effects of PKA by dephos- phorylating many common targets, its reduced activity further enhances the chondrogenesis-promoting effects of mechanical stimulation in this model. Conclusions 2. Lesperance LM, Gray ML, Burstein D. Determination of fixed charge density in cartilage using nuclear magnetic resonance. J Orthop Res. 1992;10(1):1–13. The effects of electrical, magnetic, and mechanical stimu- lation on articular cartilage are summarized in Fig. 1. Data are accumulating regarding the molecular identity of the sensors and the mechanotransduction signaling apparatus in chondrocytes that convert the effects of external forces to cellular responses. Diverse stimuli have been shown to exert chondroprotective effects, but our current knowledge is still incomplete and a better understanding of the molec- ular identity and function of mechanotransduction path- ways is of crucial importance. It is very important to em- phasize that the mechanical properties of native cartilage, and thus the responsiveness of chondrocytes to external stimuli, vary widely and depend on joint location, depth in the tissue, sample orientation, species, and donor age. These differences have important implications for cell- based regenerative approaches and should be considered during data interpretation. Further research should aim at understanding which load-induced biophysical changes are most important for cartilage ECM regeneration and main- tenance of the chondrocyte phenotype to benefit functional cartilage tissue engineering. 3. Mow VCYGW, Hui CF. Structure and function of articular carti- lage and meniscus. Basic orthopaedic biomechanics and mechanobiology. Philadelphia (PA): Lippincott Williams & Wilkins; 2005. 4. Mow VC, Ateshian GA, Spilker RL. Biomechanics of diarthrodial joints: a review of twenty years of progress. J Biomech Eng. 1993;115(4B):460–7. 4. Mow VC, Ateshian GA, Spilker RL. Biomechanics of diarthrodial joints: a review of twenty years of progress. J Biomech Eng. 1993;115(4B):460–7. 5. Maroudas AI. Balance between swelling pressure and collagen tension in normal and degenerate cartilage. Nature. 1976;260(5554):808–9. 5. Maroudas AI. Balance between swelling pressure and collagen tension in normal and degenerate cartilage. Nature. 1976;260(5554):808–9. 6. Lai WM, Mow VC, Sun DD, Ateshian GA. On the electric poten- tials inside a charged soft hydrated biological tissue: streaming potential versus diffusion potential. J Biomech Eng. 2000;122(4):336–46. 7. Bassett CA, Pawluk RJ. Electrical behavior of cartilage during loading. Science. 1972;178(4064):982–3. 8. Buschmann MD, Grodzinsky AJ. A molecular model of proteoglycan-associated electrostatic forces in cartilage mechan- ics. J Biomech Eng. 1995;117(2):179–92. 9. Chen AC, Nguyen TT, Sah RL. Streaming potentials during the confined compression creep test of normal and proteoglycan- depleted cartilage. Ann Biomed Eng. 1997;25(2):269–77. 10. Mow VC, Guo XE. Mechano-electrochemical properties of artic- ular cartilage: their inhomogeneities and anisotropies. Annu Rev Biomed Eng. 2002;4:175–209. The Role of PKA and PP2A in Mechanical Stimulation and the Chondrocyte Phenotype Interestingly, previous data also indicated a direct interplay between PKA and PP2A during chondrogenesis [145] and strongly support the important role of reversible protein phosphorylation in establishing and maintaining the chondrocyte phenotype. induced activation of protein kinases and/or phosphatases may be responsible, at least partially, for these effects. induced activation of protein kinases and/or phosphatases may be responsible, at least partially, for these effects. Calcium Signaling Is an Essential Component in Mechanotransduction Pathways in Differentiating and Mature Chondrocytes Intracellular Ca2+ signaling and changes in cytosolic Ca2+ concentration are closely related to cell proliferation and dif- ferentiation in chondroprogenitor cells, and Ca2+ release from intracellular stores and influx through plasma membrane ion channels are key factors controlling chondrogenesis [149]. Various chondrocyte plasma membrane ion channels appear to be regulated by mechanical stimuli, such as the big con- ductance Ca2+-activated K+ channel (BK-like channel) [150] or the transient receptor potential vanilloid 4 (TRPV4) cation channel [151••]. Mechanical load-induced Ca2+ influx and subsequent alterations in Ca2+ signaling have been document- ed in chondrocytes upon both compressive loading and HP [152]. Furthermore, cyclic compression is known to modulate cartilage matrix synthesis and catabolism through an autocrine/paracrine purinergic pathway; compression- induced ATP release evokes Ca2+ transients via activation of P2X and P2Y receptors that cause a combination of extra- cellular Ca2+ influx and intracellular Ca2+ release in agarose- embedded chondrocytes [101]. How exactly Ca2+ signaling is coupled to mechanosensation in chondrocytes remains an open question. Serially passaged articular chondrocytes, deprived of their ECM, rapidly lose their characteristic phenotype. Signaling events that control the re-differentiation of dedifferentiated chondrocytes have only partially been analyzed. Chondrocyte re-differentiation in micromass cultures may be mediated by PKC-dependent ERK1/2 regulation, whereas chondrocyte de- differentiation is under a separate control by PKCα and ERK1/2 [146]. In a different study, p38 MAPK along with PKCα activity was reported to be essential for chondrocyte re-differentiation [147]. Since cyclic hydrostatic pressure upregulates cartilage-specific gene expression during re- differentiation of dedifferentiated bovine articular chondrocytes [148], one can speculate that mechanical load- Fig. 1 The effects of electrical, magnetic, and mechanical stimulation on articular cartilage Fig. References Papers of particular interest, published recently, have been highlighted as: • Of importance • Of importance •• Of major importance 1. Mow VC, Ratcliffe A, Poole AR. Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures. Biomaterials. 1992;13(2):67–97. The Role of PKA and PP2A in Mechanical Stimulation and the Chondrocyte Phenotype 1 The effects of electrical, magnetic, and mechanical stimulation on articular cartilage 22 Page 8 of 12 22 Page 8 of 12 Curr Rheumatol Rep (2015) 17: 22 Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. A promising candidate for a mechanosensory organelle on chondrocytes is the primary (non-motile) cilium, first identi- fied on articular chondrocytes almost 40 years ago [153]. Tis- sue compression during joint loading can lead to deformation of the cilium, which in turn may trigger signaling involved in mechanotransduction pathways. Indeed, various extracellular matrix receptors including integrins, as well as osmo- and mechanosensitive ion channels including TRPV4, are known to be present on its surface [140•]. In particular, the primary cilium is necessary for compression-induced ATP release and Ca2+ signaling via P2X and P2Y purinergic receptors, induc- ing aggrecan mRNA expression and sulfated GAG secretion in a 3D chondrocyte culture system [154]. These findings suggest that the primary cilium does not act as the initial mechanosensor in that model, leaving several open questions regarding its specific role in chondrocyte mechanosensation. 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. Conclusions 11. Mow VC, Wang CC, Hung CT. 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https://openalex.org/W2995323718	https://europepmc.org/articles/pmc7006024?pdf=render	English	null	Influenza activity prediction using meteorological factors in a warm temperate to subtropical transitional zone, Eastern China	Epidemiology and infection	2,019	cc-by	4,588	Author for correspondence: Author for correspondence: Wendong Liu, g , E-mail: jscdclwd@sina.cn; Xiang Huo, E-mail: Huox@foxmail.com Original Paper Cite this article: Liu W et al (2019). Influenza activity prediction using meteorological factors in a warm temperate to subtropical transitional zone, Eastern China. Epidemiology and Infection 147, e325, 1–8. https://doi.org/ 10.1017/S0950268819002140 1Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China and 2Jiangsu Meteorological Service Center, Nanjing, China Epidemiology and Infection Epidemiology and Infection Epidemiology and Infection Abstract Influenza activity is subject to environmental factors. Accurate forecasting of influenza epi- demics would permit timely and effective implementation of public health interventions, but it remains challenging. In this study, we aimed to develop random forest (RF) regression models including meterological factors to predict seasonal influenza activity in Jiangsu pro- vine, China. Coefficient of determination (R2) and mean absolute percentage error (MAPE) were employed to evaluate the models’ performance. Three RF models with optimum para- meters were constructed to predict influenza like illness (ILI) activity, influenza A and B (Flu-A and Flu-B) positive rates in Jiangsu. The models for Flu-B and ILI presented excellent performance with MAPEs <10%. The predicted values of the Flu-A model also matched the real trend very well, although its MAPE reached to 19.49% in the test set. The lagged depend- ent variables were vital predictors in each model. Seasonality was more pronounced in the models for ILI and Flu-A. The modification effects of the meteorological factors and their lagged terms on the prediction accuracy differed across the three models, while temperature always played an important role. Notably, atmospheric pressure made a major contribution to ILI and Flu-B forecasting. In brief, RF models performed well in influenza activity predic- tion. Impacts of meteorological factors on the predictive models for influenza activity are type-specific. Received: 6 August 2019 Revised: 21 October 2019 Accepted: 27 November 2019 Received: 6 August 2019 Revised: 21 October 2019 Accepted: 27 November 2019 Influenza activity prediction using meteorological factors in a warm temperate to subtropical transitional zone, Eastern China Wendong Liu1 , Qigang Dai1, Jing Bao2, Wenqi Shen1, Ying Wu1, Yingying Shi1, Ke Xu1, Jianli Hu1, Changjun Bao1 and Xiang Huo1 1Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China and 2Jiangsu Meteorological Service Center, Nanjing, China Introduction Seasonal influenza has always been a major public health problem [1, 2]. It annually causes tens of millions of respiratory illnesses and hundreds of thousand deaths world- wide [3]. An accurate forecast of influenza activity in advance based on predictive models is crucial for public health authorities to predict the seasonal fluctuation and facilitate key response actions [4, 5], such as public health surveillance, deployment of emergency sup- plies and hospital resource management. However, accurate prediction remains a great challenge. A number of statistical approaches have been employed and evaluated. Random forest (RF) regression model was suggested to have enhanced prediction ability over the autoregressive integrated moving average (ARIMA), the generalized linear auto- regressive moving average time series model [6, 7] in context of animal influenza activity prediction. It performed better in identifying independent factors associated with H1N1pdm influenza infections over boosted regression trees, conventional and penalised logistic regression [8]. g g Meteorology plays an important role in the varied seasaonal patterns of influenza in temperate, subtroptical and tropical regions. Influenza activity has been reported to peak during rainy seasons in tropical climates and during dry, cold months of winter in temperate climates. The impact of climate conditions on influenza A and B could be different [9]. © Jiangsu Provincial Center for Disease Control and Prevention 2019. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Influenza like illness (ILI) has been commonly used as the index of influenza activity worldwide [3–5], however, a number of respiratory pathogenicities, including parainfluenza, adenovirus and rhinovirus, could cause ILI and thus, influence ILI activity fluctuation [10]. Recently, the positive rate of influenza virus in surveillance samples has been considered a more reliable indicator of influenza activity [11,12]. distribution, and reproduction in any medium, provided the original work is properly cited. Jiangsu Province is situated in the middle east coast of China and is a transitional district of warm temperate zone to subtropical zone. Researches conducted in this region could deliver a more comprehensive understanding of climate impact on influenza activity. In this study, we aim to develop RF models to predict the ILI activity, positive rates of Flu-A and Flu-B, respect- ively, which has been rare in published studies. Key words: Forecast; influenza activity; meteorological factor; random forest model 2 2 Wendong Liu et al. in China is conducted 3], with sentinel sites the country. Data for i.e. body temperature oat) is reported to the China Influenza Surveillance Information System (CISIS) on a weekly basis. In Jiangsu, for each sentinel site, no less than 20 nasopharyngeal swabs are collected in a week by convenience samples of ILI cases before antiviral therapy. These specimens are routinely tested for influenza virus sub- types using real-time fluorescent quantitative polymerase chain reaction (PCR) assay and the results are reported to CISIS within 48 h. a virus Fig. 1. Temporal patterns of ILI activity and influenza virus positive rates in Jiangsu province, 2011–2016. China Influenza Surveillance Information System (CISIS) on a weekly basis. In Jiangsu, for each sentinel site, no less than 20 nasopharyngeal swabs are collected in a week by convenience samples of ILI cases before antiviral therapy. These specimens are routinely tested for influenza virus sub- types using real-time fluorescent quantitative polymerase chain reaction (PCR) assay and the results are reported to CISIS within 48 h. Data sources Surveillance of ILI and influenza virus in China is conducted through a national sentinel network [13], with sentinel sites covering 2.5% of all hospitals across the country. Data for patients fitting the definition of ILI (i.e. body temperature ⩾38 °C with a cough and/or a sore throat) is reported to the Surveillance of ILI and influenza virus in China is conducted through a national sentinel network [13], with sentinel sites covering 2.5% of all hospitals across the country. Data for patients fitting the definition of ILI (i.e. body temperature ⩾38 °C with a cough and/or a sore throat) is reported to the 3 Epidemiology and Infection 3 Table 1. Summary of weekly meteorological variables in Jiangsu province, 2011–2016 Variable Min P25 P50 Mena P75 Max AP (Pa) 998.6 1006.8 1015.7 1015.2 1022.7 1034.0 Mean_T (°C) −2.191 6.953 17.092 15.621 23.551 32.648 MAX_T (°C) 1.252 11.622 22.471 20.174 27.467 37.407 MIN_T (°C) −5.997 3.44 12.698 11.938 20.434 28.199 RH (%) 45.93 67.86 74.40 73.40 80.24 91.18 PR (mm) 0 3.335 11.648 21.296 29.987 159.657 SD (hour) 2.252 27.574 37.274 37.987 48.717 82.009 Table 1. Summary of weekly meteorological variables in Jiangsu province, 2011–2016 Table 2. Cross correlation between dependent variable and meteorological factors Dependent variable Lag Correlation coefficient AP Mean_T Max_T Min_T RH PR SD Weekly ILI% 0 −0.195* 0.112* 0.106 0.119* 0.104 0.159* −0.006 1 −0.172* 0.081 0.077 0.086 0.067 0.181* −0.023 2 −0.146* 0.051 0.047 0.055 0.053 0.153* −0.061 3 −0.126* 0.026 0.025 0.028 0.033 0.142* −0.072 4 −0.113* 0.006 0.008 0.005 0.033 0.143* −0.1 Weekly positive rate of Flu-A 0 0.193* −0.225* −0.225* −0.216* −0.051 −0.059 −0.006 1 0.181* −0.221* −0.222* −0.210* −0.06 −0.041 −0.024 2 0.161* −0.204* −0.210* −0.189* −0.05 −0.013 −0.051 3 0.127* −0.173* −0.182* −0.156* 0.002 0.024 −0.103 4 0.096 −0.139* −0.152* −0.120* 0.041 0.038 −0.131 Weekly positive rate of Flu-B 0 0.375* −0.459* −0.465* −0.454* −0.271* −0.206* −0.143* 1 0.427* −0.492* −0.496* −0.486* −0.278* −0.226* −0.142* 2 0.461* −0.516* −0.519* −0.510* −0.291* −0.236* −0.144* 3 0.495* −0.539* −0.538* −0.533* −0.303* −0.256* −0.129* 4 0.509* −0.547* −0.545* −0.541* −0.299* −0.266* −0.136* *statistically significant at 0.05. Table 2. Cross correlation between dependent variable and meteorological factors In this study, weekly data of ILI percentage in outpatients (ILI %) and influenza virus positive rate in Jiangsu during 2011–2016 were obtained from CISIS. Data sources The daily meteorological data were downloaded from China Meteorological Data Sharing Service System (http://cdc.cma.gov.cn) and aggregated into weekly data. These meteorological viriables include precipitation (PR), sun- shine duration (SD), relative humidity (RH), atmospheric pres- sure (AP), minimum temperature (MIN_T), mean temperature (MEAN_T) and maximum temperature (MAX_T). the tree predictor takes on numerical values as opposed to class labels used by the RF classifier. RF regression models take the aver- age of outputs produced by the trees as the final prediction. One of the most important features of RF is to calculate the variable’s importance, which measures the association between a given variable and the prediction accuracy. RF regression approach discussed in this study uses the decrease in accuracy to assess the variable’s importance. As suggested by previous stud- ies about the good prediction capacity, we explored RF method in human seasonal influenza activity analyses, testing its forecasting and independent influence factors identifying performance. Random forest Rf is an ensemble machine learning method proposed by Breiman [14]. RF creates multiple classification and regression trees, each trained on a bootstrap sample of the original training data with a randomly selected subset of input variables. There are two para- meters to choose when running a RF algorithm: the number of trees and the number of randomly selected variables. In regression, Model evaluation Data of 2011–2015 were split as a training set to fit the RF models, reserving 2016 as testing set to evaluate the predicting accuracy. Coefficient of determination (R2) and mean absolute percentage error (MAPE) were employed to evaluate the models’ 4 Wendong Liu et al. performance both in the model fitting stage and prospective fore- casting stage. They were calculated as follows: Fig. 2. Partial autocorrelation function of time series ILI percentage, positive rate of Flu A and positive rate of Flu B. R2 = (ˆyi −y)2 (yi −y)2 MAPE = 1 n n i=1 \|ˆyi −yi\| yi × 100% where yi means the ith observation, ˆyi means the ith predication, y means average of observations and n is the number of observations. RF model fitting and forecasting Three RF models with optimum parameters were finally con- structed to predict ILI activity, Flu-A and Flu-B positive rates in Jiangsu province, including 13, 23 and 39 predictors, respectively. The dependent variable of Flu-A had undergone a natural loga- rithmic transformation before the model fitting. See Table 3. General description More than 2 million ILI cases were reported to CISIS from the sentinel sites in Jiangsu province during the study period, with an average weekly ILI of 3.92%. Totally 146 236 throat swabs were sampled from the ILI cases. Influenza viruses were detected in 16 197 swabs through real time RT-PCR, reaching a general positive rate of 11.08%. According to the typing results, Flu-A and Flu-B accounted for 64.27% and 35.73% of all influenza posi- tive samples, reaching an average positive rate of 7.12% and 3.96%, respectively. Two peaks were observed in the ILI activity and the positive rate of Flu-A in each year, one occurred in winter and the other in summer. While the positive rate of Flu-B just showed a winter peak in a year (Fig. 1). The features of the meteorological variables were summarised in Table 1. Fig. 2. Partial autocorrelation function of time series ILI percentage, positive rate of Flu A and positive rate of Flu B. Statistics analysis Descriptive statistics was used to illustrate the temporal pattern of ILI% and the influenza virus positive rate. Time series analysis methods were employed to identify the autoregressive order [15] of the dependent variables (i.e. ILI%, positive rate of Flu A and positive rate of Flu B). Cross correlation is a measure of asso- ciation of a time series with another time series at different lags [16], which is essentially a univariate correlation method. In this study, cross correlation was used to determine the lag of cli- mate variable that was most significantly associated with depend- ent variables. All the analyses in this study were completed using R version 3.5.0. Particularly, cross-correlation analyses were com- pleted using the R package ‘TSA’. RF model fitting and forecasting were done in the R package‘randomForest’ [17]. Correlation analysis As shown in Table 2, AP and PR were significantly correlated with ILI% at lag 0–4. Mean_T and Min_T were also correlated with ILI % but with no lag effect. Max_T, RH and SD presented no relationship with ILI%. As to Flu-A, AP showed correlations at lag 0–3. The three temperature variables presented correlations at lag 0–4. All the meteorological factors were identified signifi- cant correlations with Flu-B at lag 0–4. The results of autocorrel- ation analysis were displayed in Fig. 2. ILI% presented autocorrelation at lag 1, while both Flu-A and Flu-B at lag 3. The performance of the models is summarised in Table 4 and the predicting results are displayed in Fig. 3. The models for Flu-B and ILI% presented excellent performance both in model fitting stage and prospective forecasting stage, with MAPEs less than 10%. The model for Flu-A presented much worse than the other two, with MAPE up to19.49% in the test set. Nevertheless, the pre- dicted values matched the real trend very well. 5 Epidemiology and Infection 5 Table 3. Predictors in different models Table 3. Predictors in different models Model lag ILI-P Flu-A Flu-B time AP Mean_T Max_T Min_T RH PR SD RF-ILI_P 0 √ √ √ √ √ √ 1 √ √ √ 2 √ √ 3 √ √ 4 √ √ RF-Flu_A 0 √ √ √ √ √ 1 √ √ √ √ √ 2 √ √ √ √ √ 3 √ √ √ √ √ 4 √ √ √ RF-Flu_B 0 √ √ √ √ √ √ √ √ 1 √ √ √ √ √ √ √ √ 2 √ √ √ √ √ √ √ √ 3 √ √ √ √ √ √ √ √ 4 √ √ √ √ √ √ √ Table 4. Performance evaluation of different random forest models Model R2 MAPE(%) Train Test Train Test RF-ILI_P 0.79 0.50 2.48 9.95 RF-Flu_A 0.89 0.0.82 11.24 19.49 RF-Flu_B 0.95 0.80 3.20 8.58 Table 4. Performance evaluation of different random forest models trend and seasonality of influenza activity change over time, so that the ARIMA model cannot always reach a satisfactory result. that the ARIMA model cannot always reach a satisfactory result. Substantial studies have proposed that influenza activity is climate-sensitive [21–23]. Climatic factors may influence the survival and spread of influenza viruses in the environment, the host susceptibility and exposure probability [24–26]. Correlation analysis The effects of meteorological factors on epidemics of ILI have attracted considerable interest recently. Sudarat Chadsuthi, et al. [27] fitted ARIMA model with temperature and RH as cov- ariates to forecast the incidence of influenza in Thailand. N’gattia1, et al. [28] also developed ARIMA with meteorological variable rainfall to forecast influenza transmission. But the pre- diction accuracy of these models was not good enough and the climate variables did not clearly optimise the models. In this study, we employed RF algorithm fitting models to predict influ- enza activity with meteorological factors in Jiangsu province, China. In contrast with previous studies, we constructed predict- ing models not only for ILI but also for the positive rates of influenza virus (i.e. flu-A and flu-B). All the models performed very well in our dataset. Based on them, we can comprehensively and systematically evaluate the influenza activity in the future, which has significant and practical meaning for influenza pre- vention and control. Given the good performance of RF in influ- enza prediction, the models we established could be used for influenza (sub)type-specific early warning and to evoke early intervention. The key meteorological factors identified could be used for publicity, to elevate the general population’s conscious- ness and engagement in influenza prevention. Variable importance In each model, the lagged dependent variable was the most important of all predictors. The time variable presented as important in the models for ILI and Flu-A. Most of the meteoro- logical factors and their lagged terms had the potential to improve the accuracy of the models to a certain degree, but their effects dif- fered across the three models. For ILI forecasting, the weekly MEAN_T, AP and one order lagged AP were more important than the rest. For Flu-A, the lagged temperature specific variables were relatively important. With regard to Flu-B, the lagged AP and MAX_T presented greater effects than the other meteoro- logical variables to improve the model accuracy. See Fig. 4. Fig. 3. Plot of observed and predicted values via different models. In this study, humidity and PR were not recognised as major meteorological factors related to ILI activity, positive rate of flu A and B, while the temperature was identified as the main driver. This is consistent with our previous study [31]. The present study also indicates that AP plays an important role in the activity of ILI and flu B. An increased influenza risk associated with rising AP was also reported in another subtropical region in China, using distributed lag nonlinear model [32]. Our study suggests that the selected meteorological variables contributed less to the fluctuations of ILI, flu A and B, compared with the effect of autocorrelation, which has been shown as the most important of independent variables. Monamele GC, et al. also supposed that meteorological parameters could only explain no more than 30% of the influenza activity variation [33]. Although our constructed RF models showed desirable predictive ability, especially for ILI and flu B, more meteorological factors, such as specific humidity and absolute humidity, and population- specific immunity level [8] are warranted to be evaluated to improve the prediction of type/subtype-specific activity [34]. Conclusion RF model is a good method to predict the influenza activity. Three RF models were constructed to predict the positive rate of influenza viruses and ILI incidence and performed very well. The autocorrelation and seasonal variation contained in the data of the dependent variables are crucial for the prediction models. Meanwhile, the effects of meteorological factors and cumulative effects over a period of time were combined to improve the models. Further researches are warranted to explore RF model with meteorological factors as well as other variables and it has the potential to be a useful tool for predicting other major infectious diseases. Fig. 4. Variable importance in random forest regression models (just displaying the top 10 variables). Acknowledgements. This work was supported by Jiangsu Provincial Major Science & Technology Demonstration Project (No. BE2017749), Jiangsu Provincial natural science foundation (No. BK20151595), Jiangsu Provincial Medical Youth Talent (No. QNRC2016542, QNRC2016539), Preventive medicine research program (Y2018074) and Key Medical Discipline of Epidemiology (No. ZDXK A2016008). flu-B) in the previous weeks were more important than meteoro- logical factors in the models. It suggests that these models took advantage of the autocorrelation of the dependent variables. The influenza activity in Jiangsu province presented obvious seasonality which is a critical feature to fit predicting model. However, RF is unable to learn the seasonal patterns because of randomly selecting samples for each tree. In this study, we introduced a time variable into the models to fit the seasonal variance of ILI and positive rates of influenza viruses. The importance analysis shows that it played a significant role to improve the models. This strategy is worthy of flu-B) in the previous weeks were more important than meteoro- logical factors in the models. It suggests that these models took advantage of the autocorrelation of the dependent variables. The influenza activity in Jiangsu province presented obvious seasonality which is a critical feature to fit predicting model. However, RF is unable to learn the seasonal patterns because of randomly selecting samples for each tree. In this study, we introduced a time variable into the models to fit the seasonal variance of ILI and positive rates of influenza viruses. The importance analysis shows that it played a significant role to improve the models. This strategy is worthy of Author contributions. WL, HX conceived and designed the experiments. WL, HX, JH, CB performed the experiments. WL, JB, KX, QD analysed the data. Discussion Forecasting of influenza activity in human populations is crucial for influenza prevention and control [4]. Many methods have been introduced for this purpose. As a conventional univariate model, ARIMA technique has been commonly used to forecast seasonal influenza surveillance at national, regional and local levels [18–20]. ARIMA model is virtually a linear method. It can achieve good predication when the variation contained in the data is relatively stable. In practice, however, the long-term Similar to many other members of the machine learning family such as artificial neural networks, RF model cannot explain the association between risk factors and influenza activity. But RF can assess the importance of each variable on the accuracy of pre- diction [14, 29], which is essential to optimise the model and may provide clues for the further study of influenza risk factors. In this study, we found that the lagged dependent variables (i.e. the pro- portion of ILI in the outpatients and positive rates of flu-A and 6 6 Wendong Liu et al. Wendong Liu et al. icted values via different Fig. 4. Variable importance in random forest regression models (just displaying the top 10 variables). Epidemiology and Infection 7 Epidemiology and Infection reference when fitting the similar RF models. Compared with other multivariate predicting methods [27, 28], RF is not subject to multicollinearity, mainly due to randomly selecting variables for each tree in RF [29, 30]. In this study, we selected predictor variables through cross-correlation analysis. The meteorological factors and their lagged terms were incorporated into the models so long as they were identified to be significantly correlated with the dependent variables. 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https://openalex.org/W2765950247	https://researchrepository.wvu.edu/context/faculty_publications/article/2864/viewcontent/1532360.pdf	English	null	Combined Effects of Sustained Loads and Wet-Dry Cycles on Durability of Glass Fiber Reinforced Polymer Composites	Advances in materials science and engineering	2,017	cc-by	8,523	Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Combined Effects of Sustained Loads and Wet-Dry Cycles on Combined Effects of Sustained Loads and Wet-Dry Cycles on Durability of Glass Fiber Reinforced Polymer Composites Durability of Glass Fiber Reinforced Polymer Composites Mengting Li Nanjing Tech University This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/ 1941 Digital Commons Citation Digital Commons Citation Li, Mengting; Wang, Jun; Liu, Weiqing; Liang, Ruifeng; GangaRao, Hota; and Li, Yang, "Combined Effects of Sustained Loads and Wet-Dry Cycles on Durability of Glass Fiber Reinforced Polymer Composites" (2017). Faculty & Staff Scholarship. 1941. h h d f l bl https://researchrepository.wvu.edu/faculty_publications/1941 This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted for inclusion in Faculty & Staff Scholarship by an authorized administrator of The Research Repository @ WVU. For more information, please contact researchrepository@mail.wvu.edu. Authors Authors Mengting Li, Jun Wang, Weiqing Liu, Ruifeng Liang, Hota GangaRao, and Yang Li This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/ 1941 Authors Authors Mengting Li, Jun Wang, Weiqing Liu, Ruifeng Liang, Hota GangaRao, and Yang Li Research Article Combined Effects of Sustained Loads and Wet-Dry Cycles on Durability of Glass Fiber Reinforced Polymer Composites Mengting Li,1 Jun Wang,1 Weiqing Liu,1 Ruifeng Liang,2 Hota GangaRao,2 and Yang Li 1College of Civil Engineering, Nanjing Tech University, Nanjing 211816, China 2Department of Civil and Environmental Engineering, West Virginia University, Morgantown, WV, USA 3Jiangsu Wei Xin Engineering Consultants Ltd., Nanjing, China Mengting Li,1 Jun Wang,1 Weiqing Liu,1 Ruifeng Liang,2 Hota GangaRao,2 and 1College of Civil Engineering, Nanjing Tech University, Nanjing 211816, China 2Department of Civil and Environmental Engineering, West Virginia University, Morgantown, WV, USA 3Jiangsu Wei Xin Engineering Consultants Ltd., Nanjing, China 1College of Civil Engineering, Nanjing Tech University, Nanjing 211816, China 2Department of Civil and Environmental Engineering, West Virginia University, Morgantown, WV, USA 3Jiangsu Wei Xin Engineering Consultants Ltd., Nanjing, China Correspondence should be addressed to Jun Wang; wangjun3312@njtech.edu.cn and Weiqing Liu; wqliu@njtech.edu.cn Received 20 June 2017; Accepted 11 September 2017; Published 23 October 2017 Academic Editor: Fabrizio Sarasini Copyright © 2017 Mengting Li 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. This paper deals with durability of glass fiber reinforced polymer (GFRP) composites under the combined effects of sustained tensile loads and wet-dry (WD) cycles. Two different solutions (distilled water and saltwater) were used to imitate the freshwater and marine environments, respectively. Tensile properties of the unconditioned and conditioned specimens were measured to study the durability of GFRP composites under these 2 effects. The response indicated that both tensile strength and elastic modulus increased initially upon WD cycles, which was attributed to both the postcuring of resin and the sustained tensile stress allowing for fastec cure. Further exposure to WD cycles in distilled water or saltwater led to a steady decrease in tensile strength and modulus. WD cycles of saltwater and distilled water have similar effects on the degradation of the tensile properties for unstressed specimens. However, the elastic modulus and elongation at rupture of stressed specimens under WD cycles of saltwater decreased more than those specimens under WD cycles of distilled water. Moreover, increase of sustained loads led to a decrease in tensile strength. Based on Arrhenius method, a prediction model which accounted for the effects of postcure processes was developed. The predicted results of tensile strength and elastic modulus agree well with those obtained from the experiments. Authors Authors Hindawi Advances in Materials Science and Engineering Volume 2017, Article ID 1532360, 14 pages https://doi.org/10.1155/2017/1532360 Hindawi 1. Introduction In recent decades, several researchers have investigated durability issues of FRP reinforced concrete (RC) structures subjected to wet-dry (WD) cycles. Soudki et al. [2] studied the behavior of CFRP strengthened RC beams subjected to WD cycles with 3% NaCl. Their test results displayed that the critical chloride levels reached the reinforced steel bars after 200 WD cycles and visible cracks and stains appeared after 300 WD cycles. Belarbi and Bae [3] evaluated the combined effects of freeze-thaw (FT) cycles and WD cycles on the long-term behavior of RC columns strengthened with CFRP and GFRP sheets. Their studies indicated that, after 300 FT cycles, the failure load and axial stiffness of both CFRP and GFRP strengthened RC columns increased slightly due to the matrix hardening effects at extremely low temperatures. The combined FT and WD cycles have insignificant effects on mechanical properties of CFRP wrapped RC columns, while the failure loads of GFRP wrapped RC columns decreased remarkably. These results are in good agreement The use of fiber reinforced polymer (FRP) composites in the construction of waterfront and marine infrastructures has been increasing rapidly, due to their advantage of light weight, high tensile strength, and better reinforce to aqueous corro- sion. Because of economic benefits in construction, glass fiber reinforced polymer (GFRP) composites are becoming preva- lent in civil engineering structures. FRP structures are usually exposed to harsh in-service environment, such as moisture, corrosive media, sustained stress, temperature, and ultravi- olet radiation. Cyclic moisture effect near the splash-zone of marine structures is one of the important environmental factors to deteriorate the properties and behavior of polymer matrix composites [1]. Moreover, the environmental attack is combined with sustained loading prone to aggravate the deterioration of GFRP. Therefore, better understanding of the long-term behavior of GFRP under the combined effects of load and moisture cycles is helpful to promote its applications. Advances in Materials Science and Engineering 2 with former findings reported by Toutanji and EI-Korchi [4]. Yin et al. [5] investigated the mechanical properties and durability of RC columns strengthened by hybrid carbon and E-glass fiber/epoxy textile. It showed that, under the coupled effects of WD cycles and sustained stress, the bearing capacity and ductility of the columns and the reinforcement effects of textile decreased more rapidly as the sustained compression stress increased. Moreover, exposure to chloride WD cycles generated corrosive products (C3⋅CaCl2⋅10H2O) on the interface between textile and concrete. 1. Introduction strength and ductility after 400 FT and 400 WT cycles. Chen et al. [19] evaluated the durability of FRP reinforcing bars for concrete structures from accelerated aging tests. It showed that continuous immersion in solutions with a pH value of 13.6 resulted in greater degradation than exposure to WD cycling for GFRP bars, while FT cycles had insignificant effects on the strength of GFRP bars. f g Most of FRP structures are designed to resist some level of sustained load in-service engineering. The combination of environmental attacks with sustained stress can aggravate the loss of mechanical properties of FRP composites [20, 21]. Myers et al. [22] investigated the coupled effects of environmental attacks (FT cycles, moisture, high temperature cycling, and indirect ultraviolet radiation exposure) and sustained loads on the performance of interface between FRP and concrete. It is observed that the bond property of specimens under sustained load (40% of ultimate load) degraded greater than that of unstressed specimens in the same environment. Mahato et al. [23] studied the tensile behavior of GFRP composites under the coupled effects of temperature and loads. The tensile strength of the GFRP composites was observed to increase with the enhancement of loading speeds, and exposure in different temperature environments (25∘C to 110∘C) resulted in little change in glass transition temperatures (𝑇𝑔). The authors [24] investigated GFRP durability under combined effects of moisture and sustained loads and found that the tensile properties of GFRP decreased significantly as the sustained loading increased under salt water for 360 days, whereas GFRP specimens with and without sustained loads had little changes in tensile properties under tap water.f One of the major challenges to be addressed for FRP wrapped concrete is the long-term durability of externally bonded FRP [6, 7]. A number of experimental programs have been conducted to examine the behavior of FRP- concrete interface subjected to WD cycles. Silva and Biscaia [8] investigated the durability of RC beams reinforced with GFRP strips under different conditions (temperature cycles, WD cycles, complete immersion in water, and salt fogging cycles, for up to 1000 h). Their test results showed that the temperature cycles (−10 to +10∘C) were the most severe condition on the bending strength, followed by the salt fog cycles, while total immersion in water was the most severe condition on the pull-off strength, followed by WD cycles and salt fogging cycles. 1. Introduction For CFRP bonded concrete columns, WD cycles (one-week wetting at 95% RH and 30–32∘C followed by one-week drying at 20–23∘C) were the most severe condition on fracture energy compared with temperature cycles (30 to 40∘C) and outdoor environment [9]. Kim et al. [10, 11] investigated the interfacial behavior of CFRP composite sheets bonded to concrete blocks subjected to freezing-wet-dry cycles and WD cycles, respectively. The results showed that the effects of freezing-wet-dry cycles were more detrimental than those of WD cycles on the behavior of CFRP-concrete interface, especially when the temperature was below −20∘C. Biscaia et al. [12] examined the interface of GFRP and concrete subjected to salt fog cycles and WD cycles with saltwater. A 27% reduction of cohesion was found after 416 salt fog cycles and 8% reduction of the friction angle was found after 60 WD cycles due to the water attack at the interface. Wang and Amidi [13] applied environment-assisted subcritical debonding testing (EASD) program to evaluate the long-term durability of the CFRP-to-concrete interface subjected to the coupled effects of mechanical load, humidity, and temperature. They found that silane coupling agent could improve the durability of the CFRP-to-concrete interface due to introducing covalent bonds between the epoxy and concrete. Lipatov et al. [14] reported that the adding zirconia to basalt fibers contributed to improving the durability of fibers in alkali solution, and basalt fibers with 5.7 wt% ZrO2 had the best alkali resistance properties. Although many focused on single or coupled effects of environmental attacks under sustained loads in several studies, the durability of FRP composites under the combined effects of WD cycles and sustained loads has received scant attention. The coupled effects cannot be simplified by simply superposing the responses under different environmental attacks, and the mechanisms of degradation under single environmental attack are far different when compared with those under combined environmental attacks. This paper is concerned with investigating the long-term behavior of GFRP composites subjected to the combined effects of tensile loads and WD cycles. Microstructural and physical observations were performed to examine the conditioned and unconditioned samples. Moreover, a theoretical model was developed to predict the tensile properties of GFRP composites based on Arrhenius method. Advances in Materials Science and Engineering specimen was stressed to failure. The failure load was used to calculate the tensile strength of the GFRP composites in MPa. 2.4. Microstructural Observations. Electron microscopy (EM) observations and image analysis were performed to examine microstructure of specimens before and after conditions with DM4000M (Leica, Germany). The samples included uncon- ditioned specimens and conditioned specimens after 60 and 180 cycles under different sustained loads. All specimens were first cut and polished before observations. These observations were made on surfaces and sections parallel to the fiber layers for potential degradation of the glass fibers, resin, or interfaces. 2.3. Tensile Tests. After conditioning, tensile tests were con- ducted, in accordance with ASTM D638-10 [25] to collect the tensile strength, elastic modulus, and tensile elongation at the rupture of GFRP composites under different cycles. A universal test machine with 50 kN capacity was used for all specimens, as shown in Figure 2. The tensile load was applied through a stiff steel clamp and was recorded via a load cell mounted directly above the top steel clamp. Before testing, the average cross-sectional area of each specimen was measured. The strain values of the specimens were measured by an extensometer with 25.4 mm gage length which was con- nected to the middle of the specimens. Each specimen was stressed to about 50% of the tensile strength to obtain the elas- tic modulus, and then the extensometer was removed before 2. Experimental Program 2.1. Materials. The test specimens were reinforced with bidirection (0∘/90∘) E-glass plain woven fabric provided by Sinoma Nanjing Fiberglass Research & Design Institute. The fabric had an average thickness of 0.5 mm and a unit weight of 500 g/m2. Isophthalic polyester resin (Synolite 0593-I-3) supplied by DSM China was used as matrix. 3 wt% cyclo- hexanone peroxide and 2 wt% cobalt isocaprylate-styrene were used as the initiator and acceleration employed for isophthalic polyester resin curing. With the development of novel FRP structural members in recent years [15], some researchers start to pay attention to the durability of new FRP construction [16, 17]. Toutanji and Saafi [18] proposed a new hybrid concrete column consisting of an exterior PVC-FRP shell with a concrete core. The com- pressive test showed that the PVC-CFRP confined concrete columns performed well when subjected to FT and WD cycles, whereas the PVC-GFRP and PVC-AFRP confined concrete columns experienced significant reductions in both Advances in Materials Science and Engineering Advances in Materials Science and Engineering Advances in Materials Science and Engineering 3 Figure 2: Tensile test set-up. Figure 2: Tensile test set-up. Water inlet Pump Test specimens Pump Reservoir Water outlet Steel frame Load Figure 1: Schematic of the wet-dry exposure set-up. Figure 1: Schematic of the wet-dry exposure set-up. Figure 2: Tensile test set-up. Figure 2: Tensile test set-up. A flat 1 m × 2 m panel with a thickness of 2 mm was fabricated using resin infusion under flexible tooling (RIFT2) method and cut to coupons of 250 mm × 20 mm × 2 mm size. In addition, a surface layer of resin was applied to seal the specimens. The fiber volume fraction for the GFRP composites was about 60%. The specimens were cured at room temperature for 3 days, and then were cured in an oven at 60∘C for 3 hours. Table 1: Mechanical properties of GFRP under WD cycles. Table 1: Mechanical properties of GFRP under WD cycles. Specimens WD cycles Tensile strength (MPa) Elastic modulus (GPa) Elongation (mm) WWD0 0 416.75 24.70 1.813 15 457.40 26.77 1.003 30 448.47 26.95 1.123 60 431.72 25.87 0.834 90 420.36 25.43 0.725 180 410.85 23.89 0.534 360 377.67 20.86 0.515 SWD0 0 416.75 24.70 1.813 15 480.32 26.60 1.370 30 442.68 27.08 1.014 60 436.58 26.57 0.739 90 422.58 25.74 0.452 180 411.04 24.42 0.474 360 368.66 21.85 0.467 2.2. Apparatus. Figure 1 shows the environmental exposure set-up. The specimens were exposed to two environments: (1) WD cycles of distilled water; (2) WD cycles of saltwater with a salinity content of about 3.5%. Each cycle consisted of full immersion in water for 12 hours followed by air drying for 12 hours at room temperature. The numbers of cycles for the GFRP composites were 15, 30, 60, 90, 180, and 360, respectively. The applied stresses chosen for the specimens were 0%, 20%, and 40% of the tensile strength of the GFRP composites, respectively. Three duplicate specimens were connected in series and were tensioned by the weights of concrete. Both ends of the specimens were reinforced by 50 mm × 20 mm × 5 mm aluminum tab attachments. The water used to immerse the specimens was controlled by pumps. In the drying condition, the extra water was drawn back into the reservoir, while in the wetting condition, the water was poured into the specimen containers. 3. Results and Discussion 3.1. Specimens without Sustained Load. Tensile tests were conducted on GFRP composites subjected to 0, 15, 60, 90, 180, and 360 WD cycles of both distilled water and saltwater. Table 1 shows the mechanical properties of GFRP under WD Advances in Materials Science and Engineering 4 4 Advances in Materials Science and Engineering 200 300 400 500 600 0 60 120 180 240 300 360 Cycles WWD0 SWD0 Tensile strength (MPa) (a) 15 20 25 30 35 0 60 120 180 240 300 360 Cycles WWD0 SWD0 Elastic modulus (GPa) (b) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0 60 120 180 240 300 360 Cycles Elongation at rupture (%) WWD0 SWD0 (c) Figure 3: Mechanical properties of unstressed specimens exposed to WD cycles: (a) tensile strength; (b) elastic modulus; (c) elongation at rupture. Note. WWD0 is unstressed specimen exposed to WD cycles of distilled water; and SWD0 is unstressed specimen exposed to WD cycles of saltwater. 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) 200 300 400 500 0 60 120 180 240 300 360 Cycles WWD0 SWD0 Tensile strength (MPa) (a) 15 20 25 30 0 60 120 180 240 300 360 Cycles WWD0 SWD0 Elastic modulus (GPa) (b) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0 60 120 180 240 300 360 Cycles Elongation at rupture (%) WWD0 Elastic modulus (GPa) SWD0 (a) SWD0 (b) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0 60 120 180 240 300 360 Cycles Elongation at rupture (%) (c) Figure 3: Mechanical properties of unstressed specimens exposed to WD cycles: (a) tensile strength; (b) elastic modulus; (c) elongation at rupture. Note. WWD0 is unstressed specimen exposed to WD cycles of distilled water; and SWD0 is unstressed specimen exposed to WD cycles of saltwater. of tensile strength is 9% and 12% for WWD0 and SWD0, respectively, and the loss of modulus is 16% and 12% for WWD0 and SWD0, respectively, as shown in Figures 3(a) and 3(b). However, the elongation at rupture decreased initially after 90 cycles and almost remained unchanged from 90 cycles to 360 cycles. After 360 cycles, the loss of elongation at rupture is 72% and 74% for WWD0 and SWD0, respectively. 3. Results and Discussion The durability of unstressed specimens under WD cycles of distilled water has negligible differences with that of specimens under WD cycles of saltwater. The sharp decrease of elongation at rupture indicates that GFRP composites become brittle in exposure of WD cycles. cycles. The reduction in the tensile strength, modulus, and elongation at rupture of aged specimens versus the number of cycles is presented in Figure 3. Both the tensile strength and elastic modulus have an initial increase after 15 WD cycles, followed by a continued decline with the increasing number of cycles. Although the specimens were cured at room temperature for 3 days and then cured in an oven at 60∘C for 3 hours, the resin was not polymerized completely. Immersion in water led to the occurrence of postcuring of polymer, yielding an initial increase in tensile strength and modulus [24, 26, 27]. The differential scanning calorimetry (DSC) measurement of the identical samples confirmed that the resin partially cured before conditions [24]. Micro observations were conducted to examine the physical damage of GFRP composites due to exposure to WD cycles. Figure 4 shows the typical micrographs of the Further exposure to WD cycles led to a steady decrease in tensile strength and modulus. After 360 cycles, the loss Advances in Materials Science and Engineering 5 (a) (b) (c) Figure 4: Micrographs of the surfaces (×50) of the unstressed specimens: (a) unconditioned specimen; (b) SWD0 after 60 WD cycles; and (c) WWD0 after 180 WD cycles. (b) (a) (b) (a) (c) (c) Figure 4: Micrographs of the surfaces (×50) of the unstressed specimens: (a) unconditioned specimen; (b) SWD0 after 60 WD cycles; and (c) WWD0 after 180 WD cycles. (b) (a) (a) (b) (c) (d) Figure 5: Micrographs of the longitudinal section (×50) of the unstressed specimens: (a) unconditioned specimen; (b) WWD0 after 60 WD cycles; (c) SWD0 after 60 WD cycles; and (d) WWD0 after 180 WD cycles. (a) (b) (c) (d) (c) (d) Figure 5: Micrographs of the longitudinal section (×50) of the unstressed specimens: (a) unconditioned specimen; (b) WWD0 after 60 WD cycles; (c) SWD0 after 60 WD cycles; and (d) WWD0 after 180 WD cycles. Advances in Materials Science and Engineering Advances in Materials Science and Engineering 6 6 Table 2: Mechanical properties of GFRP under the combined effects of sustained loads and WD cycles. 3.2. Specimens with Sustained Loads. Table 2 shows the mechanical properties of GFRP under combined effects of 3. Results and Discussion Specimens Load level WD cycles Tensile strength (MPa) Elastic modulus (GPa) Elongation (mm) WWD2 20% 0 416.75 24.70 1.813 15 440.85 26.87 0.958 30 487.13 27.17 1.235 60 508.28 28.10 0.929 90 431.74 24.56 0.684 180 403.64 22.65 0.507 360 345.90 20.61 0.484 WWD4 40% 0 416.75 24.70 1.813 15 435.91 25.89 1.258 30 474.93 28.27 1.017 60 532.71 29.75 0.873 90 442.15 24.00 0.557 180 390.90 22.05 0.485 360 329.12 19.59 0.443 SWD2 20% 0 416.75 24.70 1.813 15 470.66 24.29 0.703 30 478.37 25.10 1.055 60 499.34 27.36 0.980 90 477.48 26.85 0.796 180 398.63 22.28 0.505 360 347.23 19.89 0.486 SWD4 40% 0 416.75 24.70 1.813 15 478.71 25.87 0.942 30 493.56 27.87 1.071 60 534.80 28.56 0.971 90 465.53 25.35 0.654 180 390.89 21.98 0.431 360 328.69 18.90 0.423 Table 2: Mechanical properties of GFRP under the combined effects of sustained loads and WD cycles. surfaces of unconditioned and conditioned specimens. It is observed that the void content of the polyester resin increases gradually with the number of WD cycles in both distilled water and saltwater. Voids and porosities not only lead to water diffusion but also lead to stress concentration. Once water molecules diffuse into a polyester matrix, they would make hydrogen-bond with the cross-linked polymer at the polar ester linkages [28]. As shown in Figure 5, hydrolysis of isophthalic polyester resins occurred due to exposure to WD cycles. Moisture absorption test of the identical samples showed that the moisture uptake content 𝑀𝑡decreased with the immersion time after 𝑀𝑡reached its peak value, which confirmed the hydrolysis of isophthalic polyester resins [24]. Moreover, chemical reaction between glass fibers and water usually cause alkali elements to leach out [29]. In addition, for specimens exposed to WD cycles of saltwater, the spatial distribution of Cl from NaCl correlates with that of Si from the E-glass fibers, yielding the aggravation of deterioration in tensile property of GFRP composites. 3.2. Specimens with Sustained Loads. 3. Results and Discussion Table 2 shows the mechanical properties of GFRP under combined effects of Advances in Materials Science and Engineering 7 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles WWD0 WWD2 WWD4 Tensile strength (MPa) (a) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles SWD0 SWD2 SWD4 Tensile strength (MPa) (b) 0 60 120 180 240 300 360 Cycles 0 100 200 300 400 500 600 WWD2 SWD2 Tensile strength (MPa) (c) 0 60 120 180 240 300 360 Cycles 0 100 200 300 400 500 600 WWD4 SWD4 Tensile strength (MPa) (d) Figure 6: Tensile strength of stressed specimens (a) subjected to WD cycles of distilled water and different loads; (b) subjected to WD cycles of saltwater and different loads; (c) subjected to WD cycles and 20% ultimate tensile strength; (d) subjected to WD cycles and 40% ultimate tensile strength. Note. WWD2 and WWD4 are specimens exposed to WD cycles of distilled water and subjected to 20% and 40% ultimate tensile strength, respectively, and SWD2 and SWD4 are specimens exposed to WD cycles of saltwater and subjected to 20% and 40% ultimate tensile strength, respectively. 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) Tensile strength (MPa) Tensile strength (MPa) ( ) 0 60 120 180 240 300 360 Cycles 0 100 200 300 400 500 600 Tensile strength (MPa) 0 60 120 180 240 300 360 Cycles 0 100 200 300 400 500 600 Tensile strength (MPa) Tensile strength (MPa) Tensile strength (MPa) (d) (c) Figure 6: Tensile strength of stressed specimens (a) subjected to WD cycles of distilled water and different loads; (b) subjected to WD cycles of saltwater and different loads; (c) subjected to WD cycles and 20% ultimate tensile strength; (d) subjected to WD cycles and 40% ultimate tensile strength. Note. WWD2 and WWD4 are specimens exposed to WD cycles of distilled water and subjected to 20% and 40% ultimate tensile strength, respectively, and SWD2 and SWD4 are specimens exposed to WD cycles of saltwater and subjected to 20% and 40% ultimate tensile strength, respectively. specimens without sustained loads, which may be attributed to the combined effects of postcuring of resin and the sustained tensile stress. 3. Results and Discussion Similar behavior was also observed by Abdel-Magid et al. [30]. The tensile stress aligns the fibers in the stress direction and endows the material with more efficiency in carrying the load after short period of prestress. From 60 cycles to 360 cycles, the tensile strength and elastic modulus of stressed specimens decreased steadily. After 360 cycles, the tensile strength decreased by 17%, 17%, 21%, and 21% for WWD2, SWD2, WWD4, and SWD4, sustained loads and WD cycles. The specimens with sustained loads have an initial increase in both tensile strength and elastic modulus after 60 cycles, as shown in Figures 6 and 7. After 60 cycles, the tensile strength increased by 22%, 20%, 28%, and 28% for WWD2, SWD2, WWD4, and SWD4, respectively. Meanwhile, after 60 cycles, the elastic modulus increased by 14%, 11%, 20%, and 16%, for WWD2, SWD2, WWD4, and SWD4, respectively. These results indicate that the initial increases of tensile strength and elastic modulus of the specimens with sustained loads are greater than those of Advances in Materials Science and Engineering 8 8 Advances in Materials Science and Engineering 0 60 120 180 240 300 360 Cycles 15 20 25 30 35 WWD0 WWD2 WWD4 Elastic modulus (GPa) (a) 0 60 120 180 240 300 360 Cycles 20 25 30 35 SWD0 SWD2 SWD4 15 Elastic modulus (GPa) (b) 0 60 120 180 240 300 360 Cycles 15 20 25 30 35 WWD2 SWD2 Elastic modulus (GPa) (c) 0 60 120 180 240 300 360 Cycles 15 20 25 30 35 WWD4 SWD4 Elastic modulus (GPa) (d) Figure 7: Elastic modulus of stressed specimens (a) subjected to WD cycles of distilled water and different loads; (b) subjected to WD cycles of saltwater and different loads; (c) subjected to WD cycles and 20% ultimate tensile strength; (d) subjected to WD cycles and 40% ultimate tensile strength. 3. Results and Discussion The previous research of authors has proven that the effect of saltwater immer- sion on tensile property is greater than that of tap water due to the chemical reaction between leaked Cl ions from saltwater and metal ions (Si, Na, and K) from E-glass fibers [24].h The micrographs of the surfaces of stressed specimens showed that the voids of polyester resins progressed in both distilled water and saltwater, as shown in Figure 9. Hydrolysis of isophthalic polyester resins was also observed under the combined effects of WD cycles and sustained loads, as shown in Figure 10. 3. Results and Discussion 0 60 120 180 240 300 360 Cycles 15 20 25 30 35 Elastic modulus (GPa) 0 60 120 180 240 300 360 Cycles 20 25 30 35 15 Elastic modulus (GPa) Elastic modulus (GPa) 0 60 120 180 240 300 360 Cycles 15 20 25 30 35 Elastic modulus (GPa) 0 60 120 180 240 300 360 Cycles 15 20 25 30 35 Elastic modulus (GPa) Elastic modulus (GPa) Elastic modulus (GPa) (c) (d) Figure 7: Elastic modulus of stressed specimens (a) subjected to WD cycles of distilled water and different loads; (b) subjected to WD cycles of saltwater and different loads; (c) subjected to WD cycles and 20% ultimate tensile strength; (d) subjected to WD cycles and 40% ultimate tensile strength. respectively, and the elastic modulus decreased by 17%, 19%, 21%, and 23% for WWD2, SWD2, WWD4, and SWD4, respectively. Meanwhile, after 360 cycles, the loss of elongation at rupture is 73%, 73%, 76%, and 77% for WWD2, SWD2, WWD4, and SWD4, respectively, as shown in Figure 8. It is obvious that the increase of sustained loads leads to a decrease in tensile property. Moreover, the effect of WD cycles of saltwater on elastic modulus and elongation at rupture is greater than that of WD cycles of distilled water. Both water diffusion and stress corro- sion are associated with the degradation of resin and the interface between fiber and matrix. The previous research of authors has proven that the effect of saltwater immer- sion on tensile property is greater than that of tap water due to the chemical reaction between leaked Cl ions from respectively, and the elastic modulus decreased by 17%, 19%, 21%, and 23% for WWD2, SWD2, WWD4, and SWD4, respectively. Meanwhile, after 360 cycles, the loss of elongation at rupture is 73%, 73%, 76%, and 77% for WWD2, SWD2, WWD4, and SWD4, respectively, as shown in Figure 8. It is obvious that the increase of sustained loads leads to a decrease in tensile property. Moreover, the effect of WD cycles of saltwater on elastic modulus and elongation at rupture is greater than that of WD cycles of distilled water. Both water diffusion and stress corro- sion are associated with the degradation of resin and the interface between fiber and matrix. 4. Prediction of Long-Term Behavior Several models have been developed to predict the long- term responses of FRP composites based on Arrhenius con- cept which assumed that the single dominant deterioration Advances in Materials Science and Engineering 9 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0 60 120 180 240 300 360 Cycles WWD0 WWD2 WWD4 Elongation at rupture (%) (a) 0 60 120 180 240 300 360 Cycles SWD0 SWD2 SWD4 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Elongation at rupture (%) (b) 0 60 120 180 240 300 360 Cycles WWD2 SWD2 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Elongation at rupture (%) (c) 0 60 120 180 240 300 360 Cycles WWD4 SWD4 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Elongation at rupture (%) (d) Figure 8: Elongation at rupture of stressed specimens (a) subjected to WD cycles of distilled water and different loads; (b) subjected to WD cycles of saltwater and different loads; (c) subjected to WD cycles and 20% ultimate tensile strength; (d) subjected to WD cycles and 40% ultimate tensile strength. 0 60 120 180 240 300 360 Cycles 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Elongation at rupture (%) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0 60 120 180 240 300 360 Cycles Elongation at rupture (%) (b) 0 60 120 180 240 300 360 Cycles WWD4 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Elongation at rupture (%) (a) 0 60 120 180 240 300 360 Cycles 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Elongation at rupture (%) Elongation at rupture (%) (d) (c) Figure 8: Elongation at rupture of stressed specimens (a) subjected to WD cycles of distilled water and different loads; (b) subjected to WD cycles of saltwater and different loads; (c) subjected to WD cycles and 20% ultimate tensile strength; (d) subjected to WD cycles and 40% ultimate tensile strength. dominance of postcure processes in environmental exposure, while a value of 𝐵= 100 indicates full polymerization of the resin. Equation (1) is only valid for 𝑡> 0. In the case of 𝑡= 0, the percent retention of 𝑃(𝑡) is assumed to be 100%. 4. Prediction of Long-Term Behavior mechanism of the material will not change with time and temperature in corrosive environment [8, 31].h The Arrhenius equation for predicting the mechanical property of FRP composites is given by [8] p For exposure in WD cycles, (1) can be transformed into 𝑃(𝑡) = 𝑃0 100 [𝐴ln (𝑡) + 𝐵] , (1) (1) 𝑃(𝑥) = 𝑃0 100 [𝐴ln (𝑥) + 𝐵] , (2) (2) where 𝑃(𝑡) and 𝑃0 are the performance attributes of FRP composites at exposure time 𝑡and zero time of exposure, respectively, 𝐴is degradation rate, and 𝐵is a material constant associated with postcure progression of polymer during exposure. A value of 𝐵> 100 relates to the initial where 𝑥is the number of WD cycles.h The tensile strength and elastic modulus of FRP com- posites tend to increase gradually with time in the initial postcuring processes and then become stable, regardless of 10 10 Advances in Materials Science and Engineering (a) (b) (c) (d) Figure 9: Micrographs of the surfaces (×50) of stressed specimens: (a) SWD2 after 60 WD cycles; (b) SWD2 after 180 WD cycles; (c) WWD2 after 180 WD cycles; and (d) SWD4 after 180 WD cycles. (a) (b) (a) (b) (b) (d) (c) (d) (c) Figure 9: Micrographs of the surfaces (×50) of stressed specimens: (a) SWD2 after 60 WD cycles; (b) SWD2 after 180 WD cycles; (c) WWD2 after 180 WD cycles; and (d) SWD4 after 180 WD cycles. (a) (b) (c) (d) Figure 10: Micrographs of the longitudinal section (×50) of stressed specimens: (a) SWD2 after 60 WD cycles; (b) SWD2 after 180 WD cycles; (c) WWD2 after 180 WD cycles; and (d) WWD4 after 180 WD cycles. (b) (a) (a) (a) (b) (d)t (c) (d) (c) Figure 10: Micrographs of the longitudinal section (×50) of stressed specimens: (a) SWD2 after 60 WD cycles; (b) SWD2 after 180 WD cycles; (c) WWD2 after 180 WD cycles; and (d) WWD4 after 180 WD cycles. Advances in Materials Science and Engineering 11 g g 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Experimental data Eq. (2) Eq. (5) Tensile strength (MPa) (a) WWD2 Experimental data Eq. (2) Eq. (5) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) (b) WWD4 Experimental data Eq. (2) Eq. 4. Prediction of Long-Term Behavior (5) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) (c) SWD2 Experimental data Eq. (2) Eq. (5) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) (d) SWD4 Figure 11: Comparison of prediction of tensile strength. 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) Tensile strength (MPa) Tensile strength (MPa) Experimental data Eq. (2) Eq. (5) (a) WWD2 (b) WWD4 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) 0 100 200 300 400 500 600 0 60 120 180 240 300 360 Cycles Tensile strength (MPa) Tensile strength (MPa) Tensile strength (MPa) Tensile strength (MPa) Figure 11: Comparison of prediction of tensile strength. For exposure in WD cycles, (3) can be transformed into For exposure in WD cycles, (3) can be transformed into For exposure in WD cycles, (3) can be transformed into the effects of environmental attacks. Thus, the effects of postcuring on the mechanical property of FRP composites can be described as a two-parameter exponential equation: 𝑃(𝑥) = 𝑃0 100 [𝐴ln (𝑥) + 100] + 𝛼(1 −𝛽−𝑥) , (5) (5) 𝑓(𝑡) = 𝛼(1 −𝛽−𝑡) , (3) (3) where 𝑥is the number of WD cycles.h where 𝑥is the number of WD cycles.h where 𝑓(𝑡) is the performance attribute of FRP composites due to postcuring, and 𝛼and 𝛽are regression constants.f The predictive equations of tensile strength and elastic modulus according to (5) and (2) are given in Tables 3 and 4, respectively. For unstressed specimens, the predicted results from (2) and (5) agree well with those obtained from experiments. However, for specimens with sustained loads, (5) provides more accurate prediction results than (2), as shown in Figures 11 and 12. Considering the combined effects of postcure processes and exposure environments, the prediction model can be given by 𝑃(𝑡) = 𝑃0 100 [𝐴ln (𝑡) + 100] + 𝛼(1 −𝛽−𝑡) . (4) (4) Advances in Materials Science and Engineering 12 12 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Experimental data Eq. (2) Eq. 4. Prediction of Long-Term Behavior (5) Elastic modulus (GPa) (a) WWD2 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Experimental data Eq. (2) Eq. (5) Elastic modulus (GPa) (b) WWD4 Experimental data Eq. (2) Eq. (5) 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) (c) SWD2 Experimental data Eq. (2) Eq. (5) 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) (d) SWD4 Figure 12: Comparison of prediction of elastic modulus. 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) Elastic modulus (GPa) Eq. (5) (b) WWD4 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) q. (5) (a) WWD2 0 5 10 15 20 25 30 35 0 60 120 180 240 300 360 Cycles Elastic modulus (GPa) Elastic modulus (GPa) Elastic modulus (GPa) Elastic modulus (GPa) Figure 12: Comparison of prediction of elastic modulus. References (3) A modified model which considered the effects of postcure processes was developed to predict the ten- sile strength and elastic modulus of GFRP composites subjected to WD cycles. It is shown that the model can capture the degradation trend of specimens under the combined effects of WD cycles and sustained loads. [1] C. Tuakta and O. B¨uy¨uk¨ozt¨urk, “Conceptual model for pre- diction of FRP-concrete bond strength under moisture cycles,” Journal of Composites for Construction, vol. 15, no. 5, pp. 743– 756, 2011. [2] K. Soudki, E. El-Salakawy, and B. Craig, “Behavior of CFRP strengthened reinforced concrete beams in corrosive environ- ment,” Journal of Composites for Construction, vol. 11, no. 3, pp. 291–298, 2007. 5. Conclusions Table 3: Predictive equations for tensile strength. Table 4: Predictive equations for elastic modulus. Exposure environment Load level Equation 𝑅-square Wet-dry cycles in distilled water 0% 0.247[−11.092 ln(𝑥) + 100] + 12.77(1 −1.091−𝑥) 0.97 0.247[−7.347 ln(𝑥) + 132.63] 0.84 20% 0.247[−14.902 ln(𝑥) + 100] + 17.33(1 −1.08−𝑥) 0.93 0.247[−8.835 ln(𝑥) + 139.158] 0.75 40% 0.247[−20.873 ln(𝑥) + 100] + 24.57(1 −1.068−𝑥) 0.88 0.247[−10.062 ln(𝑥) + 144.14] 0.57 Wet-dry cycles in saltwater 0% 0.247[−10.324 ln(𝑥) + 100] + 12.49(1 −1.08−𝑥) 0.98 0.247[−6.07 ln(𝑥) + 128.83] 0.78 20% 0.247[−19.638 ln(𝑥) + 100] + 23.57(1 −1.049−𝑥) 0.85 0.247[−5.789 ln(𝑥) + 123.24] 0.34 40% 0.247[−21.73 ln(𝑥) + 100] + 25.5(1 −1.065−𝑥) 0.98 0.247[−10.22 ln(𝑥) + 144.13] 0.63 Table 4: Predictive equations for elastic modulus. of the National Natural Science Foundation of China (Grant 51238003) is greatly appreciated. of the National Natural Science Foundation of China (Grant 51238003) is greatly appreciated. and saltwater, which indicated the GFRP compos- ites became brittle in exposure of WD cycles. The sustained loads have insignificant influences on the elongation at rupture. Conflicts of Interest [3] A. Belarbi and S.-W. Bae, “An experimental study on the effect of environmental exposures and corrosion on RC columns with FRP composite jackets,” Composites Part B: Engineering, vol. 38, no. 5-6, pp. 674–684, 2007. The authors declare that they have no conflicts of interest. 5. Conclusions of the fibers in the stress direction yield greater increases in tensile strength and elastic modulus of stressed specimens than those of unstressed spec- imens. Further exposure to WD cycles led to a steady decrease in tensile strength and modulus. For unstressed specimens, the degradation of the tensile property of specimens exposed to distilled water was almost equal to specimens exposed to saltwater after 360 cycles. However, for stressed specimens, the effect of WD cycles of saltwater on elastic modulus and elongation at rupture is greater than that of WD cycles of distilled water. Moreover, increasing sustained loads led to a decrease in tensile property. The durability of GFRP composites exposed to WD cycles of both distilled water and saltwater was investigated under three different sustained loading levels. The tensile properties of both the unconditioned and conditioned specimens were tested to study the long-term behavior of GFRP composites under different WD cycles. Based on Arrhenius concept, a model was developed to predict the tensile property of GFRP. In addition, the electron microscopy (EM) was used to characterize the aging effect on GFRP composites. The results obtained from this study are summarized as follows: (1) The tensile strength and elastic modulus of all conditioned specimens increased initially and then decreased with the increase of exposure cycle num- bers. The coupled effects of postcuring and alignment (2) The elongation at rupture decreased sharply with the increasing number of cycles in both distilled water (2)h (2) The elongation at rupture decreased sharply with the increasing number of cycles in both distilled water Advances in Materials Science and Engineering 13 Table 3: Predictive equations for tensile strength. Exposure environment Load level Equation 𝑅-square Wet-dry cycles in distilled water 0% 4.1675[−6.614 ln(𝑥) + 100] + 128.63(1 −1.16−𝑥) 0.97 4.1675[−5.794 ln(𝑥) + 126.74] 0.99 20% 4.1675[−20.479 ln(𝑥) + 100] + 427.57(1 −1.06−𝑥) 0.92 4.1675[−8.590 ln(𝑥) + 141.59] 0.51 40% 4.1675[−25.814 ln(𝑥) + 100] + 538.77(1 −1.06−𝑥) 0.91 4.1675[−9.379 ln(𝑥) + 144.51] 0.42 Wet-dry cycles in saltwater 0% 4.1675[−4.912 ln(𝑥) + 100] + 103.00(1 −1.21−𝑥) 0.83 4.1675[−7.430 ln(𝑥) + 134.38] 0.94 20% 4.1675[−20.604 ln(𝑥) + 100] + 436.32(1 −1.07−𝑥) 0.96 4.1675[−9.687 ln(𝑥) + 148.475] 0.63 40% 4.1675[−25.888 ln(𝑥) + 100] + 543.50(1 −1.07−𝑥) 0.95 4.1675[−12.266 ln(𝑥) + 160.37] 0.62 Note. 𝑅-square is the coefficient of determination. It denotes the proportion of the variance in the dependent variable which is predictable from the independent variables. Advances in Materials Science and Engineering Advances in Materials Science and Engineering 14 [21] J. U. N. Wang, H. O. T. A. Gangarao, R. Liang, and W. Liu, “Durability and prediction models of fiber-reinforced polymer composites under various environmental conditions: A critical review,” Journal of Reinforced Plastics and Composites, vol. 35, no. 3, pp. 179–211, 2016. with TRC under a Conventional and Chloride Wet-Dry Cycle Environment,” Journal of Composites for Construction, vol. 21, no. 1, Article ID 04016061, 2017. [6] V. M. Karbhari and M. A. Abanilla, “Design factors, reliability, and durability prediction of wet layup carbon/epoxy used in external strengthening,” Composites Part B: Engineering, vol. 38, no. 1, pp. 10–23, 2007. [22] J. J. Myers, S. S. Murthy, and F. Micelli, “Effect of combined environmental cycles on the bond of FRP sheets to concrete,” in Proceedings of the 2001 International Conference-Composites in Construction, Porto, Portugal, October 2001. [7] Z. Wu, Y. J. Kim, H. Diab, and X. Wang, “Recent developments in long-term performance of FRP composites and FRP-concrete interface,” Advances in Structural Engineering, vol. 13, no. 5, pp. 891–903, 2010. [23] K. K. Mahato, K. Dutta, and B. C. Ray, “High-temperature tensile behavior at different crosshead speeds during loading of glass fiber-reinforced polymer composites,” Journal of Applied Polymer Science, vol. 134, no. 16, Article ID 44715, 2017. [8] M. A. G. Silva and H. Biscaia, “Environmental effects on bond of GFRP external reinforcement to RC beams,” in Proceeding of the 8th International Symposium on Fiber Reinforced Polymer Reinforcement for Concrete Structures, FRPRCS-8, University of Patras, Patras, Greece, July 2007. [24] J. Wang, H. GangaRao, R. Liang, D. Zhou, W. Liu, and Y. Fang, “Durability of glass fiber-reinforced polymer composites under the combined effects of moisture and sustained loads,” Journal of Reinforced Plastics and Composites, vol. 34, no. 21, pp. 1739– 1754, 2015. [9] M. I. Kabir, B. Samali, and R. Shrestha, “Fracture Properties of CFRP-Concrete Bond Subjected to Three Environmental Conditions,” Journal of Composites for Construction, vol. 20, no. 4, Article ID 4016010, 2016. [25] ASTM D638-10, “Standard Test Method for Tensile Properties of Plastics,” in Proceedings of the American Society for Testing and Materials, January 2010. [10] Y. J. Kim, M. Hossain, and Y. Chi, “Characteristics of CFRP- concrete interface subjected to cold region environments including three-dimensional topography,” Cold Regions Science and Technology, vol. 67, no. 1-2, pp. 37–48, 2011. [26] M. S. Sciolti, M. Frigione, and M. A. Advances in Materials Science and Engineering Aiello, “Wet lay-up manu factured FRPs for concrete and masonry repair: influence of water on the properties of composites and on their epoxy components,” Journal of Composites for Construction, vol. 14, no. 6, pp. 823–833, 2010. [11] Y. J. Kim, M. Hossain, and J. Zhang, “A probabilistic investiga- tion into deterioration of CFRP-concrete interface in aggressive environments,” Construction and Building Materials, vol. 41, pp. 49–59, 2013. [27] B. Ghiassi, G. Marcari, D. V. Oliveira, and P. B. Lourenc¸o, “Water degrading effects on the bond behavior in FRP-strengthened masonry,” Composites Part B: Engineering, vol. 54, no. 1, pp. 11– 19, 2013. [12] H. C. Biscaia, M. A. G. Silva, and C. Chastre, “An experimental study of GFRP-to-concrete interfaces submitted to humidity cycles,” Composite Structures, vol. 110, pp. 354–368, 2014. [28] S. P. Sonawala and R. J. Spontak, “Degradation kinetics of glass-reinforced polyesters in chemical environments: Part II. Organic solvents,” Journal of Materials Science, vol. 31, no. 18, pp. 4757–4765, 1996. [13] J.-L. Wang and S. Amidi, “Subcritical debonding of FRP-to- concrete bonded interface under synergistic effect of load, moisture, and temperature,” Mechanics of Materials, vol. 92, pp. 80–93, 2016. [29] M. A. Abanilla, Y. Li, and V. M. Karbhari, “Durability character- ization of wet layup graphite/epoxy composites used in external strengthening,” Composites Part B: Engineering, vol. 37, no. 2-3, pp. 200–212, 2005. [14] Y. V. Lipatov, S. I. Gutnikov, M. S. Manylov, E. S. Zhukovskaya, and B. I. Lazoryak, “High alkali-resistant basalt fiber for reinforcing concrete,” Materials and Corrosion, vol. 73, pp. 60– 66, 2015. [30] B. Abdel-Magid, S. Ziaee, K. Gass, and M. Schneider, “The combined effects of load, moisture and temperature on the properties of E-glass/epoxy composites,” Composite Structures, vol. 71, no. 3-4, pp. 320–326, 2005. [15] P. Feng, L. Hu, P. Qian, and L. Ye, “Compressive bearing capacity of CFRP-aluminum alloy hybrid tubes,” Composite Structures, vol. 140, pp. 749–757, 2016. [16] B. Benmokrane, F. Elgabbas, E. A. Ahmed, and P. Cousin, “Characterization and Comparative Durability Study of Glass/ Vinylester, Basalt/Vinylester, and Basalt/Epoxy FRP Bars,” Jour- nal of Composites for Construction, vol. 19, no. 6, Article ID 04015008, 2015. [31] P. Marru, V. Latane, C. Puja, K. Vikas, P. Kumar, and S. Neogi, “Lifetime estimation of glass reinforced epoxy pipes in acidic and alkaline environment using accelerated test methodology,” Fibers and Polymers, vol. 15, no. 9, pp. 1935–1940, 2014. [17] J. Xu, H. Kolstein, and F. S. K. Acknowledgments [4] H. A. Toutanji and T. El-Korchi, “Tensile durability of cement- based frp composite wrapped specimens,” Journal of Composites for Construction, vol. 3, no. 1, pp. 38–45, 1999. The financial support from the National Natural Science Foundation of China (Grant 51578283), Jiangsu Province Science and Technology Support Program of Construction Industry Modernization (Grant 2017-13), and Key Program [5] S.-P. Yin, C. Peng, and Z.-Y. Jin, “Research on Mechanical Prop- erties of Axial-Compressive Concrete Columns Strengthened Advances in Materials Science and Engineering Bijlaard, “Moisture diffusion and hygrothermal aging in pultruded fibre reinforced polymer composites of bridge decks,” Materials and Design, vol. 37, pp. 304–312, 2012. [18] H. Toutanji and M. Saafi, “Durability studies on concrete columns encased in PVC-FRP composite tubes,” Composite Structures, vol. 54, no. 1, pp. 27–35, 2001. [19] Y. Chen, J. F. Davalos, I. Ray, and H. Y. Kim, “Accelerated aging tests for evaluations of durability performance of FRP reinforcing bars for concrete structures,” Composite Structures, vol. 78, no. 1, pp. 101–111, 2007. [20] P. Boer, L. Holliday, and T. H.-K. Kang, “Independent envi- ronmental effects on durability of fiber-reinforced polymer wraps in civil applications: a review,” Construction and Building Materials, vol. 48, pp. 360–370, 2013. 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https://openalex.org/W2948197228	https://zenodo.org/records/4119255/files/20%201598.pdf	English	null	Performance of europium aluminium doped polymer optical waveguide amplifier	Bulletin of Electrical Engineering and Informatics	2,019	cc-by	4,949	Bulletin of Electrical Engineering and Informatics Vol. 8, No. 4, December 2019, pp. 1343~1350 ISSN: 2302-9285, DOI: 10.11591/eei.v8i4.1598 Bulletin of Electrical Engineering and Informatics Vol. 8, No. 4, December 2019, pp. 1343~1350 ISSN: 2302-9285, DOI: 10.11591/eei.v8i4.1598 Bulletin of Electrical Engineering and Informatics Vol. 8, No. 4, December 2019, pp. 1343~1350 ISSN: 2302-9285, DOI: 10.11591/eei.v8i4.1598  1343  1343 Corresponding Author: Nur Najahatul Huda Saris, Department of Electronic Systems Engineering, Universiti Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia. Email: nnhuda3@live.utm.my Performance of europium aluminium doped polymer optical waveguide amplifier Nur Najahatul Huda Saris1, Azura Hamzah2, Sumiaty Ambran3, Osamu Mikami4, Takaki Ishigure5 1,2,3,4Department of Electronic Systems Engineering, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia 5F l f S i d T h l K i U i i 3 14 1 Hi hi K h k k Y k h 223 8522 J Nur Najahatul Huda Saris1, Azura Hamzah2, Sumiaty Ambran3, Osamu Mikami4, Takaki Ishigure5 1,2,3,4Department of Electronic Systems Engineering, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia 5Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia 5Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan Copyright © 2019 Institute of Advanced Engineering and Science. All rights reserved. ABSTRACT Article history: Received Mar 29, 2019 Revised Jun 2, 2019 Accepted Jun 28, 2019 In this paper, the graded index (GI) multimode rare-earth metal (RE-M) doped polymer optical waveguide amplifier has been prepared and tested optically. A 10-cm Europium Aluminum Benzyl Methacrylate ( was fabricated via a unique technique known as the “Mosquito Method” which utilizes a micro-dispenser machine. Optical gain from 75 to 150 µm circular core diameter waveguide of 13 wt.% concentration has been demonstrated and measured under forward pumping condition. The cladding monomer deployed in this research is Acrylate resin XCL01, which is a modified photocurable acrylate material. Fundamentally, -30 decibel (dBm) red light signal input and 23 dBm pump power of 532 nm green laser wavelength is implemented within the range of 580 to 640 nm optical amplification wavelength. A maximum gain of 12.96 dB at 617 nm wavelength has been obtained for a 100 µm core diameter of Eu-Al polymer optical waveguide. The effect of different coupler diameter for pumping and the comparison of insertion loss before and after amplification against the performance of the Eu-Al polymer waveguide amplifier are also studied. There exists an optimum core diameter of which the amplifier gain enhancement is at maximum value. Europium aluminium Forward pumping Mosquito method Optical amplifier Polymer waveguide Copyright © 2019 Institute of Advanced Engineering and Science. All rights reserved. Corresponding Author: Nur Najahatul Huda Saris, Department of Electronic Systems Engineering, Universiti Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia. Email: nnhuda3@live.utm.my Journal homepage: http://beei.org/index.php/EEI Corresponding Author: Nur Najahatul Huda Saris, Department of Electronic Systems Engineering, Universiti Teknologi Malaysia, 54100, Kuala Lumpur, Malaysia. Email: nnhuda3@live.utm.my 1. INTRODUCTION Over the past few years, utilization of lanthanide groups such as Terbium ( ), Erbium ( ), Neodymium ( ) and Europium ( ) in lasers and amplifiers has grown substantially [1-3]. This is generally due to the effective pumping system of lanthanide ions’ energy transfer process. Furthermore, lanthanide ions dissolve easily in organic hosts such as polymer by encapsulating it with organic ligand [4]. With that, higher doping concentration can be obtained. On top of that, research activities related to polymer optical fiber (POF) with low-loss visible wavelength window for short distance communications have increased recently [5, 6]. This is due to its advantages of having low production costs and processing flexibility for polymer over inorganic materials such as glass and crystal [7, 8]. Polymer generally exhibits low absorption losses in the visible wavelength region [9, 10]. Therefore, for the development of organic-dye-doped polymeric devices such as rare earth (RE) doped polymer amplifier as integrated optical waveguide devices, scaling down the size to be compact is necessary. However, this may result in interaction between ions due to high doping concentration in the active layer [11]. The transition lifetime at the metastable state will increase as well, owing to insufficient energy of the ligand surrounding the RE ion [12]. Hence, the Europium-Aluminium (Eu-Al) polymer optical Journal homepage: http://beei.org/index.php/EEI  1344  ISSN: 2302-9285 waveguide amplifier, which is a product of rare earth metal (RE-M) doping composition, is introduced by KRI Inc. waveguide amplifier, which is a product of rare earth metal (RE-M) doping composition, is introduced by KRI Inc. In this research, the Europium Aluminium Benzyl Methacrylate ( ) is used as the core monomer. However, this core monomer is yet to be commercialized. The synthesis of is relatively similar to that of a previous research conducted by KRI Inc., concerning Europium Aluminium Methyl Methacrylate [12, 13]. However, the core monomer must undergo some chemical modification by replacing the Methyl Methacrylate (MA) with Benzyl Methacrylate (BzMA) after the evaporation process of the Propylene Glycol α-Monomethyl Ether (PGME). This modification is important in order to enhance the core properties with respect to this research [14]. In the next few years, more advanced technology will be developed correlating to the demand of higher data rate. 1. INTRODUCTION Thus, it is imperative to implement the graded index (GI) multimode fiber since the fiber is a promising candidate for high bitrate transmissions in which it has the advantage of low propagation loss, low connection loss with GI multimode fiber (MMF) with wide misalignment tolerance, and low interchannel crosstalk even under a small pitch size [15]. Correspondingly, the “Mosquito Method” is adopted to create the GI circular core [16-19] of the Eu-Al polymer optical waveguide. The circular core is important as this research attempts to combine optical fiber characteristics to a planar waveguide. The optical amplification for GI multimode Eu-Al polymer optical waveguide amplifier has been reported to have satisfactory results as high as 7.1 dB/cm for 10 wt.% concentration via variable stripe length (VSL) method [14] and 3.24 dB/cm for 13 wt.% through forward pumping method [20]. Although higher optical amplification was obtained from the VSL method, the side surface excitation method seemed complicated to implement and was impractical for current application [20, 21]. Furthermore, both methods acquired core diameters of 100 μm for 5.2 and 4.9 cm. Thus, the study proceeds with Eu-Al polymer optical waveguide amplifier at 13 wt.% concentration of the core monomer. Accordingly, the research would focus on the relationship of optical gain and core diameter. The core diameter of the waveguide is precisely controlled to 75, 100, 125 and 150 μm with a deviation of only a few micrometers and tested experimentally. The length of the waveguide is fixed by doubling the length of the waveguide in the previous research, i.e., approximately 10 cm. 3.1. Optimum dispensing conditions The dispensing scanning speed, inner needle diameter and dispensing pressure are key parameters in executing the Mosquito Method [16, 19, 22]. Hence, these three dispensing criteria are investigated to create the desired core diameter. In this research, the idea of the proposed optical waveguide amplifier is based on the combination of planar waveguide and graded index (GI) optical fiber core, upon realizing the superiority of the GI multimode fiber in high-speed transmissions. For this purpose, three circular inner needle diameters (150, 170 and 190 µm) and dispensing gas pressure (210 and 420 kPa) were varied. The dependency of the core diameter on these three parameters are shown in Figure 2(a) and 2(b). The plots show the average of five core diameters created on the waveguide. From the results, the smaller cores were found to require faster dispensing scanning speed under smaller inner needle diameter of 150 µm and low pressure of 210 kPa. In this paper, four waveguides with different core diameters of 75, 100, 125, and 150 µm were fabricated based on the appropriate dispensing conditions found for XCL01 and . Table 1 presents the fabrication results of desired core diameters according to manipulated and fixed parameters as set for each core diameter. (a) (b) Figure 2. Core diameter versus dispensing scanning speed for variation of, (a) Dispensing pressure with 150 µm inner needle diameter, and (b) Inner needle diameter with 210 kPa dispensing pressure (b) (a) (b) (a) Figure 2. Core diameter versus dispensing scanning speed for variation of, (a) Dispensing pressure with 150 µm inner needle diameter, and (b) Inner needle diameter with 210 kPa dispensing pressure Table 1. Parameters of micro dispenser machine Core diameter [µm] The needle inner diameter [µm] The dispenser gas pressure [kPa] The needle scanning speed [mm/s] 75 190 284 61.8 100 190 505 61.8 125 190 603 47.3 150 190 505 27.5 2. FABRICATION METHOD The Eu-Al polymer optical waveguide amplifier was fabricated by using a unique method known as the Mosquito Method. It was performed by utilizing a micro-dispenser provided by Musashi Engineering Inc. Basically, a viscous liquid-state core monomer from a syringe connected to a dispenser was dispensed via a thin needle directly into a cladding monomer layer. The Acrylate resin, XCL01 and Europium Aluminum Benzyl Methacrylate ( ) were used as cladding and core, with refractive index of 1.501 and 1.51, respectively. The fabrication technique is illustrated in Figure 1. Figure 1. Fabrication steps of the mosquito method Figure 1. Fabrication steps of the mosquito method Firstly, the XCL01 monomer was coated on the glass substrate. The waveguide material, which is the frame of cladding monomer was made from the silicone resins supplied by ADEKA Corp. to ensure a thick layer coating of cladding monomer. After that, was inserted into the syringe connected to the micro-dispenser. Then, was dispensed directly into the cladding layer through a thin needle tip attached to the syringe. Here, the dispensing scanning speed, pressure, and the needle inner diameter were scanned throughout the process. These parameters are the key factors that control the core diameter and inter-channel pitch [19, 22-25]. In the meantime, the was discharged directly onto a liquid state of XCL01. It is very important for the cladding in its liquid state to maintain the core’s original cross-sectional shape immediately after being dispensed from the needle. Before exposure to ultra violet (UV) light, the core and cladding monomer should be diffused together since both monomers are miscible to form a concentration distribution. The copolymer was successfully formed after curing under UV Bulletin of Electr Eng and Inf, Vol. 8, No. 4, December 2019 : 1343 – 1350 1345 ISSN: 2302-9285  Bulletin of Electr Eng and Inf light and postbaked such that the concentration distribution remained fixed. The waveguide was cut depending on the desired length and then it was ready for testing. light and postbaked such that the concentration distribution remained fixed. The waveguide was cut depending on the desired length and then it was ready for testing. 3. RESULTS AND ANALYSIS In this section, the performance of rare earth metal (RE-M) using Europium Aluminum (Eu-Al) as gain medium is demonstrated with different core diameters. This section delineates the optimum parameters for dispensing conditions, experiment procedures and results, and data analyses. The performance of Eu-Al polymer optical waveguide is evaluated based on insertion loss and optical gain for a variation of core diameters with fixed length throughout the different coupler diameters used. The physical change on the surface of core waveguide are also discussed. 3.2. Near field pattern and insertion loss p Varying the waveguide core diameter is a very important aspect of this investigation in order to ascertain the optimum core diameter for best gain enhancement. Thus, each core from four waveguides was fabricated and tested multiple times at a wavelength of 617 nm, which is one of the wavelengths under visible region. In this measurement, the near field pattern (NFP) is necessary for the optical beam pattern analysis. The NFP is used to analyze the light confinement from the transceiver side (Tx) to the Receiver side (Rx). In other words, by using the NFP, the position of the launching and receiving sides can be adjusted  1346 ISSN: 2302-9285 properly to prevent any loss during measurement. In this research, the input of the red-light signal was from a light emitting diode (LED) via single mode fiber (SMF) at the Tx side. Then, the light emitted through the waveguide was captured using a charge coupled device (CCD) camera at the Rx side as shown in Figure 3. Figure 3. Near Field Pattern (NFP) measurement system Tx Rx Rx Rx Tx Figure 3. Near Field Pattern (NFP) measurement system Next, two readings were taken into consideration for the insertion loss. The first reading was made from the configuration with the waveguide and measured as in Figure 4(a) and followed by the second reading of that the configuration without the waveguide, also known as “back to back” as in Figure 4(b). The output of the light was measured via a Power Meter along the fiber to determine the insertion loss. The core cross-section, NFP and insertion loss for the best core amplification are tabulated in Table 2. (a) (b) Figure 4. Insertion loss measurement, (a) Reading with waveguide, and (b) Back to back reading (b) Figure 4. Insertion loss measurement, (a) Reading with waveguide, and (b) Back to back reading Comparing the NFP measurement results in Table 2, a distinct colour distribution of the beam (light intensity distribution) is noticed. For example, the highest light distribution in 75 µm core diameter waveguide is red, meanwhile, 100 and 150 µm core diameter waveguides are blue. It is indicated that the red-light distribution in 75 µm is more strongly confined to the core centre, whereas in other cores, it is considered that the lights spread out during propagation. 3.3. Gain enhancement for different coupler The experiment setup for optical amplification is shown in Figure 5. The green laser of 23 dBm emitting at 532 nm wavelength was used as the pump source and -30 dB red light signal emitting at 617 nm wavelength was used as the signal source. Both signal and light pump were combined using a 105 µm core graded index-multimode fiber (GI-MMF) (50:50) coupler. The amplified output signal was collected by an optical spectrum analyzer (OSA) passing through two meters of 200 µm core diameter of step index-multimode fiber (SI-MMF). Figure 5. Optical gain amplification measurement setup Figure 5. Optical gain amplification measurement setup The repetitions of the gain measurement are necessary to investigate the repeatability and the resistance to high optical power excitation. The outputs from OSA were analyzed using Microsoft Excel. For this purpose, the measurement of the signal, pumping signal and the coupled signal of input and light pump were made separately. Consequently, the gain without any noise such as Amplified Spontaneous Emission could be obtained by using mathematical subtraction. Under this condition, the optical gain measurement was repeated by substituting the 105 µm core with a 50 µm core GI-MMF coupler. Figure 6. shows the optical gain for different core diameters of the Eu-Al polymer optical waveguide amplifier using two different coupler diameters, which are 105 and 50 µm. From the graph, the optical gain is observed to have increased from 75 to 100 µm waveguide core diameter for both couplers. For the 50 µm coupler, the gain shows a slight decrease at 125 and 150 µm waveguide core diameter, which are 11.93 and 8.93 dB respectively. Figure 6. Comparison of Optical Gain for 105 and 50 µm GI-MMF Coupler Figure 6. Comparison of Optical Gain for 105 and 50 µm GI-MMF Coupler However, the gain drastically decreases from 12.96 to 3.46 dB for the 105 µm coupler at 100 to 125 µm waveguide core diameter. The decrease in gain value is believed to be due to measurement error. Surprisingly, the optical gain of 105 µm coupler for 150 µm waveguide core diameter increases sharply from 125 µm which is 7.88 dB. Overall, the trend of the optical amplification gain by using the 50 µm coupler is higher compared to that of the 105 µm. 3.2. Near field pattern and insertion loss This is believed to occur due to the difference of the refractive index distribution formed within the core. From these results, it is confirmed that light propagates through the core in any of the waveguides, although the manner in which the light is confined in the core centre is different between the waveguides. Table 2. NFP and insertion loss results of different core diameters Core Diameter [μm] 75 100 125 150 Core Cross-Section NFP Back to Back [dBm] -48.75 -46.07 -52.01 -48.64 Reading with Waveguide [dBm] -40.57 -40.81 -40.75 -40.98 Insertion Loss [dBm] 8.18 5.26 11.26 5.87 The results for insertion loss measurements showed the highest insertion loss at 125 µm, which is 11.26 dBm. In contrast, the 75 µm waveguide core diameter recorded the second lowest insertion loss, which The results for insertion loss measurements showed the highest insertion loss at 125 µm, which is 11.26 dBm. In contrast, the 75 µm waveguide core diameter recorded the second lowest insertion loss, which The results for insertion loss measurements showed the highest insertion loss at 125 µm, which is 11.26 dBm. In contrast, the 75 µm waveguide core diameter recorded the second lowest insertion loss, which Bulletin of Electr Eng and Inf, Vol. 8, No. 4, December 2019 : 1343 – 1350 1347 ISSN: 2302-9285  Bulletin of Electr Eng and Inf is 8.18 dBm. Hence, it can be concluded that the reason for the instability of the insertion loss with respect to the waveguide core diameter is due to the polished state of the end face of the waveguide or measurement error. is 8.18 dBm. Hence, it can be concluded that the reason for the instability of the insertion loss with respect to the waveguide core diameter is due to the polished state of the end face of the waveguide or measurement error. Performance of europium aluminium doped polymer optical waveguide… (Nur Najahatul Huda Saris) 3.4. Comparison of insertion loss before and after amplification Table 3 compares the surface core cross-section before and after amplification. Clearly, the appearance from post-amplification generally shows a darker shade compared to pre-amplification. This could be attributed to higher heat emitted by the laser pump applied to the waveguide during amplification as supported by the measurement of insertion loss after the amplification in Figure 7. As illustrated in the graph, the 125 µm core recorded the highest increasing insertion loss after amplification, which is approximately 2.91 dBm followed by the 100 µm core of 1.73 dBm. Based on the graph, the insertion loss after the amplification increases for all core diameters except for the 75 µm core diameter. The decrease in insertion loss value after the amplification is shown as 0.02 dBm. Even with decreasing values in insertion loss, the gap remains very close. As a result, it is thought that the value could be due to measurement error. Table 3. Comparison of core cross section before and after amplification Core Diameter [μm] 75 100 125 150 Pre Amplification Post Amplification Figure 7. Insertion loss comparison before and after amplification Figure 7. Insertion loss comparison before and after amplification 3.3. Gain enhancement for different coupler This could be related to the higher energy density of the launching side provided by the 50 µm coupler as compared to the 105 µm coupler; i.e., and respectively, as proven from (1): (1)  1348  8  ISSN: 2302-9285 Although the gain attained by the 105 µm coupler at 100 µm is 12.96 dB, which is higher compared to the 50 µm coupler, the difference is not substantial, of which amounts to only 0.33 dB gap at the wavelength of 617 nm. Bulletin of Electr Eng and Inf, Vol. 8, No. 4, December 2019 : 1343 – 1350 4. CONCLUSION The Mosquito Method has been adopted in this study to prepare four different 10-cm polymer optical waveguides amplifiers with graded index multimode core diameters of 75, 100, 125, and 150 µm. It is confirmed that the fabrication of the desired core diameter using this method was dependent on the controllability of needle inner diameter, dispensing scanning speed and pressure. Optical amplification had been observed at 617 nm with a green laser at 532 nm. Based on observation, the surface of the waveguide core turned darker-an effect from the heat emitted by the laser during amplification-which has caused the increase in insertion loss. Meanwhile, the highest gain recorded is 12.96 and 12.63 dB for 100 µm waveguide core diameter using 105 and 50 µm coupler respectively by forward pumping. The results show a potential for signal gain when incorporating , a rare earth metal (RE-M) doped polymer, as an active optical device. Considerably, it is expected that this compact device could be integrated into many applications such as in-vehicle optical interconnect, medicine, and communication network in the near future. ulletin of Electr Eng and Inf, Vol. 8, No. 4, December 2019 : 1343 – 1350 1349  ISSN: 2302-9285 Bulletin of Electr Eng and Inf ACKNOWLEDGEMENTS This research is a collaboration of Malaysia Japan International Institute of Technology (MJIIT) and Keio University and supported by Q.K130000.2543.17H26. We would like to show our gratitude to Keio University for their assistance in providing the facilities and instruments to conduct the research. We are also immensely grateful to Takaaki Ishigure Informatics and Optical Device System Laboratory members for their comments on earlier versions of the manuscript. REFERENCES ] Slooff, L.H., et al., ''Rare-earth doped polymers for planar optical amplifiers" Journal of Applied Physics, 200 91(7): p. 3955-3980. [2] Zhou, B., et al., "Controlling upconversion nanocrystals for emerging applications", Nature Nanotechnology, 2015, 10(11): p. 924. [2] Zhou, B., et al., "Controlling upconversion nanocrystals for emerging applications", Nature Nanotechnology, 2015, 10(11): p 924 [3] Johnson, N.J., et al., "Direct evidence for coupled surface and concentration quenching dynamics in lanthanide-doped nanocrystals", Journal of the American Chemical Society, 2017, 139(8): p. 3275-3282. [4] Kuriki, K., Y. Koike, and Y. Okamoto, 'Plastic optical fiber lasers and amplifiers containing lanthanide complexes", Chemical Reviews, 2002. 102(6): p. 2347-2356. ] Tsang, K.C., C.-Y. Wong, and E.Y.B. Pun, "High-gain optical amplification in Eu 3+-doped polymer", Opti letters, 2010, 35(4): p. 520-522. ( ) p [6] Koike, Y. and K. Koike, "Progress in low‐loss and high‐bandwidth plastic optical fibers", Journal of Polymer Science Part B: Polymer Physics, 2011. 49(1): p. 2-17. [7] Ribeiro, T., C. Baleizão, and J. Farinha, "Functional films from silica/polymer nanoparticles", Materials, 2014. 7(5): p. 3881-3900. ] Koike, Y. and A. Inoue, "High-speed graded-index plastic optical fibers and their simple interconnects for 4K/8 video transmission", Journal of Lightwave Technology, 2016, 34(6): p. 1551-1555. , f g gy, , ( ) p [9] Makino, K., et al., Low loss graded index polymer optical fiber with high stability under damp heat conditions. Optics express, 2012. 20(12): p. 12893-12898. p p , ( ) p [10] Kaino, T., "Optical Absorption of Polymers", Encyclopedia of Polymeric Nanomaterials, S. Kobayashi and K. Müllen, Editors. 2014, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 1-14. [11] Koeppen, C., et al., Rare-earth organic complexes for amplification in polymer optical fibers and waveguides. JOSA B, 1997. 14(1): p. 155-162. 2] Mataki, H., et al., "High-Gain Optical Amplification of Europium–Aluminum (Eu3+–Al)-Nanocluster-Dop Planar Polymer Waveguides", Japanese journal of applied physics, 2007, 46(1L): p. L83. [13] H. Mataki and T. Fukui, "Organic/inorganic optical nanocomposite with highly-doped rare-earth nanoclusters: novel phosphors for white LEDs," 5th IEEE Conference on Nanotechnology, 2005., Nagoya, Japan, 2005, pp. 772-775 vol. 2. [14] Mitani, M., et al., "Polymer optical waveguide composed of europium-aluminum-acrylate composite core for compact optical amplifier and laser", Integrated Optics: Devices, Materials, and Technologies XIX, 2015, International Society for Optics and Photonics. y p [15] Rasel, O.F., A. Yamauchi, and T. Ishigure, "Low-loss 3-dimensional shuffling graded-index polymer o waveguides for optical printed circuit boards", IEICE Transactions on Electronics, 2018. BIOGRAPHIES OF AUTHORS Nur Najahatul Huda Saris is a Postgraduate student at Malaysia-Japan International Institute of Technology (MJIIT), Universiti Teknologi Malaysia. She received the Bachelor’s degree in Electronics System Engineering from MJIIT in 2016. Currently, she is a member of Ministry Board of Technologist (MBOT) for graduate technologist. Her current research interests include optical waveguide amplifier and laser as well as rare-earth-doped optical devices. Nur Najahatul Huda Saris is a Postgraduate student at Malaysia-Japan International Institute of Technology (MJIIT), Universiti Teknologi Malaysia. She received the Bachelor’s degree in Electronics System Engineering from MJIIT in 2016. Currently, she is a member of Ministry Board of Technologist (MBOT) for graduate technologist. Her current research interests include optical waveguide amplifier and laser as well as rare-earth-doped optical devices. Azura Hamzah is a Senior Lecturer at Malaysia-Japan International Institute of Technology (MJIIT), Universiti Teknologi Malaysia. In MJIIT, she is a Full Member for Optical Devices and Systems Ikohza (ODESY) and an Associate Member for Disaster Preparedness and Prevention Centre (DPPC). She received her Bachelor’s degree in Communication Engineering from International Islamic University Malaysia, Malaysia in 2005 and the Master’s and PhD degree from Universiti Malaya, Malaysia in 2008 and 2012. Her research interests are in optical amplifier and laser, pulse laser, optical sensor, optical interconnect and rare-earth-doped optical devices. Currently, she is a member for The Institution of Electrical and Electronics Engineers and one of the Secretariat for IEEE Photonic Society Malaysia Chapter. Sumiaty Ambran is a faculty member of Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia (UTM). She has joined UTM since 2006 as an academic staff. She received the Bachelor’s degree in Electronics Telecommunication from Universiti Teknologi Malaysia, in 2005 and the Master’s degree from Universiti Teknologi Mara, in 2008. She obtained her PhD in 2013 from the Optoelectronics Research Centre, University of Southampton, United Kingdom in the area of planar integrated optical devices. Her research interests include optical sensor, optical telecommunication and rare-earth-doped optical devices for laser and amplifier application. Osamu Mikami is Guest Professor at Tokai University, Japan. He was Professor at Malaysia-Japan International Institute of Technology, UTM-KL for 3 years until April 2018. He received Bachelor, Master and PhD degrees from Osaka University, in 1969, 1971 and 1978, respectively. After working at NTT Research Laboratories for 23 years, he moved to Tokai University as Professor. His research interest is optical device for optical interconnection technology and optical fiber sensing. REFERENCES Soma and T. Ishigure, "Fabrication of polymer parallel optical waveguide with GI-circular-core using dispenser," 2012 4th Electronic System-Integration Technology Conference, Amsterdam, Netherlands, 2012, pp. 1-6. REFERENCES 101(7): p. 509-517. [16] Ishigure, T., "Graded-index core polymer optical waveguide for high-bandwidth-density optical printed circuit boards: fabrication and characterization", Optical Interconnects XIV, 2014, International Society for Optics and Photonics. [17] S. Morikawa and T. Ishigure, "Fabrication of GI-core polymer optical waveguide using a dispenser and its application to optical printed circuit boards," 2010 23rd Annual Meeting of the IEEE Photonics Society, Denver, CO, 2010, pp. 108-109. 8] Kinoshita, R., et al., "Polymer optical waveguides with GI and W-shaped cores for high-bandwidth-densi on-board interconnects", Journal of Lightwave Technology, 2013, 31(24): p. 4004-4015. [19] Kinoshita, R., D. Suganuma, and T. Ishigure, "Accurate interchannel pitch control in graded-index circular-core polymer parallel optical waveguide using the Mosquito method", Optics Express, 2014, 22(7): p. 8426-8437. [20] Y. Yoshida, T. Fukui and T. Ishigure, "Polymer waveguide incorporated with europium-aluminum polymer composite for compact and high-gain optical amplification devices," 2017 Conference on Lasers and Electro-Optics Pacific Rim (CLEO-PR), Singapore, 2017, pp. 1-4. [21] Y. Yoshida, T. Fukui and T. Ishigure, "Polymer waveguide incorporated with europium-aluminum polymer composite for compact and efficient amplification devices," 2017 IEEE CPMT Symposium Japan (ICSJ), Kyoto, 2017, pp. 181-184. [22] Soma, K. and T. Ishigure, Fabrication of a graded-index circular-core polymer parallel optical waveguide using a microdispenser for a high-density optical printed circuit board. IEEE Journal of Selected Topics in Quantum Electronics, 2013, 19(2): p. 3600310-3600310. [23] Takahashi, A. and T. Ishigure, "Bend insensitive graded index multimode polymer optical waveguides fabricated using the Mosquito method", Smart Photonic and Optoelectronic Integrated Circuits XVII. 2015, International Society for Optics and Photonics. Performance of europium aluminium doped polymer optical waveguide… (Nur Najahatul Huda Saris)  0  1350  1350 ISSN: 2302-9285 ISSN: 2302-9285 [24] A. Yamauchi, Y. Morimoto, T. Enomoto, K. Suzuki, H. Masuda and T. Ishigure, "Graded-Index Multimode Polymer Optical Waveguide Enabling Low Loss and High Density 3D On-Board Integration," 2016 IEEE 66th Electronic Components and Technology Conference (ECTC), Las Vegas, NV, 2016, pp. 490-496. [24] A. Yamauchi, Y. Morimoto, T. Enomoto, K. Suzuki, H. Masuda and T. Ishigure, "Graded-Index Multimode Polymer Optical Waveguide Enabling Low Loss and High Density 3D On-Board Integration," 2016 IEEE 66th Electronic Components and Technology Conference (ECTC), Las Vegas, NV, 2016, pp. 490-496. p gy f g pp [25] K. Soma and T. Ishigure, "Fabrication of polymer parallel optical waveguide with GI-circular-core using dispenser," 2012 4th Electronic System-Integration Technology Conference, Amsterdam, Netherlands, 2012, pp. 1-6. [25] K. BIOGRAPHIES OF AUTHORS He is Fellow member of The Institute of Electronics, Information and Communication Engineers, member of The Japanese Society of Applied Physics and Japanese Institute of Electronics Packaging. Takaaki Ishigure (M’00) was born in Gifu, Japan, on July 30, 1968. He received the B.S. degree in applied chemistry and the M.S. and Ph.D. degrees in material science from Keio University, Yokohama, Japan, in 1991, 1993, and 1996, respectively. He is currently an Assistant Professor with Keio University. He has been, concurrently, a Group Leader of the Japan Science and Technology (JST) Agency Exploratory Research for Advanced Technology (ERATO)-Solution-Oriented Research for Science and Technology (SORST) project. In 2005, he stayed at the Department of Electrical Engineering, Columbia University, New York, NY, as a Visiting Research Scientist. His current research interests are in the preparation of high-bandwidth graded-index plastic optical fibers (GI POF) and their system design. Bulletin of Electr Eng and Inf, Vol. 8, No. 4, December 2019 : 1343 – 1350
https://openalex.org/W4225078855	https://nhess.copernicus.org/articles/23/1029/2023/nhess-23-1029-2023.pdf	English	null	Characterizing the evolution of mass flow properties and dynamics through analysis of seismic signals: Insights from the 18 March 2007 Mt. Ruapehu lake-breakout lahar	null	2,022	cc-by	13,460	Correspondence: Braden Walsh (braden_walsh@sfu.ca) Received: 26 January 2022 – Discussion started: 10 February 2022 Revised: 15 January 2023 – Accepted: 1 February 2023 – Published: 6 March 2023 Received: 26 January 2022 – Discussion started: 10 February 2022 Revised: 15 January 2023 – Accepted: 1 February 2023 – Published: 6 March 2023 Abstract. Monitoring for mass ﬂows on volcanoes can be challenging due to the ever-changing landscape along the ﬂow path, which can drastically transform the properties and dynamics of the ﬂow. These changes to the ﬂows re- quire the need for detection strategies and risk assessments that are tailored not only between different volcanoes but at different distances along ﬂow paths as well. Being able to understand how a ﬂow event may transform in time and space along the channel is of utmost importance for haz- ard management. While visual observations and simple mea- suring devices in the past have shown how volcanic mass ﬂows transform along the ﬂow path, these same features for the most part have not been described using seismological methods. On 18 March 2007, Mt. Ruapehu produced the biggest lahar in Aotearoa / New Zealand in over 100 years. At 23:18 UTC the tephra dam holding the Crater Lake wa- ter back collapsed causing 1.3 × 106 m3 of water to ﬂow out and rush down the Whangaehu channel. We describe here the seismic signature of a lake-breakout lahar over the course of 83 km along the Whangaehu River system using three three-component broadband seismometers installed < 10 m from the channel at 7.4, 28, and 83 km from the Crater Lake source. Examination of three-component seismic amplitudes, frequency content, and directionality, combined with video imagery and sediment concentration data, was carried out. The seismic data show the evolution of the lahar as it trans- formed from a highly turbulent out-burst ﬂood (high peak frequency throughout), to a fully bulked-up multi-phase hy- perconcentrated ﬂow (varying frequency patterns depending on the lahar phase), to a slurry ﬂow (bedload dominant). Es- timated directionality ratios show the elongation of the lahar with distance down the channel, where each recording sta- tion depicts a similar pattern but for differing lengths of time. Furthermore, using directionality ratios shows extraordinary promise for lahar monitoring and detection systems where streamﬂow is present in the channel. Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 https://doi.org/10.5194/nhess-23-1029-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Volcanic mass ﬂows (e.g., debris ﬂows, pyroclastic density currents, debris avalanches, hyperconcentrated ﬂows) are one of the greatest threats to communities, industry, recreation, etc. on and around volcanoes. Volcanic mass ﬂows are par- ticularly dangerous as they are fast-moving turbulent ﬂows that can occur without any warning or an eruption transpiring (Capra et al., 2010). These ﬂows can move a sizable amount of liquid and debris great distances which can critically im- pact locations hundreds of kilometers from the volcano or source. Lake breakout or outburst ﬂood events can be par- ticularly destructive because they tend to be larger and can cause long-lasting changes to the landscape and surround- ing ecosystems (O’Connor et al., 2022; Procter et al., 2021). Furthermore, unlike eruption or rain-triggered mass ﬂows, outburst ﬂoods have very little to no warning. Eruption trig- gered ﬂows can be prepared for by the onset of the eruption Characterizing the evolution of mass ﬂow properties and dynamics through analysis of seismic signals: insights from the 18 March 2007 Mt. Ruapehu lake-breakout lahar Braden Walsh1, Charline Lormand2, Jon Procter3, and Glyn Williams-Jones1 1Department of Earth Sciences, Simon Fraser University, Burnaby, V5A 1S6, British Columbia, Canada 2Department of Earth Sciences, University of Durham, Durham, DH1 3LE, UK 3Volcanic Risk Solutions, Institute of Agriculture and Environment, Massey University, Palmerston North, 4442, Aotearoa / New Zealand Braden Walsh1, Charline Lormand2, Jon Procter3, and Glyn Williams-Jones1 1Department of Earth Sciences, Simon Fraser University, Burnaby, V5A 1S6, British Columbia, Canada 2Department of Earth Sciences, University of Durham, Durham, DH1 3LE, UK 3Volcanic Risk Solutions, Institute of Agriculture and Environment, Massey University, Palmerston North, 4442, Aotearoa / New Zealand 1.1 Anatomy of lahars Furthermore, discharge is maximum at the transition between phase 1 and phase 2 (Cronin et al., 1999) and is described as the head of the ﬂow. Phase 2 is described as a mixing zone between streamﬂow and increasing sedi- ment content, where the peak sediment concentration usually occurs at the end of phase 2 or at the beginning of phase 3 (e.g., Pierson and Scott, 1985). Cronin et al. (1999) deﬁned phase 3 as the lahar body, which has the least amount of the original streamﬂow contained within. Phase 3 is also char- acterized by coarse sediment suspensions and is the most likely location for debris ﬂow rheology. Finally, phase 4 is the tail of the lahar where debulking and dilution occurs trans- forming the lahar back into a hyperconcentrated, mixed, or streamﬂow. In order to better characterize and understand these ﬂow events, many in situ applications and instruments have been used in the past (e.g., trip wires, stage gauge, load cells, pore pressure). While many of these tools can yield quick assessments and provide ample warning (e.g., current me- ters, trip wires), they can sometimes be at risk of false detec- tions and equipment damage or loss, and/or they can lack the capability to evaluate multiple pulses or ﬂow events (Arat- tano et al., 1999). Geophysical instruments (e.g., seismome- ters, geophones, infrasound) on the other hand can be in- stalled at a safe distance away from the channel and have not only shown signs of being capable warning systems (e.g., Coviello et al., 2019), but of having the ability to accu- rately estimate ﬂow properties (e.g., Arattano and Marchi, 2005; Doyle et al., 2010; Schimmel et al., 2021) as well as ﬂow dynamics (e.g., Gimbert et al., 2014; Coviello et al., 2018; Walsh et al., 2020). However, in order to fully utilize these instruments, improved interpretation, compre- hension, assessment, and universality is needed. One tech- nique to increase the ability to predict, warn, and estimate the properties and dynamics of ﬂow events is to use all three components of the seismic recording. 1.1 Anatomy of lahars In Aotearoa / New Zealand, there have been numerous cases of large damaging mass ﬂows in modern times. For example, in October 2012, a lake-breakout lahar originating from Te Maari, destroyed hiking trails and forestry, even- tually ﬂowing over 4.5 km to damage and block off High- way 46 (Procter et al., 2014; Walsh et al., 2016). Moreover, on 24 December 1953, the deadliest volcanic mass ﬂow in Aotearoa / New Zealand history occurred, killing 151 peo- ple when a lahar struck a train crossing at the Tangiwai rail bridge, 39.8 km from the Crater Lake on top of Mt. Ruapehu (O’Shea, 1954). The ability to predict and investigate the changing dynamics and properties of large volcanic mass ﬂows as they progress down channel is the ﬁrst step in be- ginning to understand ﬂow mechanisms better and ultimately address the hazards involved to mitigate the risk. When a lahar is created from a lake breakout or outburst ﬂood event, the transition from ﬂood or streamﬂow torrent depends on the erosivity of the channel and the supply of sediment being entrained within the ﬂow (e.g., Scott, 1988; Doyle et al., 2011). An event may start as a highly turbulent low- sediment ﬂow, then transform into a hyperconcentrated ﬂow, and may even eventually “bulk up” to exhibit characteris- tics of a debris ﬂow with the possibility of plug-like (limited internal motion and collisions) or laminar behavior (Scott, 1988, Pierson et al., 1990). At Mt. Ruapehu, the propaga- tional differences of lahars down channel have been observed and characterized in the past (e.g., Cronin et al., 1996, 1999, 2000; Manville et al., 2000; Procter et al., 2010a; Lube et al., 2012). From these studies, models of how lahars bulk up and transition throughout the run-out distance have been pos- tulated. For the lahars in the Whangaehu channel, Cronin et al. (1999) created three four-phase conceptual models based on source distances of 23.5, 42, and > 55 km. The ﬁrst two models are for lahar regimes, whereas the third model de- scribes a lahar almost at its peak run-out distance. In each model, the ﬁrst phase consists of a super charged streamﬂow pulse that ﬂows ahead of the head of the ﬂow and is consid- ered the front of the lahar. This phenomenon has also been noted for debris ﬂows interacting with streamﬂow (Arattano and Moia, 1999). 1.1 Anatomy of lahars Recently, several stud- ies have shown that using all three components is effective in characterizing ﬂow events (e.g., snow-slurry lahars, Cole et al., 2009; snow avalanches, Kogelnig et al., 2011; stream- ﬂow, Roth et al., 2016; landslides, Surinach et al., 2005; la- hars, Walsh et al., 2020; rockfalls, Kuehnert et al., 2021; hy- perconcentrated ﬂows, Walsh et al., 2016). Using the hor- izontal components along with the vertical component can yield additional information about the ﬂow that is not uti- lized if only the vertical component is used. Notably, direc- tionality (cross-channel over channel-parallel) analysis (e.g., Doyle et al., 2010; Walsh et al., 2020) can provide informa- B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1030 and/or the monitoring of the volcano through various meth- ods (e.g., seismology, infrasound, gravity, and gas and wa- ter chemistry). Likewise, for rain-induced ﬂows, using tech- niques such as measuring the amount or intensity of rain (e.g., Capra et al., 2010, 2018) or by monitoring the amount of available material (e.g., Iguchi, 2019) can help forecast when an event may occur. tion about the wetted perimeter, sediment concentration, and number of particle collisions. Furthermore, differing energies and frequency outputs from channel-parallel and channel- perpendicular signals can point to speciﬁc changes within the ﬂow that can provide insights into the internal dynamics (Burtin et al., 2010; Roth et al., 2016). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2 Data tions and surrounding ring plain volcaniclastics (Carrivick and Manville, 2009; Pardo et al., 2012). On top of the vol- cano, above the currently active vent sits a 1 × 107 m3 acidic crater lake (Procter et al., 2010a). The Whangaehu channel is the preferred outlet for Crater Lake water and lahars in recent history (Procter et al., 2012, 2021). The Whangaehu channel is on the eastern ﬂank of Mt. Ruapehu where it runs down across the volcanic ring plane and eventually heads south- west for ∼200 km reaching the Tasman Sea (Fig. 1). The seismic data for the 18 March 2007 lahar were recorded on three seismometers installed at various distances (7.4, 28, 83 km) along the Whangaehu channel (Fig. 1). The data from the three three-component broadband Guralp 6T sen- sors (COLL, RTMT, TRAN) were recorded using a sampling rate of 100 Hz and GPS time stamps (Walsh et al., 2022). For each site, the seismometer axes were installed to true north and the recorded data were rotated to align north as ﬂow parallel (P) and east as the cross-channel direction (T). The seismic data were rotated to align with the channel in order to determine the differences in energy output be- tween the cross-channel and ﬂow-parallel directions (e.g., directionality). The monitoring station Round the Moun- tain Track (RTMT) was installed 4 m from the channel and 7.4 km downstream from the source of the lahar. The lahar arrived at RTMT at 23:36 UTC and had an average velocity of 9.3 ms−1 (Fig. 2a). The Trans Rail Gauge (TRAN) station was installed 28 km from source and 10 m from the chan- nel, which also included a video camera that captured an im- age every 30 s. The lahar arrived at TRAN at 24:35 UTC and had an average velocity of 5.6 ms−1 (Fig. 2d). The Colliers Bridge (COLL) station was installed 10 m from the channel and 83 km from the source. The lahar arrived at COLL at 04:13 UTC and had an average velocity of 4.8 ms−1 (Fig. 2f). Arrival times are based off of images and eye witnesses at each of the monitoring stations. The ﬂow velocity was esti- mated at RTMT and COLL from imagery and at TRAN from a ﬂow meter. Sediment concentration at COLL was measured manually through dip buckets. 2 Data Prior to the events that took place in the morning local time on 18 March 2007, a heavy rainstorm occurred accumulating about 256 mm of water over the 10 h prior to the dam breach that led to the outburst ﬂood (Massey et al., 2010). The in- tense rain caused the Crater Lake level to rise an extra 6.4 m, which caused seepage and extra water to overtop the natural lava formation ledge and enter the Whangaehu channel (Car- rivick and Manville, 2009). At ∼23:18 UTC, the tephra dam collapsed causing 1.3 × 106 m3 of water to ﬂow out of the lake and into the Whangaehu channel (Procter et al., 2010a). The dam was eroded and undercut in multiple stages result- ing in a series of retrogressing landslides along with the main lahar. Since the lahar was caused by lake-breakout dynam- ics and thus contained an abundance of water, the event was classiﬁed as a hyperconcentrated streamﬂow (Procter et al., 2010b). At ∼8.0 km from source, the lahar veloc- ity was recorded at ∼9.5 ms−1 and had an estimated 6 m of downcutting, showing the capability of the lahar to de- posit and erode massive amounts of material (Procter et al., 2010a, b). Furthermore, the 18 March 2007 lahar was one of the most thoroughly monitored lahars ever (Manville and Cronin 2007). In total there were 21 monitoring locations (only three of which had three-component seismometers) set up to measure various lahar properties (e.g., ﬂow monitor, camera, stage height, ﬂow sampling, pore pressure, seismic) along the channel (Keys and Green, 2008; Lube et al., 2012), with the lahar taking over 16 h to eventually travel out to the Aotearoa / New Zealand coast, ∼200 km from the original crater lake source. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 3 Results To examine the multi-component dynamics of the 18 March 2007 lake-breakout event along the Whangaehu channel at three monitoring locations, the data were corrected for in- strument response and split into 10 s time windows. At each recording location, peak spectral frequency (PSF), root mean squared (rms) amplitude, and directionality ratios (DRs) are estimated for each of the 10 s time windows (Table S1 in the Supplement). At each monitoring station the ﬁrst hour of the lahar including 5 min prior to the arrival is shown in all the results except when indicated. Here, we delve into the properties of the 18 March 2007 lake-breakout hyperconcentrated streamﬂow that bulked up to a volume of ∼4.4×106 m3 (Procter et al., 2010a) over the course of 83 km along the Whangaehu channel, originating from Mt. Ruapehu, Aotearoa / New Zealand. The combina- tion of seismic analysis (frequency and directionality) with supplementary measurements (e.g., video, sediment concen- tration) shows how a lahar transforms over time and distance and how using these seismic techniques can help monitor the ever-changing dynamics and properties of a ﬂow event. Fur- thermore, we examine previous models of the evolution of a lahar and compare the model with the seismic data available. 1.2 18 March 2007 lake-breakout event Mt. Ruapehu (2797 ma.s.l.) is the largest stratovolcano in the central North Island of Aotearoa / New Zealand (Fig. 1), which sits at the southwestern end of the Taup¯o Volcanic Zone (TVZ). The volcano has a volume of 110 km3, which is composed of several overlapping cone-building forma- https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 1031 https://doi.org/10.5194/nhess-23-1029-2023 3.1 Frequency analysis In order to examine the PSFs for all three components at each site along the channel, we use the frequency recorded at the maximum amplitude of the frequency spectra for each 10 s running time window (i.e., non-overlapping windowed FFT). The PSF for RTMT (7.4 km from source) shows similar pat- terns between all three components (Fig. 3). The 5 min prior to the arrival of the front of the lahar is characterized by scat- tered PSFs between 20–40 Hz for the cross-channel (Fig. 3a) https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1032 Mt. Ruapehu and the surrounding area located on the central North Island of Aotearoa / New Zealan angaehu channel and the path the 18 March 2007 lahar traveled down. Red triangles denote the three ehu channel at 7.4, 28, and 83 km. Figure 1. Map of Mt. Ruapehu and the surrounding area located on the central North Island of Aotearoa / New Zealand. The blue outline represents the Whangaehu channel and the path the 18 March 2007 lahar traveled down. Red triangles denote the three monitoring stations along the Whangaehu channel at 7.4, 28, and 83 km. and parallel (Fig. 3b) directions, while in the vertical direc- tion (Fig. 3c) the PSF is ∼30 Hz. When the front (streamﬂow pulse/bow wave) of the lahar arrives at the station, the PSF in all three components decreases to ∼5–10 Hz for about 1 min before increasing again to higher frequencies. After the head (peak seismic amplitude) of the lahar passes the station, the PSF in the cross-channel and parallel directions remain be- tween 30 and 40 Hz for the rest of the recording window. In the vertical component, the PSF is scattered between 20 and 40 Hz for ∼15 min after the arrival of the head of the lahar and then becomes narrower, similar to both the cross-channel and parallel components with PSFs between 30 and 40 Hz. https://doi.org/10.5194/nhess-23-1029-2023 https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 1033 h et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals Figure 2. Images from the 18 March 2007 lake break-out lahar from RTMT (a), TRAN (b–e), and COLL (f). Note the transformation of the lahar at TRAN from streamﬂow (b), increased discharge pre-lahar phase 1 pulse/ﬂow front (bow wave) (c), head of the lahar (peak seismic amplitude) (d), and low PSF beginning of the lahar body (see Fig. 4 after 15 min mark) (e). Figure 2. Images from the 18 March 2007 lake break-out lahar from RTMT (a), TRAN (b–e), and COLL (f). Note the transformation of the lahar at TRAN from streamﬂow (b), increased discharge pre-lahar phase 1 pulse/ﬂow front (bow wave) (c), head of the lahar (peak seismic amplitude) (d), and low PSF beginning of the lahar body (see Fig. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 4 after 15 min mark) (e). Further down the channel at station TRAN (28 km from source), the PSFs for all three components show a similar overall pattern (Fig. 4). The pre-lahar PSF distribution in all three components is between 20 and 32 Hz. Like RTMT higher up the channel, the PSF for the front of the lahar at TRAN drops down to around 10 Hz, and when the lahar head arrives (∼10 min, Fig. 4) the PSF increases to ∼30 Hz for parallel (Fig. 4b) and cross-channel (Fig. 4a) directions and between 20 and 30 Hz in the vertical component (Fig. 4c). This decrease to lower frequencies before the head of the lahar at TRAN lasts for about 5 min. After the head of the lahar passes the recording station, the PSF content decreases for ∼15 min to 10–20 Hz for the parallel and cross-channel components and between 10 and 25 Hz for the vertical com- ponents. The PSF after the 30 min mark in Fig. 4 displays a bimodal pattern with frequencies between 10 and 35 Hz, with PSF time windows concentrating most at ∼30 Hz. At the COLL recording station (83 km from source), the PSF distribution shows differing patterns for all three com- ponents (Fig. 5). The PSF in the cross-channel direction (Fig. 5a) depicts a bimodal pattern throughout with a strong lower concentration of time windows at ∼18 Hz and a higher PSF at ∼25 Hz. For the parallel component (Fig. 5b), the pre-lahar signal has a wide PSF range between 12 and 30 Hz. When the lahar arrives, the PSF becomes concentrated at ∼22 Hz for ∼8 min before transforming into a bimodal pat- tern similar to that of the cross-channel PSF, with frequencies between 20 and 30 Hz. In the vertical component (Fig. 5c), the pre-lahar PSF is scattered between 22 and 30 Hz, then as the front of the lahar passes the station the PSF stabilizes around 28 Hz for about 12 min. When the lahar head arrives, the PSF again transitions to more of a scattered pattern dur- ing the highest energy stage of the lahar (Fig. 5, 25–40 min). https://doi.org/10.5194/nhess-23-1029-2023 https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1034 1034 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals Figure 3. Peak spectral frequencies for RTMT (7.4 km from source) for (a) cross-channel (blue), (b) ﬂow-parallel (red), and (c) vertical (green) directions. Bottom row (d) depicts the rms amplitude of the lahar color coded to the same colors as the PSF. The dashed vertical line marks the timing of the lahar front passing the monitoring station. All PSFs and rms amplitudes were calculated using 10 s time windows. Figure 3. Peak spectral frequencies for RTMT (7.4 km from source) for (a) cross-channel (blue), (b) ﬂow-parallel (red), and (c) vertical (green) directions. Bottom row (d) depicts the rms amplitude of the lahar color coded to the same colors as the PSF. The dashed vertical line marks the timing of the lahar front passing the monitoring station. All PSFs and rms amplitudes were calculated using 10 s time windows. Figure 4. Peak spectral frequencies for TRAN (28 km from source) for (a) cross-channel (blue), (b) ﬂow-parallel (red), and (c) vertical (green) directions. Bottom row (d) depicts the rms amplitude of the lahar color coded to the same colors as the PSF. The dashed vertical line marks the timing of the lahar front passing the monitoring station. All PSFs and rms amplitudes were calculated using 10 s time windows. Figure 4. Peak spectral frequencies for TRAN (28 km from source) for (a) cross-channel (blue), (b) ﬂow-parallel (red), and (c) vertical (green) directions. Bottom row (d) depicts the rms amplitude of the lahar color coded to the same colors as the PSF. The dashed vertical line marks the timing of the lahar front passing the monitoring station. All PSFs and rms amplitudes were calculated using 10 s time windows. 3.2 Directionality either installed so that the north component is aligned to be parallel and the east component aligned as perpendicular to the ﬂow, or the components are rotated during the data pro- cessing stage to align with the channel orientation. Further- more, with the channel side installations, attenuational fac- tors can mostly be ignored due to the close proximity to the channel and energy output of the ﬂow event. The directional- ity ratio (DR) can be deﬁned as the cross-channel amplitude divided by the ﬂow-parallel amplitude. A DR > 1 indicates When recording mass ﬂows with three-component sensors, the directionality may be examined due to the sensor being able to record signals in the two horizontal directions. The di- rectionality ratio allows for the determination of which hori- zontal component has stronger energy over the course of the recording window. This is possible because, in channel side deployments for mass ﬂow monitoring systems, the sensor is https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1035 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1035 Figure 5. Peak spectral frequencies for COLL (83 km from source) for (a) cross-channel (blue), (b) ﬂow-parallel (red), and (c) vertical (green) directions. Bottom row (d) depicts the rms amplitude of the lahar color coded to the same colors as the PSF. The dashed vertical line marks the timing of the lahar front passing the monitoring station. All PSFs and rms amplitudes were calculated using 10 s time windows. Figure 5. Peak spectral frequencies for COLL (83 km from source) for (a) cross-channel (blue), (b) ﬂow-parallel (red), and (c) vertical (green) directions. Bottom row (d) depicts the rms amplitude of the lahar color coded to the same colors as the PSF. The dashed vertical line marks the timing of the lahar front passing the monitoring station. All PSFs and rms amplitudes were calculated using 10 s time windows. that the cross-channel amplitude is larger than that of the ﬂow parallel, and vice versa for a DR < 1. 3.2 Directionality Directionality ratios have been used in the past to show rheology changes within ﬂows, where the DR increases when streamﬂow transitions into a lahar (Walsh et al., 2020), and have been hypothesized to be an indicator for ﬂow properties such as sediment con- centration, wetted perimeter, and/or the number of particle collisions within a lahar (Doyle et al., 2010). Figure 6. Directionality ratio plots over time for RTMT (a), TRAN (b), and COLL (c). Vertical rms seismic signals for the three stations are plotted in panel (d), where blue is RTMT, red is TRAN, and green represents COLL. The dashed vertical lines mark the tim- ing of the lahar front passing the monitoring station. All DRs and rms amplitudes were calculated using 10 s time windows. ( y ) The directionality ratios estimated from 10 s non- overlapping running time windows of the rms amplitudes at each seismic station for the 18 March 2007 lake-breakout la- har are shown in Fig. 6. The DR for RTMT (Fig. 6a) dis- plays a DR ≤1 (0.8–1.0) pre-lahar, then decreases (0.7–0.8) as the lahar arrives at the recording station (Fig. 6, dashed line), then as soon as the lahar head arrives, the DR increases to above DR = 1 for ∼2 min. After the peak lahar ﬂood pulse passes RTMT, the DR then proceeds to decrease be- low a DR = 1 for the rest of the recording window. Similar to RTMT, the DR for TRAN starts out with a DR < 1 (0.7– 0.8) and as the lahar front passes, the DR similarly decreases to 0.6–0.7 before increasing to a DR > 1 for ∼5 min when the lahar is at peak energy output starting at about the 10 min mark (Fig. 6d, red line). After the passing of the peak energy, the DR for TRAN decreases below 1 again for the remain- der of the recording window. Further down the channel at COLL (Fig. 6c), the DR before the lahar arrives has a wide range of values between 0.8 and 1.2. When the front of the lahar passes (Fig. 6, dashed line), the DR stabilizes between 0.8 and 1, before increasing slightly when the peak energy of the lahar passes the monitoring site at about the 25 min mark. Figure 6. Directionality ratio plots over time for RTMT (a), TRAN (b), and COLL (c). 3.2 Directionality Vertical rms seismic signals for the three stations are plotted in panel (d), where blue is RTMT, red is TRAN, and green represents COLL. The dashed vertical lines mark the tim- ing of the lahar front passing the monitoring station. All DRs and rms amplitudes were calculated using 10 s time windows. 4.2.1 Phase-1 evolution The ∼10 Hz PSF may be explained by ﬂow pro- cesses (e.g., frictional resistance of the ﬂow by the chan- nel, waves at free surface) (Schmandt et al., 2013; Barriere SCF = Pf 2 f 1f · A(f ) Pf 2 f 1A(f ) , (1) (1) where f is the frequency and A(f ) is the spectral ampli- tude associated with each frequency bin. The spectral spread measures the width of the spectral energy around the SCF (i.e., standard deviation), thus yielding information about the quality of the PSFs (e.g., Rubin et al., 2012; Giannakopoulos and Pikrakis, 2014; Saló et al., 2018). Spectral spread can be estimated by where f is the frequency and A(f ) is the spectral ampli- tude associated with each frequency bin. The spectral spread measures the width of the spectral energy around the SCF (i.e., standard deviation), thus yielding information about the quality of the PSFs (e.g., Rubin et al., 2012; Giannakopoulos and Pikrakis, 2014; Saló et al., 2018). Spectral spread can be estimated by SS = v u u t Pf 2 f 1(f −SCF)2 · A(f ) Pf 2 f 1A(f ) . (2) (2) The computed normalized spectrograms along with SCFs and spectral spreads for each of the three monitoring stations are shown in Fig. 7. For simplicity and comparison, only the ﬂow-parallel data are shown. The normalized spectrograms for every station and component can be seen in Figs. S1–S3 in the Supplement, as well as the values in Table S1. The computed normalized spectrograms along with SCFs and spectral spreads for each of the three monitoring stations are shown in Fig. 7. For simplicity and comparison, only the ﬂow-parallel data are shown. The normalized spectrograms for every station and component can be seen in Figs. S1–S3 in the Supplement, as well as the values in Table S1. The normalized spectrogram for RTMT (Fig. 7a) yields very similar results to that of the PSF (Fig. 3b), where most of the higher spectral amplitudes are at the same frequencies as those of the PSF. Notably, the low ∼10 Hz signal imme- diately before the arrival of the head of the lahar is not only seen in the dominant normalized spectra, but also through the decrease in SCF. Additionally, the PSFs at these time win- dows are contained within the spectral spread (Fig. 7a, black lines). For TRAN, the normalized spectrogram (Fig. 4 Discussion displaying the only time frame with increased spectral ampli- tude distributed throughout the spectral spread (Fig. 7b, after 30 min). This most likely explains the bimodal distribution of PSFs for TRAN in Fig. 4 after the ∼30 min mark. Con- tinuing, the normalized spectrogram for COLL (Fig. 7c) also shows similarities to that of the PSFs in Fig. 5b. The PSFs for COLL range between ∼20 and 30 Hz with a slight bimodal pattern. This same pattern can be seen where the higher spec- tral amplitudes are located (Fig. 7c). Furthermore, the SCF for COLL splits the PSF range and stays at ∼25 Hz during the bimodal phase of the PSF. Overall, with the analysis of the normalized spectrograms, SCFs, and spectral spreads, we conﬁrm that the use of PSFs to describe mass ﬂow dynamics is concise for the 18 March 2007 lake-breakout lahar. displaying the only time frame with increased spectral ampli- tude distributed throughout the spectral spread (Fig. 7b, after 30 min). This most likely explains the bimodal distribution of PSFs for TRAN in Fig. 4 after the ∼30 min mark. Con- tinuing, the normalized spectrogram for COLL (Fig. 7c) also shows similarities to that of the PSFs in Fig. 5b. The PSFs for COLL range between ∼20 and 30 Hz with a slight bimodal pattern. This same pattern can be seen where the higher spec- tral amplitudes are located (Fig. 7c). Furthermore, the SCF for COLL splits the PSF range and stays at ∼25 Hz during the bimodal phase of the PSF. Overall, with the analysis of the normalized spectrograms, SCFs, and spectral spreads, we conﬁrm that the use of PSFs to describe mass ﬂow dynamics is concise for the 18 March 2007 lake-breakout lahar. 4.1 Frequency constraints In order to obtain an understanding of if PSFs are able to properly describe the lahar dynamics (i.e., the weight of the spectral amplitude at the PSF), frequency constraints must be analyzed. To complete this, normalized spectro- grams along with spectral centroidal frequency (SCF) and spectral spreads are computed (e.g., Rubin et al., 2012; Saló et al., 2018). The normalized spectrograms are estimated by normalizing (using the maximum) the spectral amplitude for each 10 s time window of the lahar individually. By nor- malizing each time window, ranges of dominant frequen- cies can be visualized. SCFs are used because they represent the weighted average of the spectra and yield the location (i.e., frequency) of the center of the spectral mass. The SCF of each time window is estimated similar to that of Saló et al. (2018), in which 4.2.1 Phase-1 evolution A lahar propagating down channel can bulk up by collect- ing material from erosion or through the coalescing of mul- tiple pulses to shorten the total length of the lahar (Procter et al., 2010b; Doyle et al., 2011). Lahars can also debulk by depositional means or by the natural elongation of the la- har as it progresses down channel (Doyle et al., 2011; Lube et al., 2012). Considering the 18 March 2007 lake-breakout lahar was a large pulse of water that only mixed with the existing streamﬂow and contained no juvenile material, ex- amining the seismic signatures along the ﬂow path can be used to characterize the evolution and transformation of a lake-breakout event from outburst ﬂood to hyperconcentrated ﬂow and beyond. At RTMT, the seismic signature is domi- nated by the ﬂow-parallel direction (Fig. 3d) with > 30 Hz PSF (Fig. 3b). The exception to this is the time frame im- mediately before the head of the lahar passes, when the PSF decreases to ∼10 Hz. This low-frequency signal can be seen also at TRAN (Fig. 4a–c) and in the ﬂow-parallel direction at COLL (Fig. 5b). However, at COLL the PSF is ∼20 Hz in- stead of 10 Hz as recorded at RTMT and TRAN, most likely due to differing ﬂow properties at 83 km from source. This low PSF before the head of the lahar arrives at each station probably represents the supercharged stream ﬂow pulse (bow wave, Fig. 2c) that is pushed in front of the head of the la- har (i.e., phase 1; see Sect. 1.1) as described by Cronin et al. (1999), where they noticed these same pulses in front of lahar heads for three lahars on Mt. Ruapehu in 1995. Con- versely, this frontal pulse could be from the uplift of stream- ﬂow from the faster moving underﬂow of the lahar (Manville et al., 2000). Furthermore, the low-frequency zone before the head of the ﬂow lengthens as the lahar progresses down- stream, suggesting that lahar elongation can also be seen in the seismic frequency domain (∼1 min at RTMT, ∼5 min at TRAN). B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1036 https://doi.org/10.5194/nhess-23-1029-2023 https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 4.2.1 Phase-1 evolution 7b) is, again, very similar to the PSF in Fig. 4b. The SCF mirrors the pattern of the PSF with higher frequencies for the stream- ﬂow, a decrease for the front of the lahar, increase for the head of the lahar, decrease after the passing of the head, and ﬁnally a slight increase later in the lahar body. The normal- ized spectra yield this same pattern, with the late lahar body https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1037 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1037 Figure 7. Normalized spectrograms for the ﬂow-parallel direction for each of the three monitoring sites along the Whangaehu channel. Red dots represent the spectral centroidal frequency, and black lines show the range of the spectral spread. Note that normalized spectrograms for the other directions can be seen in Figs. S1–S3. Figure 7. Normalized spectrograms for the ﬂow-parallel direction for each of the three monitoring sites along the Whangaehu channel. Red dots represent the spectral centroidal frequency, and black lines show the range of the spectral spread. Note that normalized spectrograms for the other directions can be seen in Figs. S1–S3. Figure 7. Normalized spectrograms for the ﬂow-parallel direction for each of the three monitoring sites along the Whangaehu channel. Red dots represent the spectral centroidal frequency, and black lines show the range of the spectral spread. Note that normalized spectrograms for the other directions can be seen in Figs. S1–S3. amplitude for RTMT (Fig. 3d) is accompanied by an increase to higher PSFs > 30 Hz (Figs. 3a–c and 7a). PSFs > 30 Hz have been shown in the past to either be dominated by turbu- lence or bedload transport (e.g., Gimbert et al., 2014; Roth et al., 2016). The 2007 lake-breakout lahar has been de- scribed as a hyperconcentrated streamﬂow (e.g., Procter et al., 2010b) with low sediment concentration, especially early on before the lake water captured enough material to bulk up and transform. At RTMT, which was only 7.4 km from source, the lahar had not fully bulked up yet and was in a net depositional regime (Procter et al., 2010a). Due to the conditions of the lahar at RTMT (e.g., Fig. 4.2.1 Phase-1 evolution 2a), we surmise the higher PSF content for the peak seismic amplitude is dominated by turbulent-ﬂow-induced noise. Furthermore, the higher PSF content at RTMT (> 30 Hz) compared to TRAN and COLL (∼30 Hz) could be due to the angle of the slope at the recording stations. Gimbert et al. (2014) noted that turbulence noise will dominate over bedload-induced noise on steeper slopes due to an increase in shear velocity. If we use the average ﬂow velocities as a comparison, the lahar at RTMT (9.3 ms−1) ﬂowed faster than at the other two sta- tions (TRAN, 5.6 ms−1; COLL, 4.8 ms−1). Further down the channel at TRAN, the PSF for the peak seismic amplitude is ∼30 Hz for all three components (Fig. 4a–c, Table S1). Again, this high PSF may be attributed to turbulence, as seen by the images taken at TRAN (Fig. 2d). The difference at TRAN is the duration of the higher PSF, where at RTMT the high PSF stays throughout the entirety of the record- ing window, at TRAN the high PSF only lasts for ∼5 min et al., 2015; Bartholomaus et al., 2015) and could be due to the ﬂow at this stage being more sensitive to discharge (e.g., increase in shear velocity and/or ﬂow depth) (Gimbert et al., 2014; Schmandt et al., 2017; Anthony et al., 2018) or, in the case of the underﬂow hypothesis, frictional slid- ing on the channel bed (Huang et al., 2004). The frontal surge or phase 1 of the lahar can be seen in the DR (Fig. 6) as well. For every station along the channel the DR has a slight drop when phase 1 passes the recording station (Fig. 6, dashed line). The elongation of phase 1 also has a correla- tion with distance from source, where the dip in the DR lasts for only ∼1 min at RTMT, ∼5 min at TRAN, and approxi- mately 20 min at COLL. The reason the DR decreases dur- ing phase 1 for the 2007 lahar could be due to the parallel component being more sensitive to ﬂow processes than bed- load forces (Barriere et al., 2015; Roth et al., 2016). During phase 1, discharge increases, sediment concentration is low (Cronin et al., 1999), and streamﬂow dominates resulting in a low DR (e.g., Doyle et al., 2010). 4.2.1 Phase-1 evolution The low DR can also be seen before the arrival of phase 1 due to streamﬂow al- ready occurring in the channel. The higher ﬂow-parallel am- plitude over cross-channel amplitude for streamﬂow has also been noted in the past for lahars at Volcán de Colima, Mexico (Walsh et al., 2020). 4.2.2 Phase-2 evolution Following the low PSF phase 1 (i.e., front of the lahar), the peak seismic amplitude occurs (ﬂow head). The peak seismic 4.2.3 Development of ﬂow phases at TRAN While the lahar at RTMT had not fully developed yet, and at COLL lost energy, at TRAN the 18 March 2007 lahar was a dynamic bulked-up lahar (see Fig. 2b–e). The evi- dence for this is in the PSF content for TRAN (Fig. 4a–c) compared to the other two monitoring sites (Table S1). At TRAN the PSF has a step-up step-down pattern for the ﬁrst 30 min of the lahar passing and then transitions to a bimodal or wide PSF range for the rest of the recording window. As noted above, the low PSF preceding the lahar head arrival is thought to be due to a sensitivity to water transport proper- ties (Fig. 2c, phase 1). The increase to higher PSFs during the peak seismic amplitude may be from particle collisions and/or higher turbulence (Fig. 2d, transition from phase 1 to 2). After the maximum seismic amplitude at TRAN (Fig. 4, ∼10–15 min), the PSF decreases to 10–20 Hz. This drop in PSF after the highest stage and amplitude could be from a more water-ﬂow-dominated regime (seen in the increased parallel amplitude, Fig. 4d, and decrease in DR, Fig. 6b), where turbulence decreases (Fig. 2e), discharge is still high, and the peak sediment concentration has not occurred yet (e.g., Cronin et al., 1999). Likewise, the decrease may also be from greater frictional sliding on the channel bed (Huang et al., 2004). After the decrease to 10–20 Hz PSFs, the PSF displays a bimodal or wide frequency range at ∼28 min (Figs. 4a–c and 7b). As aforementioned for COLL, this PSF pattern could be from both bedload- and water-ﬂow-induced noise. This time frame (phase 3) is also where the peak sed- iment concentration would be (not recorded at TRAN), as noted by Cronin et al. (1999), and thus the PSF would show more high bedload-induced PSFs. This hypothesis also com- pares well with the DR (Fig. 6b), where the cross-channel energy increases starting at ∼25 min, indicating that the sed- iment concentration may be increasing (Doyle et al., 2010). Finally, the wide PSF range later in the recording window (Fig. 4) could also result from the lahar having two distinct The DR at the peak seismic amplitude for all three record- ing stations increases (Fig. 6). The DR for both RTMT and TRAN increases to DR > 1. Doyle et al. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1038 in DR overall when the PSF increases (Fig. 8a). The DR for COLL (Fig. 6c) during this same time frame probably is not due to the number of particle collisions due to the plug-like ﬂow (Fig. 2f), but rather the increase in sediment concen- tration (Fig. 8c). As the sediment concentration increases at COLL the DR starts to increase as well (Fig. 8b). Similar to Doyle et al. (2010), COLL yields a correlation between DR and sediment concentration (R2 = 0.95, Fig. 8b), where higher DRs indicate higher concentrations of sediment con- tained in the ﬂow. Lastly, as noted above, DRs may correlate with PSF or at least indicate differing processes taking place within the ﬂow (Fig. 8a). Lower PSF would produce lower DRs because low PSFs are more sensitive to water-ﬂow pro- cesses (hence higher parallel energy), whereas higher PSFs would produce higher DRs due to higher PSFs being domi- nated by sediment, particle collisions, and turbulence (higher cross-channel energy) (Fig. 8a). (Fig. 4a–c, ∼11–16 min). The difference at TRAN could be from the evolution of the lahar. By the time the lahar reached the monitoring station at TRAN (28 km from source), the la- har was bulked up and had fully developed four phases as described by Scott (1988) or Cronin et al. (1999) (Fig. 2c– e; see Sect. 4.2.3). By time the lahar reached COLL 82 km from source (Fig. 5), the peak seismic amplitude was asso- ciated with PSFs between 15 and 30 Hz, with bimodal pat- terns in the horizontal components and a tighter spread in the vertical component (∼27–29 Hz). At COLL, the lahar had converted into a plug-like ﬂow with lower turbulence, and hence the higher PSFs are most likely associated with bed- load transport (Fig. 2f). Furthermore, Burtin et al. (2010) and Roth et al. (2016) noted that when the vertical component has greater seismic amplitudes than the horizontal compo- nents, bedload dominates. This same amplitude feature can be seen at COLL (Fig. 5d, past ∼25 min), where the verti- cal energy is greater than each of the horizontal components. The bimodal frequency pattern of the horizontal components (Fig. 5a and b) is likely from the recording of both water- ﬂow noise (lower PSF) and bedload transport (higher PSF). This also explains why the vertical component does not show the same bimodal frequency pattern. Barriere et al. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals (2015) described the parallel component as being more sensitive to ﬂow properties (e.g., discharge, depth, shear velocity), and Doyle et al. (2010) noted that the cross-channel component is likely dominated by the amount of turbulence (e.g., water and particles acting on the channel walls), thus the reason- ing behind the differing PSF patterns between components. This PSF feature is similar to the lahars recorded by Walsh et al. (2020), where the cross-channel PSF is conﬁned within a narrow band of around 15–20 Hz and the ﬂow-parallel PSF is more bimodal (10–40 Hz). At COLL, the cross-channel PSF (Fig. 5a) is dominated by PSFs at ∼18 Hz (lower than ver- tical component at ∼28 Hz, Fig. 5c), with the ﬂow parallel between 20 and 30 Hz (Fig. 5b). https://doi.org/10.5194/nhess-23-1029-2023 https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 4.2.3 Development of ﬂow phases at TRAN (2010) noted that higher wetted perimeters will increase the DR, which can be seen at TRAN for the 18 March 2007 lake-breakout la- har (Figs. 2d and 6 peak DR and/or rms amplitude). Con- versely, the DR decreases after the peak seismic amplitude, while the wetted perimeter is still high (Fig. 2d and e). While the wetted perimeter may be a factor in increasing cross- channel energy and thus the DR, the more likely explana- tion for the 18 March 2007 lahar might be the higher level of particle collisions and turbulence at the peak seismic am- plitude. More turbulent particle collisions would increase the DR (e.g., Doyle et al., 2010) due to more lateral excitation within the ﬂow and against the channel walls increasing the cross-channel signal. The increase in collisional energy also relates well with the PSF, as higher PSF correlates to an increase in the number of interﬂow collisions as shown by Huang et al. (2004), and may also explain the slight increase https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1039 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1039 Figure 8. Plots of (a) comparing PSF and DR at TRAN, (b) sediment concentration and DR at COLL (R2 = 0.95), and (c) seismic amplitude (black line) with sediment concentration (purple line) depicting the lag in sediment at COLL. Note on panel (a) parallel (red dots) and cross- channel (blue dots) PSFs display three different zones (black circles). Also note that at COLL the ﬁrst sediment concentration measurement did not occur until the 30 min mark. Figure 8. Plots of (a) comparing PSF and DR at TRAN, (b) sediment concentration and DR at COLL (R2 = 0.95), and (c) seismic amplitude (black line) with sediment concentration (purple line) depicting the lag in sediment at COLL. Note on panel (a) parallel (red dots) and cross- channel (blue dots) PSFs display three different zones (black circles). Also note that at COLL the ﬁrst sediment concentration measurement did not occur until the 30 min mark. layers as described by Cronin et al. (2000), where there is a wide, more dilute, ﬁner-grain top layer and a channelized sediment-rich layer on the bottom. The two-layer model can apply to TRAN because the lahar at this monitoring station overtook the channel (Fig. 4.2.3 Development of ﬂow phases at TRAN 2d and e) and proceeded to ﬂow horizontally outward forming the surface layer described by Cronin et al. (2000). into a plug-like slurry ﬂow. Each of these ﬂow types yields differing PSF ranges and patterns due to the relationship be- tween the channel geometry, sediment concentration, turbu- lence, and bedload transport. While the lahar at different sta- tions along the channel may have differing PSF content, we also show that the lahar elongates, and a predictable model (e.g., Cronin et al., 1999) can be used with and shown in the seismic data. Being able to apply such a model may yield some relevance of universality in terms of warning systems at different distances away from the mass ﬂow source, whereas, shown above, the ﬂow phases at each monitoring station can be seen, but at differing lengths and times in the seismic sig- nal (e.g., Fig. 6). To better visualize this concept, conceptual models based off of the Cronin et al. (1999) models are cre- ated for each of the three seismic stations for the 18 March 2007 lahar (Fig. 9). In the conceptual models for the 2007 lahar, the aforementioned elongation of the frontal pulse or bow wave (phase 1) and head of the lahar (phase 2) is shown, along with the differences and similarities between the prop- erties of the lahar at the three seismic monitoring sites. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1040 Figure 9. Conceptual models for the 18 March 2007 lahar at each of the three monitoring stations along the Whangaehu channel, depicting ﬂow type and the estimated seismic properties at each ﬂow phase. (a) RTMT 7.4 km from source, (b) TRAN 28 km from source, and (c) COLL 83 km from source. Flow types (FTs) are as follows: streamﬂow (SF), bow wave streamﬂow (BW), mixed ﬂow (MF), hyperconcentrated ﬂow (HF), transitional ﬂow (TF), and sediment-laden streamﬂow (SLF). Note that decreased (D), in- creased (I), high (H), low (L), and mixed (M) are notations for direc- tionality ratios and peak spectral frequency estimates. See Table S1 for value ranges for each property. the lag in sediment concentration, or differences in peak amplitude with peak discharge. Phase 1 (frontal streamﬂow surge) of the model proposed by Cronin et al. (1999) was based on a hyperconcentrated ﬂow interacting with stream- ﬂow, but has also been shown for debris ﬂows as well (e.g., Arattano and Moia, 1999). Arattano and Moia (1999) showed at Moscardo Torrent, Italy, through a hydrograph that there was a precursory surge ahead of the debris ﬂow that was not seen in the seismic record. Similarly, at Ruapehu, for the 18 March 2007 lahar, at each of the three stations there is little evidence or rise in the seismic amplitude that would in- dicate that there was a precursory surge or phase 1 (Figs. 3– 5, bottom panel), which could be problematic for detec- tion methods that use amplitude thresholds or short-time- average vs. long-time-average (STA/LTA) algorithms. Con- versely, the surge ahead of the lahar can be seen in both the PSF analysis (drop to low frequencies) and in the DR (de- crease in DR) right before the peak seismic amplitude ar- rives. This shows that when monitoring for future events not only the amplitude should be used but other analysis (e.g., PSF, DR) as well, otherwise there could be a delay in the detection of an event. Figure 9. Conceptual models for the 18 March 2007 lahar at each of the three monitoring stations along the Whangaehu channel, depicting ﬂow type and the estimated seismic properties at each ﬂow phase. (a) RTMT 7.4 km from source, (b) TRAN 28 km from source, and (c) COLL 83 km from source. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals This same feature can be seen at each of the three monitoring sites for the 18 March 2007 event (Fig. 6), indicating that differing ﬂow types will still show this DR pattern within the same ﬂow and at other channels. To fur- ther show this, there were three natural non-lake-breakout eruption-based lahars that occurred in the Whangaehu chan- nel in September 2007 (for more details on the lahars see Cole et al., 2009; Kilgour et al., 2010) and recorded on the seismometer at RTMT. The DR for the September events starts with streamﬂow with a DR < 1, and when the ﬁrst lahar arrives the DR increases to > 1 and as the lahar fully passes, the DR decreases to < 1 again (Fig. 10a). As the second la- har arrives at RTMT (Fig. 10, second dashed line), the DR increases to > 1 again. After the second lahar passes the DR deceases once again back below DR < 1. Finally, as the third lahar arrives (Fig. 10, third dashed line) the DR yet again increases above 1 for the entirety of the event. DR for streamﬂow is < 1 and then increases when the head of the lahar arrives. This same feature can be seen at each of the three monitoring sites for the 18 March 2007 event (Fig. 6), indicating that differing ﬂow types will still show this DR pattern within the same ﬂow and at other channels. To fur- ther show this, there were three natural non-lake-breakout eruption-based lahars that occurred in the Whangaehu chan- nel in September 2007 (for more details on the lahars see Cole et al., 2009; Kilgour et al., 2010) and recorded on the seismometer at RTMT. The DR for the September events starts with streamﬂow with a DR < 1, and when the ﬁrst lahar arrives the DR increases to > 1 and as the lahar fully passes, the DR decreases to < 1 again (Fig. 10a). As the second la- har arrives at RTMT (Fig. 10, second dashed line), the DR increases to > 1 again. After the second lahar passes the DR deceases once again back below DR < 1. Finally, as the third lahar arrives (Fig. 10, third dashed line) the DR yet again increases above 1 for the entirety of the event. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals Flow types (FTs) are as follows: streamﬂow (SF), bow wave streamﬂow (BW), mixed ﬂow (MF), hyperconcentrated ﬂow (HF), transitional ﬂow (TF), and sediment-laden streamﬂow (SLF). Note that decreased (D), in- creased (I), high (H), low (L), and mixed (M) are notations for direc- tionality ratios and peak spectral frequency estimates. See Table S1 for value ranges for each property. Using all three components of the seismometer can be very beneﬁcial in lahar monitoring. The above-mentioned DR analysis can only be completed with horizontal record- ing, and analyzing PSF in each component can yield crit- ical information about the ﬂow properties and dynamics. Examining the seismic amplitude differences can generate signiﬁcant discoveries; for example, when the vertical com- ponent is stronger than the horizontal components, bedload may dominate over turbulence noise (Burtin et al., 2010). Greater ﬂow-parallel signals may indicate higher water trans- port noises (Barriere et al., 2015), while higher cross-channel signals could be caused by increased interﬂow particle colli- sions and ﬂow-channel wall interactions (Doyle et al., 2010). While using the differences in each component can be use- ful, there are also some concerns. Channel geometry and bed conditions can alter the seismic signal (e.g., Coviello et al., 2019; Marchetti et al., 2019). Additionally, the ﬂow- parallel direction can be inﬂuenced by the lahar that has al- ready passed, the lahar at the station, and the lahar arriving. Furthermore, the tilt of the seismometer may play a large role in determining which component is stronger (e.g., Anthony et al., 2018). In the case of the 18 March 2007 lahar, a large pulse of water passed the monitoring stations which may ex- plain why the parallel component is stronger than the other two components at RTMT (Fig. 3d) and TRAN (Fig. 4d). At COLL, the lahar had elongated, lost energy, and thus showed more decreased ﬂow-parallel energy compared to the previ- ous two stations (Fig. 5d). In the cross-channel direction, if a ﬂow overtops the channel, the amplitude would presum- ably be dampened. This may be the case at TRAN where both the ﬂow-parallel and vertical directions are more ener- getic than the cross-channel amplitude after the passing of the head and breaking out of the channel occurred (Figs. 2d DR for streamﬂow is < 1 and then increases when the head of the lahar arrives. 4.3 Implications for monitoring The main goal of this research is to contribute to deﬁning bet- ter monitoring criteria for dangerous mass ﬂow events. The data described above are part of a larger collection of moni- toring data collected over the entire length of the Whangaehu channel, consisting of 21 monitoring sites and years of prepa- ration (e.g., Manville and Cronin, 2007; Keys and Green, 2008). Due to this, the ability to accurately estimate the prop- erties of the lahar at various stages along its path is possible. When it comes to ﬂow events of any size, the ability to un- derstand how the dynamics change with distance along the channel is important for warning and future hazard mitiga- tion. We show here that a lake-breakout event can start out as an outburst ﬂood, bulk up into a hyperconcentrated ﬂow, then eventually elongate and entrap enough sediment to transform Another implication for future warning is the implementa- tion of three-component sensors and the use of DRs for chan- nels that have streamﬂow. Walsh et al. (2020) showed that for lahars ﬂowing in La Lumbre channel at Volcán de Colima the https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals For many mass ﬂows and especially those that ﬂow into channels with preexisting streamﬂow, the peak seismic am- plitude does not always coincide with the arrival of the mass ﬂow, and thus may not be the most reliable for event de- tection or warning (e.g., Arattano and Moia, 1999; Cole et al., 2009). These observations may be due to a frontal surge, https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals 1041 Figure 10. (a) Directionality ratio for the time sequence of the three lahars that occurred on 25 September 2007. (b) Rms amplitude of the seismic record at RTMT during the timing of the three September lahars. Note that the dashed black lines represent the timing of each lahar arriving at the monitoring site. Figure 10. (a) Directionality ratio for the time sequence of the three lahars that occurred on 25 September 2007. (b) Rms amplitude of the seismic record at RTMT during the timing of the three September lahars. Note that the dashed black lines represent the timing of each lahar arriving at the monitoring site. and 4d). Another concern when using the horizontal com- ponents of a seismometer is the effects shallow layers may have on the site response of the sensor. This is especially true when a sensor is installed on soft or loose sediment (e.g., soil, ﬂuvial/alluvial deposits). To test for potential effects by shal- low layer fundamental frequencies, H/V analysis of ambi- ent noise (streamﬂow dominant) was conducted (see Supple- ment). For RTMT, the H/V results depict a broad-frequency peak between 5 and 15 Hz with a local maximum at ∼8 Hz (Fig. S4a). Comparing the H/V frequency with the PSF of RTMT (Fig. 3), the only overlap is when the front of the la- har passes the station where the PSF decreases for ∼1 min before the head of the lahar arrives. The H/V analysis for TRAN has a multi-broad-peak shape, with frequency peaks at ∼14 and ∼28–35 Hz (Fig. S4b). While these frequencies are similar to PSF values for TRAN (Fig. 4), the H/V analysis has no distinguishable fundamental frequency and contains large errors, and no frequency peak has a H/V ampliﬁca- tion > 2. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals In order for a H/V frequency peak to be considered ideal, generally the ampliﬁcation must be greater than 2 and the standard deviation lower than a factor of 2 (SESAME, 2004). The H/V ampliﬁcation for COLL displays a broad- frequency peak between 13 and 18 Hz, with a local maximum at ∼18 Hz (Fig. S4c). Comparing the PSFs at COLL (Fig. 5), only the cross-channel direction has signiﬁcant PSF values in the same frequency range (∼18 Hz band). With all three sta- tions not yielding distinct H/V fundamental frequencies, we surmise that the PSF content for the 18 March 2007 lake- breakout lahar is most likely dominated by the large ﬂow passing by the seismic sensor rather than large site ampli- ﬁcation effects from a shallow layer. While this may be the case, there is still the possibility that some of the PSF values could be due to local effects and should not be considered in the lahar analysis, e.g., the low PSFs at RTMT between 15 and 20 min (Fig. 3a and b), at TRAN contributing to some of the “jumping” in PSF content (Fig. 4a–c), or in the mostly dominant 15–20 Hz PSF in the cross-channel direction at COLL (Fig. 5a). Conversely, SCF values at each station do not reside in the broad H/V frequency range at any station (Fig. 7), which may further support the hypothesis that al- most all of the recorded frequencies are indeed produced by the lahar. With the use of horizontal components becoming common in mass ﬂow monitoring, future three-component analyses of mass ﬂows should consider estimating H / V ra- tios or use other site response methods (e.g., spectral ratio analysis) in order to identify whether near-surface structures may affect the recorded ﬂow data. Overall, all these con- cerns can and should be tested to estimate potential error in three-component methods. Nevertheless, using all three com- ponents of the seismometer can enhance the productivity of warning systems and, if possible, should be used instead of single-component sensors. and 4d). Another concern when using the horizontal com- ponents of a seismometer is the effects shallow layers may have on the site response of the sensor. This is especially true when a sensor is installed on soft or loose sediment (e.g., soil, ﬂuvial/alluvial deposits). B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals To test for potential effects by shal- low layer fundamental frequencies, H/V analysis of ambi- ent noise (streamﬂow dominant) was conducted (see Supple- ment). For RTMT, the H/V results depict a broad-frequency peak between 5 and 15 Hz with a local maximum at ∼8 Hz (Fig. S4a). Comparing the H/V frequency with the PSF of RTMT (Fig. 3), the only overlap is when the front of the la- har passes the station where the PSF decreases for ∼1 min before the head of the lahar arrives. The H/V analysis for TRAN has a multi-broad-peak shape, with frequency peaks at ∼14 and ∼28–35 Hz (Fig. S4b). While these frequencies are similar to PSF values for TRAN (Fig. 4), the H/V analysis has no distinguishable fundamental frequency and contains large errors, and no frequency peak has a H/V ampliﬁca- tion > 2. In order for a H/V frequency peak to be considered ideal, generally the ampliﬁcation must be greater than 2 and the standard deviation lower than a factor of 2 (SESAME, 2004). The H/V ampliﬁcation for COLL displays a broad- frequency peak between 13 and 18 Hz, with a local maximum at ∼18 Hz (Fig. S4c). Comparing the PSFs at COLL (Fig. 5), only the cross-channel direction has signiﬁcant PSF values in the same frequency range (∼18 Hz band). With all three sta- tions not yielding distinct H/V fundamental frequencies, we surmise that the PSF content for the 18 March 2007 lake- breakout lahar is most likely dominated by the large ﬂow passing by the seismic sensor rather than large site ampli- ﬁcation effects from a shallow layer. While this may be the Finally, implementation of these new results into new or existing mass ﬂow warning systems must be discussed. In an ideal setup, to remove any doubt about the recorded signal, machine learning techniques should be used to separate the mass ﬂow noise from other non-ﬂow noises (e.g., environ- mental, human induced, earthquakes). For instance, recently Wenner et al. (2021) used a supervised random forest algo- rithm to classify differing sources in a debris ﬂow setting. Once the mass ﬂow source has been classiﬁed, integrating automated DR and PSF analysis would be quick and straight- B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. This work was supported by the Resilience to Nature’s Challenges – New Zealand National Science volcano pro- gram of research. We would also like to thank all the people from Massey University, Horizons Regional Council, NIWA, and the De- partment of Conservation that collected data and set up monitoring locations all along the channel in preparation for and during the la- har. A ﬁnal special thanks to Kate Arentsen for editorial support. 5 Conclusions At 23:18 UTC on 18 March 2007, Mt. Ruapehu produced the biggest lahar in Aotearoa / New Zealand in over 100 years, causing 1.3 × 106 m3 of water to ﬂow out of the Crater Lake and rush down the Whangaehu channel, ﬂowing for over 200 km to the Tasman Sea. Seismic analysis at three monitoring locations along the channel (7.4, 28, and 83 km) yielded an understanding of how ﬂow type and processes of the lahar evolve with distance. The proximal lahar was a highly turbulent outburst ﬂood, which generated high PSF content in all three components. Further along the channel after the lahar had bulked up, the PSF content was vari- able and showed changes in the ﬂow regime. Finally, at the most distal monitoring station, the lahar had lost energy and transformed into a slurry-type ﬂow where the PSF content became more bedload-dominant. Additionally, directionality ratios from all three sites along with data from additional la- hars yielded strong evidence that DRs can be used for warn- ing systems when there is streamﬂow present in the channel. Furthermore, PSFs and DRs show evidence of a pre-lahar water pulse that may be concealed in the raw seismic data but has been observed visually. Ultimately, the use of three- component broadband seismic analysis for the 18 March 2007 lahar at Mt. Ruapehu may lead to more accurate and Review statement. This paper was edited by Giovanni Macedonio and reviewed by Emma Surinach and three anonymous referees. B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals advanced real-time warning systems for mass ﬂows through the use of frequency and directionality around the world. forward. Implementation of these techniques would be sim- ilar to other seismic analysis or detection methods, such as a STA/LTA (e.g., Coviello et al., 2019) or a number of fre- quency detection algorithms (e.g., Rubin et al., 2012) where real-time analysis of set time windows is used to determine if there has been a change in the seismicity along the chan- nel. The system could be programmed to identify changing features in the ﬂow automatically by analyzing the content of each window, as well as comparing previous time win- dows. The analysis of continual data could then be fed into machine learning algorithms (e.g., Rubin et al., 2012; Wen- ner et al., 2021) to increase not only the conﬁdence of de- tection, but also the characterization of ﬂow behavior. One of the main discoveries of this research was the evolution of seismic signals produced by the lahar as the ﬂow moved fur- ther from its source. The changes in the seismic signal along with the ﬂow characteristics may be able to help hazard and forecast modeling through the use of numerical models (e.g., Mead et al., 2021). Modern ﬂow hazard assessment is based on numerical models that use potential energy equations of a large non-changing mass sliding down slope with limited in- puts for how the ﬂow may evolve over time as starting inputs. This can lead to errors in risk mitigation and hazard assess- ments. The ﬁndings shown above on how the 18 March 2007 lahar evolved over 83 km at Mt. Ruapehu will help to im- prove mass ﬂow modeling in the future by enabling modelers to add constraints or more inputs on how a mass ﬂow might evolve, leading to improved forecasts and hazard assessment. Data availability. The data used in this publication can be found at: https://theghub.org/resources/4890 (last access: 10 February 2023; Walsh et al., 2022). Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/nhess-23-1029-2023-supplement. Author contributions. BW performed seismic analysis and drafted the paper, CL organized and prepared data, and JP created the vi- sual location representation of the event. All participating authors contributed to the discussions and editing of the draft of the paper, as well as approving the ﬁnal edition. Competing interests. https://doi.org/10.5194/nhess-23-1029-2023 https://doi.org/10.5194/nhess-23-1029-2023 Nat. Hazards Earth Syst. Sci., 23, 1029–1044, 2023 1042 B. Walsh et al.: Characterizing the evolution of mass ﬂows through seismic signals Lett., 36, L09405, https://doi.org/10.1029/2009GL038030, 2009. Manville, V. and Cronin, S.: Breakout lahar from New Zealand’s crater lake, EOS T., 88, 441–456, 2007. 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https://openalex.org/W2589703983	https://europepmc.org/articles/pmc5400616?pdf=render	English	null	ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy	Oncotarget	2,017	cc-by	11,146	ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy p Keywords: ATM kinase, breast cancer stem cells (BCSCs), mammospheres, autophagy, ATG4 Received: July 05, 2016 Accepted: January 23, 2017 Published: February 20, 2017 ABSTRACT The efficacy of Ataxia-Telangiectasia Mutated (ATM) kinase signalling inhibition in cancer therapy is tempered by the identification of new emerging functions of ATM, which suggests that the role of this protein in cancer progression is complex. We recently demonstrated that this tumor suppressor gene could act as tumor promoting factor in HER2 (Human Epidermal Growth Factor Receptor 2) positive breast cancer. Herein we put in evidence that ATM expression sustains the proportion of cells with a stem-like phenotype, measured as the capability to form mammospheres, independently of HER2 expression levels. Transcriptomic analyses revealed that, in mammospheres, ATM modulates the expression of cell cycle-, DNA repair- and autophagy-related genes. Among these, the silencing of the autophagic gene, autophagy related 4C cysteine peptidase (ATG4C), impairs mammosphere formation similarly to ATM depletion. Conversely, ATG4C ectopic expression in cells silenced for ATM expression, rescues mammospheres growth. Finally, tumor array analyses, performed using public data, identify a significant correlation between ATM and ATG4C expression levels in all human breast cancer subtypes, except for the basal-like one. Overall, we uncover a new connection between ATM kinase and autophagy regulation in breast cancer. We demonstrate that, in breast cancer cells, ATM and ATG4C are essential drivers of mammosphere formation, suggesting that their targeting may improve current approaches to eradicate breast cancer cells with a stem-like phenotype. www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ ATM expression determines the ability to form mammospheres and promotes breast cancer- stem like phenotype Recent works suggest that HER2 is a driver of cancer stem-like phenotype in luminal estrogen receptor-positive (ERp) breast cancers, in the absence of HER2 gene amplification, and in breast cancers with amplification of HER2 receptor [21, 22]. In order to analyse the impact of ATM kinase on formation of cells with a stem-like phenotype in breast cancer, we choose as cancer model system: 1) mammospheres derived from luminal estrogen receptor-positive HER2- low breast cancer cell line (MCF7); 2) mammospheres derived from luminal estrogen receptor-positive HER2- overexpressing breast cancer cell line (MCF7-HER2). To avoid off-target effects, ATM expression was genetically downregulated by two specific shRNA interference in both cell lines, using lentiviral vectors, (Figure 1A) and in vitro mammospheres formation was assayed by measuring the ability to grow in low serum and anchorage independent conditions [20] (Figure 1B). In these experiments, we observed that the downregulation of ATM led to a decrease in mammospheres number and size in both MCF7 cell lines with the two ATM target sequences (Figure 1B). On average, 200 mammospheres formed from 8000 MCF7 cells (~2,5% of Sphere Forming Efficiency, SFE) seeded under serum-free suspension conditions and 400 mammospheres formed from 8000 MCF7-HER2 cells (~5 %, SFE) (Figure 1B), which is in accordance with the crucial role of HER2 receptor as promoter of mammospheres formation. Interestingly, the silencing of ATM caused a ~50% reduction in mammospheres number and diameter in both cell lines (Figure 1B). In addition to sphere formation, the colony formation capacity of dissociated mammospheres seeded in 2D adherent plate was analysed. As shown in Figure 1C, silencing of ATM expression significantly reduced the number of colonies grown in both cell lines. It is well established that, cells grown as mammospheres, compared to cells grown in adherent conditions, significantly upregulate the expression of genes driving the stem like phenotype [23]. In line with this observation, we could show that the expression of HER2 and of several stem cell markers such as SRY-box 2 (SOX2), POU class 5 homeobox 1 (POU5F1; best known as OCT4) and Nanog homeobox (NANOG) was elevated in mammospheres, derived from both cell lines, compared to adherent cells (Figure 2A). The role of autophagy in carcinogenesis remains elusive. Autophagy may promote or counteract tumor initiation and progression depending on the specific context [14]. RESULTS Ataxia-Telangiectasia Mutated (ATM) is a multifunctional kinase that plays complex and controversial roles in cancer. ATM is historically considered a tumor suppressor gene for its central role in the DNA damage response (DDR) [4, 5]. Indeed, this kinase is considered as a good target for cancer therapies and, consistently with its DDR function, also drives the therapeutic resistance of CSCs [6-8]. We have recently identified ATM as a promoter of HER2 tumorigenicity in breast cancer [9], suggesting a dual function of ATM in cancer. In particular, we showed that abrogating ATM function significantly impaired HER2-dependent tumorigenicity in vitro and in vivo also uncovering a novel cancer-related function of ATM as regulator of HER2 receptor stability [9]. Importantly, ATM plays non-nuclear functions in addition to the DDR signalling that may contribute to its dual, opposing role in cancer [6-8]. The emerging role of ATM in the regulation of autophagy is intriguing [10-12], although its impact on cancer progression has been poorly investigated so far. Interestingly, by using Atm-/-Becn1+/- mice model, it was demonstrated that the genetic inactivation of the autophagic gene Beclin 1 results in a significant delay of lymphoma and leukaemia onset normally occurring in Atm-deficient mice, due to the rescue of mitochondrial abnormalities and not of the DDR [13]. This observation supports the hypothesis that ATM kinase and autophagy could talk each other in the regulation of cancer progression. INTRODUCTION breast cancer, and they were shown to be particularly resistant to conventional anticancer therapy, which may contribute to treatment failure and tumor relapse [2]. The fate of cancer stem cells is determined by the “stem cell niche” in the tumor, which comprises stromal cells, cytokines, and growth factors. Moreover this niche is characterized by starvation and hypoxic conditions that are considered critical niche factors to promote invasive growth of tumors. The observation that CSCs have a specific regulation by the microenvironment suggests the The cancer stem cell hypothesis proposes that cancers arise from and are maintained by a small population of cancer-initiating cells residing within tumor mass. These cells are characterized by the re-expression of stem cell markers and self-renewal potential, and have therefore been named cancer stem cells (CSCs) or more exactly cancer stem-like cells. [1]. In recent years, CSCs have been identified in multiple cancers, including www.impactjournals.com/oncotarget Oncotarget 21692 novel insights into the molecular mechanisms underlying mammosphere formation attributing a previously unrecognised role in this process to the crosstalk between ATM kinase and ATG4C autophagic gene in breast cancer progression. presence of a peculiar metabolic demand and a specific response to environmental stresses in these cells compared to the bulk tumor [3]. Molecular mechanisms involved in their regulation, maintenance and resistance to therapies are peculiar and specific for this subpopulation of cells in the tumor and so targeting strategies for the destruction of CSCs specific signalling pathways provides a novel opportunity for cancer research [3]. ATM expression determines the ability to form mammospheres and promotes breast cancer- stem like phenotype Recent evidence support the idea that autophagy could act as a cytoprotective process to augment CSC survival under conditions of nutrient or growth factor starvation, metabolic stress, and hypoxia within the “stem cell niche” in the tumor [15, 16]. Consistently, autophagy is an important driver of stem- like phenotype in breast cancer [17-19] and autophagic genes such as ATG4A and Beclin 1 play a role in breast (B)CSC maintenance, further supporting the idea that BCSCs employ autophagy for promoting their survival and growth [18, 19]. Overall these evidences led us to investigate the role of ATM kinase in the regulation of the stem-like phenotype in breast cancer. Herein, we utilised the preparation of 3D spheroid cultures, also known as “mammospheres” [20], as functional assay to enrich for a population of cells with a stem-like phenotype to investigate the role of ATM in the regulation of Breast Cancer Stem-like cells. Our results give www.impactjournals.com/oncotarget Oncotarget 21693 genes in mammospheres shATM versus shCTR, involved in these pathways (Supplementary Table 2). Validation of the expression pattern of 9 genes (4 upregulated and 5 downregulated), from our 20 selected genes, obtained by microarray analysis was performed by qRT-PCR (Figure 3C and Supplementary Figure 1A-1B). In all cases, the trend of fold change of expression identified in the microarrays studies was confirmed by qRT-PCR experiments (Figure 3C). Interestingly, when we compared the expression of our selected genes in mammospheres with respective adherent parental cell lines, we observed that silencing ATM in mammospheres resulted in an expression profile more similar to that of adherent parental cells, supporting the idea that ATM expression could modulate essential genes involved in mammospheres formation (Supplementary Figure 1A-1B) [19]. Similarly, the level of ATM mRNA was upregulated in mammospheres, when compared to adherently cultured cells (Figure 2A). However, the selective silencing of ATM expression, with both target sequences using lentiviral vectors, resulted in the impairment of SOX2 , but not of OCT4 and NANOG mRNA expression in mammospheres (Figure 2B). To further investigate the role of ATM expression in driving the stem-like phenotype in breast cancer, we evaluated, by flow cytometry, the percentage of aldehyde dehydrogenase (ALDH) activity-positive cell population in our cell lines; indeed ALDH activity is validated as a well-known marker of the sub-population of cells with stem-like characteristics [24]. Consistently with previous reports [10], HER2 overexpression enhanced ALDH activity (Figure 2C) [10]. ATM expression determines the ability to form mammospheres and promotes breast cancer- stem like phenotype More interestingly, we could show that the genetic downregulation of ATM expression, with both shRNA sequences, resulted in the reduction of ALDH activity in both cell lines (Figure 2C and Supplementary Table 1). Overall, while these data indicate a role of ATM expression in the modulation of mammospheres formation, probably this is independent of HER2 expression levels. They also suggest that ATM exerts a mild effect on the expression of stemness markers. Autophagic gene ATG4C promotes mammospheres formation It was recently reported that some autophagic regulator genes, such as Beclin-1 and ATG4A, modulated the autophagic flux in mammospheres derived from MCF7 cells, and that the deregulation of this feature impaired mammospheres forming capacity [18, 19]. By analyzing our microarray and qRT-PCR expression data, we observed that the expression of the autophagic gene ATG4C was significantly downregulated in mammospheres interfered for ATM expression, compared to control ones (Supplementary Table 2 and Figure 3C). Moreover, ATG4C mRNA expression was upregulated in cells grown as mammosphere compared to the ones grown in adherent conditions (Supplementary Figure 1A). Overall, these data suggest that ATG4C may promote mammospheres formation. Identification of ATM-dependent regulated gene set in mammospheres To further uncover the molecular mechanism involved in ATM-mediated regulation of mammospheres formation, we performed a gene expression analysis. We used two different RNA interference sequences targeting ATM and we performed the experiment as biological triplicate: three independent lentiviral infections were carried out on both cell lines (MCF7 and MCF7-HER2) silenced or not for ATM, grown as mammospheres. Microarray based expression profiling, pooling all the experiments, revealed that 550 probes for MCF7 and 196 probes for MCF7-HER2 were significantly expressed in mammospheres derived from cells silenced for ATM in a differential fashion compared to those obtained from control cell lines, interfered with shGFP (Figure 3A). As ATM impairs mammospheres formation independently of HER2 expression (Figure 1), we focused our analysis on the differential expression of 115 genes intersected in MCF7 and MCF7-HER2, which are downregulated or upregulated upon ATM expression silencing (Figure 3A). We hypothesized that these genes could be responsible for ATM-dependent reduction of mammospheres formation irrespective of HER2 expression levels. Functional annotation of these 115 genes using DAVID (Database for Annotation, Visualization and Integrated Discovery) revealed that they are mainly associated to mitosis, regulation of protein kinase, cell cycle, DNA repair and cell death (Figure 3B). In Supplementary Table 2, we selected the first 10 upregulated and first 10 downregulated p To further validate our hypothesis we downregulated ATG4C expression by specific RNA interference selective for this isoform (Figure 4A and Supplementary Figure 2C). Remarkably, we could show that the downregulation of ATG4C expression impairs mammospheres forming ability (Figure 4B), similarly to what previously observed upon ATM expression silencing (Figure 1A-1B). ATG4C is one on the four members of ATG4s protease family (including ATG4A, B, C and D); the family members share similar structure but have very well established different functions [25]. Recently, ATG4A has been identified as a modulator of mammospheres formation [18]. Accordingly, we could show that the expression of all ATG4 family members is enhanced, although to different extent, in mammospheres compared to adherent cells. Importantly ATG4A was the ATG4 family members most strongly up regulated in our models (Figure 5A). More interestingly, ATM does not affect the mRNA levels of ATG4A, B and D neither in MCF7 nor in MCF7-HER2 cells (Figure 5B) driving the conclusion that ATM selectively impinges on ATG4C expression. www.impactjournals.com/oncotarget Oncotarget 21694 Figure 1: ATM expression promotes mammospheres formation. Identification of ATM-dependent regulated gene set in mammospheres MCF7 and MCF7-HER2 breast cancer cell lines w infected with lentivirusexpressing two different RNA interference for ATM (shATM #1 and shATM #3) or a control sequence (shGF A. Representative Western Blot analysis of ATM and HER2 protein levels in mammospheres (MS); GAPDH was used as loading con (upper panel). The graph represent quantification of ATM protein levels and it was determined using the ImageJ software. Results indicated as mean±s.d. for three independent experiments B. Single cells were plated in ultralow attachment plates as described Materials and methods section, so that cells with stem cell properties were allowed to grow as non-adherent spheroids (mammospher Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown. Bars den 50 µm. Numbers of the mammospheres (diameter>50 µm) were counted, and the % of Sphere Forming Efficiency (%SFE) was calcula based on the numbers of cells that were initially seeded as mean±s.d. for three independent experiments performed with both target sequences for ATM (shATM#1 and shATM#3). The diameter of mammosphere (in µm) was quantified using I.A.S software (Delta Siste Italy). C. Mammospheres were dissociated by trypsin digestion and 600 cells/well were seeded in 6-well plates in differentiating me (mammospheres medium + 5% FBS serum). Colony formation was assessed 7 days later and stained with MTT (left panel). The numbe colonies are expressed as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM #1 shATM #3) . Student’s t-test P<0.01, P<0.001) (right panel). gure 1: ATM expression promotes mammospheres formation. MCF7 and MCF7-HER2 breast cancer cell lines w fected with lentivirusexpressing two different RNA interference for ATM (shATM #1 and shATM #3) or a control sequence (shGF Figure 1: ATM expression promotes mammospheres formation. MCF7 and MCF7-HER2 breast cancer cell lines wer infected with lentivirusexpressing two different RNA interference for ATM (shATM #1 and shATM #3) or a control sequence (shGFP) A. Representative Western Blot analysis of ATM and HER2 protein levels in mammospheres (MS); GAPDH was used as loading contro (upper panel). The graph represent quantification of ATM protein levels and it was determined using the ImageJ software. Results ar indicated as mean±s.d. for three independent experiments B. Identification of ATM-dependent regulated gene set in mammospheres Single cells were plated in ultralow attachment plates as described in Materials and methods section, so that cells with stem cell properties were allowed to grow as non-adherent spheroids (mammospheres) Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown. Bars denot 50 µm. Numbers of the mammospheres (diameter>50 µm) were counted, and the % of Sphere Forming Efficiency (%SFE) was calculated based on the numbers of cells that were initially seeded as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3). The diameter of mammosphere (in µm) was quantified using I.A.S software (Delta Sistemi Figure 1: ATM expression promotes mammospheres formation. MCF7 and MCF7-HER2 breast cancer cell lines were infected with lentivirusexpressing two different RNA interference for ATM (shATM #1 and shATM #3) or a control sequence (shGFP). A. Representative Western Blot analysis of ATM and HER2 protein levels in mammospheres (MS); GAPDH was used as loading control (upper panel). The graph represent quantification of ATM protein levels and it was determined using the ImageJ software. Results are indicated as mean±s.d. for three independent experiments B. Single cells were plated in ultralow attachment plates as described in Materials and methods section, so that cells with stem cell properties were allowed to grow as non-adherent spheroids (mammospheres). Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown. Bars denote 50 µm. Numbers of the mammospheres (diameter>50 µm) were counted, and the % of Sphere Forming Efficiency (%SFE) was calculated based on the numbers of cells that were initially seeded as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3). The diameter of mammosphere (in µm) was quantified using I.A.S software (Delta Sistemi, Italy). C. Mammospheres were dissociated by trypsin digestion and 600 cells/well were seeded in 6-well plates in differentiating media (mammospheres medium + 5% FBS serum). Colony formation was assessed 7 days later and stained with MTT (left panel). The number of colonies are expressed as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM #1 and shATM #3) . Student’s t-test P<0.01, *P<0.001) (right panel). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 21695 Figure 2: ATM expression promotes stem-like phenotype. A. Identification of ATM-dependent regulated gene set in mammospheres www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 21696 Figure 3: Gene expression profiling of mammospheres with or without interfering ATM expression. A. Number o differentially expressed mRNAs between mammospheres silenced for ATM and control mammospheres obtained from three independen Figure 3: Gene expression profiling of mammospheres with or without interfering ATM expression. A. Number of differentially expressed mRNAs between mammospheres silenced for ATM and control mammospheres, obtained from three independent experiment performed with both targeting sequences for ATM (shATM#1 and shATM#3) and selected by the following conditions: \|Log2FC\|>0.58, T-test<0.05. In the Figure are shown the number of differentially expressed genes in mammospheres derived from MCF7 and from MCF7-HER2 cells. The intersection area encloses 115 common genes between mammospheres derived from both cell lines. B. Gene Ontology analysis of intersection set (115 mRNAs), obtained by the DAVID online tool. C. Microarray and qRT-PCR comparison. Log2 Fold Change of 9 genes between mammospheres shATM vs shCTR detected by microarray (black lines) were compared with those measured by qRT-PCR (grey lines). Positive values represent gene expression upregulation and negative values downregulation in mammospheres silenced for ATM gene (shATM) compared to control cells (shCTR). qRT-PCR results were normalized with TBP. Error bars indicate standard deviations of at least three independently performed experiments. Figure 3: Gene expression profiling of mammospheres with or without interfering ATM expression. A. Number of diff i ll d RNA b h il d f ATM d l h b i d f h i d d Figure 3: Gene expression profiling of mammospheres with or without interfering ATM expression. A. Number of differentially expressed mRNAs between mammospheres silenced for ATM and control mammospheres, obtained from three independent experiment performed with both targeting sequences for ATM (shATM#1 and shATM#3) and selected by the following conditions: \|Log2FC\|>0.58, T-test<0.05. In the Figure are shown the number of differentially expressed genes in mammospheres derived from MCF7 and from MCF7-HER2 cells. The intersection area encloses 115 common genes between mammospheres derived from both cell lines. B. Gene Ontology analysis of intersection set (115 mRNAs), obtained by the DAVID online tool. C. Microarray and qRT-PCR comparison. Log2 Fold Change of 9 genes between mammospheres shATM vs shCTR detected by microarray (black lines) were compared with those measured by qRT-PCR (grey lines). Positive values represent gene expression upregulation and negative values downregulation in mammospheres silenced for ATM gene (shATM) compared to control cells (shCTR). qRT-PCR results were normalized with TBP. Identification of ATM-dependent regulated gene set in mammospheres Error bars indicate standard deviations of at least three independently performed experiments. www.impactjournals.com/oncotarget Identification of ATM-dependent regulated gene set in mammospheres The expression of HER2, ATM and SOX2, OCT4 and NANOG mRNA levels in non-infected Mammospheres (MS) and Adherent (Adh) cells was investigated using by quantitative Real-Time PCR. Relatives quantities (RQ) were calculated to TBP (TATA-box Binding Protein) and are relative to MCF7 adherent parental cells (Adh). Results are expressed as the mean±s.d. for at least three independent experiments and analyzed using Student’s t-test (P<0.05, **P<0.001). B. The expression of SOX2,OCT4 and NANOG mRNA levels was investigated as in infected Mammospheres (MS) with lentivirus expressing control interference (shGFP) or shATM. C. Indicated cells were assayed for ALDH activity utilizing the ALDEFLUORTM assay (STEMCELLTM TECHNOLOGIES). Cells incubated with ALDEFLUOR substrate (BAAA) and the specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), were used to establish the baseline fluorescence of these cells (R1) and to define the ALDEFLUOR- positive region (R2). Incubation of cells with ALDEFLUOR substrate in the absence of DEAB (DEAB-) induces a shift in BAAA fluorescence defining the ALDEFLUOR-positive population (R2). The quantification of ALDH-positive cells in each breast cancer cell line is shown (lower panel). Error bars indicate the s.d. from three independent experiments with both targeting sequences for ATM (shATM#1 and shATM#3). Figure 2: ATM expression promotes stem-like phenotype. A. The expression of HER2, ATM and SOX2, OCT4 and NANOG mRNA levels in non-infected Mammospheres (MS) and Adherent (Adh) cells was investigated using by quantitative Real-Time PCR. Relatives quantities (RQ) were calculated to TBP (TATA-box Binding Protein) and are relative to MCF7 adherent parental cells (Adh). Results are expressed as the mean±s.d. for at least three independent experiments and analyzed using Student’s t-test (P<0.05, **P<0.001). B. The expression of SOX2,OCT4 and NANOG mRNA levels was investigated as in infected Mammospheres (MS) with lentivirus expressing control interference (shGFP) or shATM. C. Indicated cells were assayed for ALDH activity utilizing the ALDEFLUORTM assay (STEMCELLTM TECHNOLOGIES). Cells incubated with ALDEFLUOR substrate (BAAA) and the specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), were used to establish the baseline fluorescence of these cells (R1) and to define the ALDEFLUOR- positive region (R2). Incubation of cells with ALDEFLUOR substrate in the absence of DEAB (DEAB-) induces a shift in BAAA fluorescence defining the ALDEFLUOR-positive population (R2). The quantification of ALDH-positive cells in each breast cancer cell line is shown (lower panel). Error bars indicate the s.d. from three independent experiments with both targeting sequences for ATM (shATM#1 and shATM#3). ATM modulates ATG4C levels and sustains autophagic flux in mammospheres showed that the downregulation of ATM expression in mammospheres triggers the reduction of ATG4C protein levels (Figure 6A), and the impairment of LC3-I conversion into LC3-II monitored through quantification of LC3-II/ LC3-I ratio (Figure 6A). ATG4C is a member of ATG4s proteases that are responsible for the cleavage of Microtubule-associated protein 1A/1B-light chain 3 (LC3-I) into LC3-II protein, which is then lipidated and recruited to autophagosomal membranes [25]. Tracking the conversion of LC3-I to LC3-II is indicative of ATG4s activity and thus of autophagic activity [26]. We therefore investigated whether the loss of ATM-dependent regulation of ATG4C expression level may result in a defect in autophagosome formation in mammospheres upon ATM silencing. To quantify autophagosome formation we analysed the expression and the processing of LC3 protein, a well- known marker of autophagy [27]. Western blot analysis To further assess the effect of ATM-dependent regulation of ATG4C on autophagic response we checked the levels of the autophagic receptor p62/SQSTM1, an indicator of the autophagic flux which is known to be degraded following autophagy induction [28, 29]. Interestingly the downregulation of ATM expression in mammospheres, resulted in the accumulation of p62 compared to control cells (Figure 6A). Since autophagosome formation can result either from increased de novo autophagosome biosynthesis or from the inhibition of the autophagic flux, we distinguished between Figure 4: ATG4C expression promotes mammospheres formation. MCF7 and MCF7-HER2 breast cancer cell lines were transfected with ATG4C siRNA or a control sequence (SignaliSilence ®,Cell Signaling) using Lipofectamine ® 3000 Reagent (Life Technologies). A. Representative Western Blot analysis of ATG4C protein levels in mammospheres (MS); Tubulin was used as loading control (upper panel). B. Single cells were plated in ultralow attachment plates as described in Materials and Methods section, so that cells with stem cell properties were allowed to grow as mammospheres. Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown (upper). Bars denote 50 µm. Numbers of the mammospheres (diameter>50 µm) were counted, and the %SFE was calculated based on the numbers of cells that were initially seeded (Bottom). mean±s.d. for three independent experiments and analysed using Student’s t-test (P<0.05, P<0.01, P<0.001). The diameter of mammosphere (in µm) was quantified using I.A.S software (Delta sistemi, Italy). Figure 4: ATG4C expression promotes mammospheres formation MCF7 and MCF7 HER2 breast cancer cell lines were Figure 4: ATG4C expression promotes mammospheres formation. www.impactjournals.com/oncotarget Oncotarget 21697 ATM modulates ATG4C levels and sustains autophagic flux in mammospheres MCF7 and MCF7-HER2 breast cancer cell lines were transfected with ATG4C siRNA or a control sequence (SignaliSilence ®,Cell Signaling) using Lipofectamine ® 3000 Reagent (Life Technologies). A. Representative Western Blot analysis of ATG4C protein levels in mammospheres (MS); Tubulin was used as loading control (upper panel). B. Single cells were plated in ultralow attachment plates as described in Materials and Methods section, so that cells with stem cell properties were allowed to grow as mammospheres. Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown (upper). Bars denote 50 µm. Numbers of the mammospheres (diameter>50 µm) were counted, and the %SFE was calculated based on the numbers of cells that were initially seeded (Bottom). mean±s.d. for three independent experiments and analysed using Student’s t-test (P<0.05, P<0.01, P<0.001). The diameter of mammosphere (in µm) was quantified using I.A.S software (Delta sistemi, Italy). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 21698 these two possibilities using the lysosomal inhibitor chloroquine (CQ). Interestingly, in control condition, as expected, the CQ treatment blocks autophagic flux after LC3-II formation and before p62 degradation, resulting in an increased in LC3-II and p62 levels, (Figure 6B). Conversely, CQ treatment failed to induce an increase in LC3-II and p62 levels in ATM silenced mammospheres (Figure 6B) suggesting a block in the autophagic flux in shATM cells.i derived mammospheres, were treated with Chloroquine (CQ). Efficacy of this treatment was confirmed by an increase in LC3-II dots number/cells in response to CQ in control adherent cells and control mammospheres (Figure 6C and Supplementary Figure 2A). Conversely, shATM did not increase LC3 dots formation after chloroquine treatment in mammospheres, indicating that the downregulation of ATM expression induces a decrease in the autophagic flux in this context (Figure 6C), which confirmed data obtained with western blot analysis. We confirmed these results by performing a confocal microscopy analysis of LC3 protein in mammospheres downregulated or not for ATM. In normal condition LC3 protein is cytosolic whereas it appears as “puncta” when autophagy is induced (LC3-II form). We could show a marked increase of LC3 dots in mammospheres compared to adherent cells in basal condition (Supplementary Figure 2A). Interestingly, consistently with western blot analysis (Figure 6A-6B), silencing of ATM expression drives a dramatic reduction in the formation of LC3-II dots in mammospheres (Figure 6C). To further investigate whether the reduction in the number of LC3 dots may be due to differences in the autophagic flux, MCF7 cells and We next decided to check for autophagosome degradation. To this end, we quantified LC3-II dots co-localized with the lysosomal protease Cathepsin D, following CQ treatment. As expected, we found a significant reduction of LC3-II dots fused with lysosomes in shATM cells compare to control cells (Figure 6D). Of note, ATG4A, ATG7 and ATG5-12 are stable following shATM or siATG4C treatments (Supplementary Figure 2B-2C). These data indicate a specific and unique effect of ATM on ATG4C protein. Overall these results suggest that ATM expression sustains ATG4C levels and thus the autophagic response in mammosphere context. Figure 5: Downregulation of ATM expression selectively impinges on ATG4C expression. A.The expression of ATG4A, B, C and D mRNA levels in non-infected Mammospheres (MS) and Adherent (Adh) cells was investigated using by quantitative Real-Time PCR. www.impactjournals.com/oncotarget Relatives quantities (RQ) were calculated to TBP (TATA-box Binding Protein) and are relative to MCF7 adherent parental cells (Adh). Results are expressed as the mean±s.d. for at least three independent experiments and analyzed using Student’s t-test (P<0.05, *P<0.01). B. The expression of ATG4A, B, C and D mRNA levels was investigated as in (A) in infected Mammospheres (MS) with lentivirus expressing control interference (shGFP) or shATM. Figure 5: Downregulation of ATM expression selectively impinges on ATG4C expression. A.The expression of ATG4A, B, C and D mRNA levels in non-infected Mammospheres (MS) and Adherent (Adh) cells was investigated using by quantitative Real-Time PCR. Relatives quantities (RQ) were calculated to TBP (TATA-box Binding Protein) and are relative to MCF7 adherent parental cells (Adh). Results are expressed as the mean±s.d. for at least three independent experiments and analyzed using Student’s t-test (P<0.05, *P<0.01). B. The expression of ATG4A, B, C and D mRNA levels was investigated as in (A) in infected Mammospheres (MS) with lentivirus expressing control interference (shGFP) or shATM. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 21699 Figure 6: ATM expression regulates ATG4C levels and autophagic flux in mammospheres (MS). A. Representative Western Blot analysis of ATM, ATG4C, p62 and LC3 protein levels in mammospheres (MS) derived from MCF7 cell lines, with or without shATM. Actin was used as loading control. The graphs represented quantification of LC3 II/I ratio, LC3II/actin and p62/actin ratio was determined using the ImageJ software. Results are indicated as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test (P<0.05, P<0.01, P<0.001). B. Representative Western Blot analysis of p62 and LC3 proteins in mammospheres (MS) with or without shATM and treated with CQ (20 µM, 30 min). Actin was used as loading control. The graphs represented quantification of LC3 II/I ratio, LC3II/actin and p62/actin ratio was determined using the ImageJ software. Results are indicated as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test (P<0.05). C. Representative image, using confocal microscopy, ofthe formation of autophagosome assayed by immunofluorescence for endogenous LC3 protein in MCF7 cells seeded from dissociated mammospheres. Mammospheres were treated or not with choloroquine CQ (20 µM, 30 min) for analysing autophagic flux. The graph show the accumulation of LC3 dots per infected cells. Results are expressed as the mean±s.d. for at least three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test (P<0.05). D. Representative image, using confocal microscopy, of the lysosomal degradation of autophagosomes assayed by immunofluorescence for endogenous LC3 and Cathepsin D proteins in MCF7 cells seeded from dissociated mammospheres. Mammospheres were treated with choloroquine CQ (20 µM, 30 min) in order to visualize the autophagosome degradation (co-localization LC3-II dots with Cathepsin D). The graph indicates a clear reduction of LC3-II dots co-localizing with Cathepsin D in ShATM infected cells. Results are expressed as the mean±S.D of at least three i d d t i t f d ith b th t ti f ATM ( hATM#1 d hATM#3) d l d i St d t’ t t t Figure 6: ATM expression regulates ATG4C levels and autophagic flux in mammospheres (MS). A. Representative Western Blot analysis of ATM, ATG4C, p62 and LC3 protein levels in mammospheres (MS) derived from MCF7 cell lines, with or without shATM. Actin was used as loading control. DISCUSSION Although our knowledge concerning new functions of ATM kinase has greatly increased during the last decade, their exact role in carcinogenesis and cancer therapies remains elusive. We previously demonstrated that ATM promotes HER2-dependent tumorigenesis in breast cancer, reporting also a context-dependent role of ATM targeting in cancer [9]. In this study, we described for the first time a mechanism through which ATM regulates autophagy in a fashion independent on HER2, which has crucial implications in breast cancer progression. In particular, we showed that: (1) ATM expression is induced in mammospheres culture as compared to parental adherent breast cancer cell lines, and (2) ATM depletion with a small interfering RNA leads to impaired mammospheres formation. Interestingly, we put in evidence that ATM expression sustains SOX2 expression and ALDH activity without significantly impinging on other stemness markers such as NANOG and OCT4 (Figure 2) suggesting the existence of mechanisms of modulation of mammosphere formation by ATM other than the regulation of the stemness genes. To clarify this issue, we performed microarray experiments revealing that, in mammospheres, ATM regulates the expression of genes involved in the control of DNA repair and cell division, which is consistent with the central role of ATM as guardian of the genome [4]. Moreover, in this experiment we showed that other ATM-dependent function could be involved in the regulation of Breast cancer stem-like phenotype. In particular, we focused our attention on the autophagic gene ATG4C. Indeed, autophagy is a well-established pro-survival mechanism for BCSCs maintenance [17] [18, 19] suggesting the hypothesis that ATM may modulate BCSCs via ATG4C. Accordingly, we observed by western blot and real time PCR that the depletion of ATM leads to the decrease in mRNA and protein levels of ATG4C. The ATM-dependent downregulation of ATG4C expression correlates very well with an impairment of the autophagic flux in mammospheres silenced for ATM. Consistently, we showed that silencing of ATG4C impairs mammospheres formation similarly to the silencing of ATM. More interestingly, ATG4C overexpression rescues mammospheres defects induced by ATM down regulation. These findings provide the first evidence on a link between the expression of ATM and the autophagic gene ATG4C. In previous studies, ATM and ATG4C were reported to have a tumor-suppressive www.impactjournals.com/oncotarget The graphs represented quantification of LC3 II/I ratio, LC3II/actin and p62/actin ratio was determined using the ImageJ software. Results are indicated as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test (P<0.05, P<0.01, P<0.001). B. Representative Western Blot analysis of p62 and LC3 proteins in mammospheres (MS) with or without shATM and treated with CQ (20 µM, 30 min). Actin was used as loading control. The graphs represented quantification of LC3 II/I ratio, LC3II/actin and p62/actin ratio was determined using the ImageJ software. Results are indicated as mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test (P<0.05). C. Representative image, using confocal microscopy, ofthe formation of autophagosome assayed by immunofluorescence for endogenous LC3 protein in MCF7 cells seeded from dissociated mammospheres. Mammospheres were treated or not with choloroquine CQ (20 µM, 30 min) for analysing autophagic flux. The graph show the accumulation of LC3 dots per infected cells. Results are expressed as the mean±s.d. for at least three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test (P<0.05). D. Representative image, using confocal microscopy, of the lysosomal degradation of autophagosomes assayed by immunofluorescence for endogenous LC3 and Cathepsin D proteins in MCF7 cells seeded from dissociated mammospheres. Mammospheres were treated with choloroquine CQ (20 µM, 30 min) in order to visualize the autophagosome degradation (co-localization LC3-II dots with Cathepsin D). The graph indicates a clear reduction of LC3-II dots co-localizing with Cathepsin D in ShATM infected cells. Results are expressed as the mean±S.D of at least three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test ( P<0.001). www.impactjournals.com/oncotarget Oncotarget 21700 Restoration of ATG4C expression rescues ATM ability to form mammospheres Restoration of ATG4C expression rescues ATM ability to form mammospheres negative [lacking expression of ER, progesterone receptor (PR), and HER2] tumors (basal-like). Positive correlation between ATM and ATG4C expression was statistically significant in all subtypes, except for the basal like subtype, suggesting that the functional link between ATM and ATG4C genes could have a clinical significance (Table 1). To further validate the functional link between ATM and ATG4C expression in the modulation of autophagy and mammospheres formation, we overexpressed ATG4C in mammospheres previously silenced for ATM expression. In Figure 7A we confirmed by RT-PCR the overexpression of ATG4C in MCF7 and MCF7-HER2 cell lines interfered with shCTR and shATM constructs (Figure 7A). Importantly, the overexpression of ATG4C in cells silenced for ATM expression, significantly rescued the percentage of spheres formation (%SFE) of shATM cells which turn to be comparable to the one of shCTR cells (Figure 7B). This result demonstrated that there is a strong correlation between ATM- dependent regulation of ATG4C expression level and ATM- dependent regulation of mammospheres formation. This connection was independent of HER2 receptor expression levels as it can be observed in both cell lines. We cannot exclude that other proteins could be directly or indirectly involved in this regulation. Interestingly, re-expression of ATMwt in shATM mammospheres rescues ATG4C levels and mammosphere formation (Supplementary Figure 3A and 3B) further confirming the correlation between mammosphere formation and expression levels of ATM and ATG4C mRNA. ATM and ATG4C gene expression correlation in breast cancer human samples To further evaluate the clinical significance of our findings, we asked whether we could identify a significant correlation between ATM and ATG4C expression in breast cancer human samples. To this aim we took advantage of The Cancer Genome Atlas (TCGA) microarray data on of breast cancer samples. To quantify the correlation, we downloaded normalized genes microarray data from the TCGA Data Coordination Center [30] and computed the Pearson correlation coefficients and corresponding P values (Table 1). Highlighting ATM expression showed a significant positive correlation with the expression of autophagic gene ATG4C among microarray data derived from 511 breast cancer samples (Table 1). This observation suggests that the functional correlation between ATM and ATG4C genes in mammospheres could have also a significant relevance in breast cancer patients. Breast cancer is a very heterogeneous disease. To further explore the relationship between ATM and ATG4C genes we calculated the correlations between ATM and ATG4 genes in all four subtypes of breast cancer based on published classifications [30]: two estrogen receptor (ER)-positive subtypes separated mainly by relatively low (luminal A) and high (luminal B) expression of proliferation-related genes, a subtype enriched for HER2- amplified tumors [human epidermal growth factor receptor 2 (HER2)-enriched], a subtype associated with triple- www.impactjournals.com/oncotarget Oncotarget 21701 role [31, 32]. Here, we demostrated that these proteins promote stem-like phenotype in breast cancer, suggesting that ATM kinase and autophagy could play also a tumorigenic role in breast cancer. without affecting other members of ATG4 family, even though the precise molecular mechanism deserves further investigation. Little information is reported about the transcriptional regulation of the ATG4 proteases. Some observations indicated that ATG4C could be transcriptionally regulated by p53 upon DNA damage In addition, in this study, we demonstrated that ATM specifically regulates ATG4C isoform in mammospheres, specifically regulates ATG4C isoform in mammospheres, transcriptionally regulated by p53 upon DNA damage Figure 7: ATG4C expression rescues mammospheres formation ability in ATM interfered cells. MCF7 and MCF7-HER2 breast cancer cell lines transfected with construct overexpressing human pCMV3-hATG4C-HA (ATG4C) and control GFP overexpressing construct, using Lipofectamine ® 3000 Reagent (Life Technologies) A. The expression of ATG4C mRNA levels in Mammospheres (MS), was investigated by quantitative Real-time PCR. Relatives quantities (RQ) were calculated to TBP and are relative to control MCF7 mammospheres. Results are expressed as the mean±s.d. for at least three independent experiments. B. ATM and ATG4C gene expression correlation in breast cancer human samples Single cells were plated in ultralow attachment plates as described in Materials and methods section, so that cells with stem cell properties were allowed to grow as non- adherent spheroids (mammospheres). Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown (upper). Bars denote 50 µM. Numbers of the mammospheres (diameter>50 µM) were counted, and the %SFE was calculated based on the numbers of cells that were initially seeded (Bottom). Mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test ( P<0.01, *P<0.001). The diameter of mammosphere (in µM) was quantified using I.A.S software (Delta sistemi, Italy). Figure 7: ATG4C expression rescues mammospheres formation ability in ATM interfered cells. MCF7 and MCF7-HER2 breast cancer cell lines transfected with construct overexpressing human pCMV3-hATG4C-HA (ATG4C) and control GFP overexpressing construct, using Lipofectamine ® 3000 Reagent (Life Technologies) A. The expression of ATG4C mRNA levels in Mammospheres (MS), was investigated by quantitative Real-time PCR. Relatives quantities (RQ) were calculated to TBP and are relative to control MCF7 mammospheres. Results are expressed as the mean±s.d. for at least three independent experiments. B. Single cells were plated in ultralow attachment plates as described in Materials and methods section, so that cells with stem cell properties were allowed to grow as non- adherent spheroids (mammospheres). Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown (upper). Bars denote 50 µM. Numbers of the mammospheres (diameter>50 µM) were counted, and the %SFE was calculated based on the numbers of cells that were initially seeded (Bottom). Mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student’s t-test ( P<0.01, **P<0.001). The diameter of mammosphere (in µM) was quantified using I.A.S software (Delta sistemi, Italy). www.impactjournals.com/oncotarget Oncotarget 21702 Table 1: Correlation of ATM with ATG4C gene in BRCA samples from TCGA cancer atlas data TCGA sample group (n) Pearson correlation P-value Bonferroni corrected P-value All BRCA (511) 0.26 2.70E-09 1.35E-08 Basal (95) 0.15 1.26E-01 6.30E-01 HER2 (58) 0.37 3.55E-03 1.78E-02 Luminal A (231) 0.17 9.60E-03 4.80E-02 Luminal B (127) 0.3 3.29E-04 1.65E-03 We computed the Pearson correlation coefficients and corresponding uncorrected and Bonferroni corrected P-values between ATM and ATG4C genes in expression data from 511 breast cancer samples extracted from the TCGA Cancer Atlas. ATM and ATG4C gene expression correlation in breast cancer human samples Statistically significant results shown in bold font (P<0.05). le 1: Correlation of ATM with ATG4C gene in BRCA samples from TCGA cancer atlas data We computed the Pearson correlation coefficients and corresponding uncorrected and Bonferroni corrected P-values between ATM and ATG4C genes in expression data from 511 breast cancer samples extracted from the TCGA Cancer Atlas. Statistically significant results shown in bold font (P<0.05). correlation coefficients and corresponding uncorrected and Bonferroni corrected P values genes in expression data from 511 breast cancer samples extracted from the TCGA Cancer Atlas. ults shown in bold font (P<0.05). mainly rely on ATG4C, even though the elucidation of the molecular mechanism requires further experimental investigations.i activation, and that ATG4C mRNA is modulated by miR- 376b upon starvation and rapamycin-induced autophagy [33, 34]. Future experiment will be launched to clarify whether ATM regulates ATG4C expression through p53 or via the modulation of miR-376b. g Interestingly, the findings reported in this study suggest that ATM may represent a novel candidate target to impair the autophagic activity in Breast Cancer Stem- like cells independently of HER2. Indeed, our results suggest that ATM targeting severely impinges not only on the DDR, as previously reported [41-43], but also on autophagy functionality, which is required for the homeostasis of the specific subset of breast cancer cells. Along similar lines, we surmise that ATG4C could also represent a valuable molecular target, as demonstrated by a large set of evidences suggesting that anti-autophagy compounds are effective in suppressing tumor growth and countering tumor resistance to chemotherapies [44][45][46]. Moreover, the inhibition of autophagy is reported to sensitize CSCs to several anticancer treatment [47]. Unfortunately, the relevance of autophagy inhibition in cancer treatment remains controversial because of the limited availability of chemical modulators. ATG4s are the only cysteine proteases among ATG genes proposed as an attractive candidate to efficiently achieve autophagy inhibition in cancer so far [48]. As an example, ATG4B has been recently proposed has a novel target for leukemic stem cells, supporting the idea that ATG4s proteases are good target for CSCs eradication [49]. Moreover, we cannot rule out that ATM may regulate also ATG4 protein levels and activity. Interestingly, ATG4s proteins are reported to be regulated by ROS during starvation [35]. According to this study, the Cys81 residue near the catalytic site of ATG4 is a direct oxidation target by H2O2 and the oxidation of this residue inhibits ATG4 protease activity. ALDH activity assay To measure and isolate cells with high ALDH activity, the Adelfluor assay was performed according to manufacturer’s (Stemcell Technologies, Durham, NC) guidelines. Dissociated single cells were suspended in Aldefluor assay buffer containing the ALDH substrate, Bodipyaminoacetaldehyde (BAAA) at 1,5 μM and incubated for 40 minutes at 37 °C. To distinguish between ALDH-positive and -negative cells, a fraction of cells was incubated under identical condition in the presence of a 10-fold molar excess of the ALDH inhibitor, diethylamino benzaldehyde (DEAB). This results in a significant decrease in the fluorescence intensity of ALDH-positive cells and was used to compensate the flow cytometer (FACScanto, Becton Dickinson). Cell cultures, transfection and infection Human breast cancer cell lines MCF-7 and MCF- 7HER2, described in Stagni et al 2015 [6], were cultured in RPMI-1640 containing 2 mM L-glutamine and supplemented with 10% HyClone Fetal Bovine Serum (Invitrogen) at 37°C in a CO2 incubator (5%) . ATM was silenced in MCF7 and MCF7-HER2 cells by lentivirus mediated expression of short-hairpin RNA using lentivirus produced in HEK 293T cells by cotransfecting pSIN18.cPPT.RNAi. p.EGFP.WPRE lentiviral vector with targeting sequences together with respective plasmids encoding for gag-pol and VSV-G proteins. Viral supernatant was collected 48 h post-transfection, filtered through a 0.45 μm pore size filter and added to the cells (MCF7 and MCF7-HER2 breast cancer cell lines) in the presence of 2 μg/ml polybrene [8]. Cells were transiently transfected using Lipofectamine 3000 (Life Technologies) essentially following the manufacturer’s instruction. RNA extraction and analysis Total RNA was extracted from breast cancer cell lines MCF7 and MCF7-HER2 and derived mammospheres with TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA quantitation was performed via quantitative real-time PCR (RT-PCR). The total RNA was reverse-transcribed with SuperScript III reverse transcriptase (Invitrogen), and amplified by using the Power SYBR Green PCR Master Mix (Applied Biosystems) and the 7900HT Fast Real- Time PCR System (Applied Biosystems). Primers were designed from the Roche Universal Probe Library and were as follows: DNA constructs, antibodies and reagents The shATM construct, generously provided by Y Lerenthal and Y Shiloh, had the following sequence: shATM construct (#1 position 912 5’-GACTTTGGCTGTCAACTTTCG-3’) and shATM (#3 position 8538 5’ GGA GCG CAC CAT CTT CTT C 3’) shRNA and control shGFP 5’- GGAGCGC ACCATCTTCTTC-3’ [50]. The ATG4C siRNA was from Signaling Silence ®,Cell Signaling. ATG4C expression construct pCMV3-C-HA was from Sino Biological Inc (HG16060-CY). pEGFP-C3(Clontech).The following antibodies and reagents were used: anti-ATM (2C1; Santa Cruz Biotechnology), anti-tubulin (Sigma, St. Louis, MO, USA), Mouse anti-c-ErbB-2 protein monoclonal antibodies, clone 3B5 (Ab-3, Oncogene Science,Uniondale, NY), anti-ATG4C (Cell Signaling, Beverly, MA, USA), anti-Hsp90 (F8, Santa Cruz Biotechnology), anti-LC3 (Cell Signaling, Beverly, MA, USA), anti-p62 (Santa Cruz Biotechnology), anti-Actin (Sigma, St. Louis, MO, USA), anti-GAPDH (Chemicon). Cloroquine (Sigma, St. Louis, MO, USA), anti-ATG4A (Biorbyt United Kingdom), anti-ATG7 (Cell Signaling, Beverly, MA, USA), Anti-ATG5 (Cell Signaling, Beverly, MA, USA), anti-Cathepsin D (Scripps laboratories, San Diego CA). ATM and ATG4C gene expression correlation in breast cancer human samples This, in turn, prevents the delipidation of LC3 without affecting the C-Terminal processing of LC3 by ATG4, thus leading to increased autophagosome formation [35]. It was proposed that cytosolic ATM regulates autophagy via its activation upon ROS rather than DNA damage induction [36-39]. This evidence prompts us to speculate that the selective ATM-dependent regulation of ATG4C and autophagy in mammospheres may be ascribed to differences in ROS regulation between cells grown in adherent conditions or as mammospheres. Interestingly, ATM dependent regulation of ROS plays a critical role in hematopoietic stem cell (HSC) maintenance [40]. In this study, the treatment of Atm-/- mice with antioxidant N-acetylcystine (NAC) restored the HSC pool, confirming the critical role of ROS regulation by ATM for stemness [40]. So, we could speculate that ATM could act as a ROS sensor modulating the autophagic flux according to ROS levels in different populations of cells within the tumor. Further experiments are required to clarify whether ATM activity is involved in the regulation of the stem-like phenotype, and whether ROS- and/or DNA damage-mediated ATM activation are essential for the regulation of this phenotype. Finally, we showed that ATG4A, ATG7 and ATG5-ATG12 protein levels are unaffected by shATM treatment (Supplementary Figure 2B), supporting the idea that defects in autophagic flux Remarkably, we were able to show a correlation between ATM and ATG4C expression in all breast cancer subtypes except for the basal-like one (Table 1) underscoring a clinical impact of our findings. This evidence suggests also that the elucidation of the molecular mechanism whereby ATM regulates ATG4C and autophagy in breast cancer can be relevant as it may pave the way for the development of new biomarkers for diagnostic and/or prognostic evaluation and for the design of novel therapeutic strategies. www.impactjournals.com/oncotarget Oncotarget 21703 MATERIALS AND METHODS insulin (Sigma), B27 (Invitrogen), 20 ng/ml epidermal growth factor (GIBCO), 10 ng/ml basic fibroblast growth factor (GIBCO) and 0,4% Bovine Serum Albumine (Sigma)] as described in Dontu et al. 2003 [20]. After 7 days, the diameter of mammospheres were measured in phase contrast picures using the I.A.S. software (Delta Sistemi, Rome, Italy). Numbers of the mammospheres (diameter>50 µM) were counted and the efficiency of mammosphere formation was evalueted (%SFE= number of mammospheres / number of plated cells * 100). Mammospheres pellet was collected by gentle centrifugation (900 rpm, 5 min) to further analysis or dissociated into single cell by trypsin addiction (5 min 37°C) and mechanical pipetting. Single cells were plated at a density of 600 cell/well in mammosphere medium supplemented with 5% Fetal Bovine Serum (HyClone, Invitrogen) to test the clonogenic activity. After 10 days the cells were stained with 3-[4,5-Dimethylthiazol-2-yl]-2,5- Diphenyltetrazolium Bromide (MTT Vitality Stain, promega) 5 mg/ml for 16 hours and the colony number enumerated. Mammopheres diameter was measured usig I.A.S. software (Delta Sistemi, Rome, Italy). Immunofluorescence Cells were washed in PBS and fixed with 4%paraformaldehyde in PBS for 30 min. After permeabilization with 0.4% TritonX-100 in PBS for 5 min or with Digitonin 50µg/ml (in the case of Cathepsin D staining) for 5 min, cells were blocked in 3% normal goat serum in PBS and incubated overnight at 4°C with primary antibodies. We used the antibodies directed against LC3. Cells were then washed in blockingbuffer and incubated for 1 h with labelled anti-rabbit (FITC or Cy3, Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody. Nuclei were stained with 1 mg/ ml DAPI and examined under a Zeiss LSM 700100 oil- immersion objective (Zeiss, Oberkoechen, Germany). We used ‘ZEN2009 Light edition’ software for image analysis. All measurements in this workwere performed by a blind approach. All analyses were performed innonsaturated single z-confocal planes. Mammosphere culture Single cell suspensions of breast cancer cell lines, MCF7 and MCF7-HER2, were grown in ultralow attachment 6-well plates (Corning) at a density of 4000 cell/mL in mammosphere medium [Dulbecco’s modified Eagle’s medium/F- 12, containing 5 ug/mL www.impactjournals.com/oncotarget Oncotarget 21704 Gene name hsa-ANL For hsa-ANL Rev hsa-ATG4A For hsa-ATG4A Rev hsa-ATG4B For hsa-ATG4B Rev hsa-ATG4C For hsa-ATG4C Rev hsa-ATG4D For hsa-ATG4D Rev hsa-ATM For hsa-ATM Rev hsa-ECT2 For hsa-ECT2 Rev hsa-HER2 For hsa-HER2 Rev hsa-HMOX1 For hsa-HMOX1 Rev hsa-HSPA6 For hsa-HSPA6 Rev hsa-NEDD9 For hsa-NEDD9 Rev hsa-SPRR2A For hsa-SPRR2A Rev hsa-PLK4 For hsa-PLK4 Rev hsa-RAD51 For hsa-RAD51 Rev hsa-SOX2 For hsa-SOX2 Rev hsa-TBP For hsa-TBP Rev hsa-NANOG For hsa-NANOG Rev hsa-OCT4 For hsa-OCT4 Rev Primer sequence 5' - 3' TCCCTAGAAGAAGCTGAAGCAG TTCAATTCATCAATCAAAAGTGTTC ACAGATGAGCTGGTATGGATCCTT AGACGAGCACTTATATCAGACAACA ATTGGTGCCAGCAAGTCAA GCAGGCCAGATGTGAAGG GCATAAAGGATTTCCCTCTTGA GCTGGGATCCATTTTTCG ACGTTTCTCAGGACTGCACA ACAGACTTCCACTCGGCTGT TTGTTGTCCCTACTATGGAAATTAAG AGCGAAATTCTGCTTTAAATGAC AGTAAAAGATCTTCCCTTTGAACCT CTCGGGCATCCATTTGAA TCCTGTGTGGACCTGGATGAC CCAAAGACCACCCCCAAGA GGCAGAGGGTGATAGAAGAGG AGCTCCTGCAACTCCTCAAA CCGCCTATTTCAATGACTCG ATTGATGATCCGCAACACG GAGCTGGATGGATGACTACGA AGCTCTTTCTGTTGCCTCTCA TCAACAGCAGCAGTGCAAG CTGTGGACACTTTGGTGGTG GAAAACCAAAAAGGCTGTGGT TGAGATGCATACTCCTTTACAAGC AATCCAAATGTAATGCTTTGGTG AGGACTGAGATTCTGATTTGACG GGCAGCTACAGCATGATGCAGGAGC CTGGTCATGGAGTTGTACTGCAGG TGCCCGAAACGCCGAATATAATC TGGTTCGTGGCTCTCTTATCCTC CAGCTGTGTGTACTCAATGATAGATT ACACCATTGCTATTCTTCGGCCAGTTG GACAACAATGAAAATCTTCAGGAG CTGGCGCCGGTTACAGAACCA hsa-HMOX1 For hsa-HSPA6 For hsa-HSPA6 Rev hsa-NEDD9 For hsa-SPRR2A For reference gene (TBP, the endogenous control) and the target genes in each sample set. All reactions were performed in triplicate. Numerical data were expressed as mean±s.d. Relative changes in gene expression were quantified by applying the comparative threshold method, also called 2−ΔΔCt method, after determining the Ct values for the www.impactjournals.com/oncotarget Oncotarget 21705 Protein extraction and western blot analysis according to the manufacturer’s instructions. RNA quality was assessed with an Agilent Bioanalyzer RNA 6000 Nano kit; 200 ng of RNA was labeled with Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies), purified and hybridized overnight onto, the Agilent 8X60K whole human genome oligonucleotide microarrays (Grid ID 039494) according to the manufacturer’s instructions for one-color protocol. The Agilent DNA microarray scanner (model G2505C) was used for slide acquisition and spot analysis was performed with Feature Extraction software ver 10.7 (Agilent Technologies). Cells pellet were incubated in RIPA buffer (50 mM Tris-Hcl pH 7.4, 1% NP-40, 0,5% Sodium Deoxycholate, 0,1% SDS, 150mM NaCl, 2mM EDTA, 1mM phenylmethylsulfonyl fluoride, 25mM NaF, 1mM orthovanadate, 40 mM beta-glycerophosphate, 10 mg/ ml TPCK, 5 mg/ml TLCK) 30’ on ice and centrifugated at 12000 rpm 10’ a 4°C. For immunoblotting, 20 μg of protein extract were separated by SDS–polyacrylamide gel electrophoresis, blotted onto nitrocellulose membrane and detected with specific antibodies. All immunoblots were revealed by enhanced chemiluminescence (Amersham). Quantification of western blot were determinated using ImageJ software. All statistical analyses were performed with GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA),using the Student’s t-test, P<0.05 being considered significant. Comparative analysis with cancer genome atlas From the TCGA data portal (http://cancergenome. nih.gov.) we downloaded all breast cancer expression data files (in the aggregated gene symbol format, for 17814 genes in total) , including all metadata [30]. The whole dataset includes 511 primary tumours samples (metastatic and normal tissue samples were excluded), grouped into subtypes according to the Pam50 signature [29] : basal- like n=95, HER2-enriched n=58, luminal A n=231, luminal B, n=127). Within this dataset, we computed the Pearson correlation index, using R-Bioconductor, between ATG4C and ATM genes and all expression TCGA data divided into tumours subtypes. Data analysis Data filtering and analysis were performed using Microsoft Excel and R-Bionconductor. All the features with the flag gIsWellAboveBG=0 (too close to background) were filtered out and excluded from the following analysis. Filtered data were normalized by aligning samples to the 75th percentile. Differentially expressed genes were selected by a combination of fold change and 1-tail T-test thresholds (p-value<0.05; fold-change ratio >1.5 or <1/1.5 in linear scale and FCR is >0.58 in logaritmic scale). The analysis of over- and under- represented functional annotations was performed using the DAVID web tool [51]. Hierarchical sample clustering and heatmaps were obtained by TM4 MultiexperimentViewer [52]. Updated microarray probe annotations were downloaded from the official Agilent website (https://earray.chem.agilent. com). Array data are deposited in private status on Gene Expression Omnibus database https://www.ncbi.nlm.nih. gov/geo, record GSE81360. Microarray hybridization and data analysis The microarray assay was conducted using a biological triplicate and two different target sequence for ATM to avoid off targets effects. 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https://openalex.org/W2596833428	https://europepmc.org/articles/pmc5417826?pdf=render	English	null	Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions	Molecular & cellular proteomics	2,017	cc-by	16,810	1 The abbreviations used are: RBE, relative biological effectiveness; AA, acetic acid; ABC, ammonium bicarbonate; ATM, ataxia telangi- ectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; CDK1, cyclin-dependent kinase 1; CHK2, checkpoint kinase 2; DDR, DNA damage response; DSB, double-strand break; FA, formic acid; FLNB, Filamin-B; GAPDH, glyceraldehyde-3-phosphate dehydrogen- ase; GOBP, gene ontology biological process; GOCC, gene ontology cellular compartment; GOMF, gene ontology molecular function; Martin Winter‡§¶, Ivana Dokic§¶, Julian Schlegel§¶, Uwe Warnken‡, Ju¨ rgen Debus§¶, Amir Abdollahi§¶§§, and Martina Schno¨ lzer‡ ‡‡§§ versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected find- ing was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected find- ing was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first com- prehensive proteomic and phosphoproteomic study ap- plying stable isotope labeling by amino acids in cell cul- ture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expres- sion level limited alterations were observed 2 h post irra- diation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attrib- uted to radiation response as revealed by crosscheck with the PhosphoSitePlus database. In summary, the comprehensive insight into the radia- tion-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se. Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. Molecular & Cellu- lar Proteomics 16: 10.1074/mcp.M116.066597, 855–872, 2017. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because ra- diation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was ob- served for 55 phosphorylation sites indicating the exist- ence of distinct signaling circuitries induced by X-ray More than 50% of all cancer patients receive radiotherapy during their course of treatment (1). Author contributions: M.W., A.A., J.D., and M.S. designed research, M.W., I.D. and J.S. performed experiments, M.W. analyzed and inter- preted data, U.W. contributed analytical tools, M.W., A.A., and M.S. wrote the paper. All authors reviewed the manuscript. From the ‡Functional Proteome Analysis, German Cancer Re- search Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidel- berg, Germany; §Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany; ¶German Cancer Consortium (DKTK), Heidelberg, Germany; Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, Uni- versity of Heidelberg Medical School, Im Neuenheimer Feld 450, D-69120 Heidelberg, Germany; Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany Received December 20, 2016, and in revised form, March 15, 2017 Published, MCP Papers in Press, March 16, 2017, DOI 10.1074/ mcp.M116.066597 Received December 20, 2016, and in revised form, March 15, 2017 Research crossmark Research crossmark ssmark © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Martin Winter‡§¶, Ivana Dokic§¶, Julian Schlegel§¶, Uwe Warnken‡, Ju¨ rgen Debus§¶, Amir Abdollahi§¶§§, and Martina Schno¨ lzer‡ ‡‡§§ Conformal X-ray irradia- tion techniques such as intensity-modulated radiation therapy (IMRT) combine the application of a curative dose to the tumor while sparing adjacent radiosensitive organs keeping side-effects to healthy surrounding tissue within tolerable lim- its (2). Contrary to X-rays, which show an exponential dose decrease with increasing tissue depth, charged particles de- posit most of their energy to a small region within the tissue with a sharp dose fall-off after the so-called Bragg peak (3). This precise dose localization enables further dose escalation within the tumor while sparing healthy tissue (4). Worldwide, more than 135.000 patients have been treated with particles until the end of 2014 and the number is growing steadily (5). From the ‡Functional Proteome Analysis, German Cancer Re- search Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidel- berg, Germany; §Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany; ¶German Cancer Consortium (DKTK), Heidelberg, Germany; Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, Uni- versity of Heidelberg Medical School, Im Neuenheimer Feld 450, D-69120 Heidelberg, Germany; Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany An important term in the field of radiobiology is the relative- biological effectiveness (RBE)1 being defined as the ratio of Molecular & Cellular Proteomics 16.5 855 Radiation Proteomics Radiation Proteomics haved similar upon proton and carbon irradiation, however a distinct number of sites responded differentially to X-rays versus particle based treatments. To validate selected candi- dates, we used synthetic isotope labeled phosphopeptides in a targeted spike-in experiment as recently proposed by Ken- nedy et al. as an alternative to extensive Western blotting, which requires phosphosite specific antibodies (16). By this approach, we validated the prevailing pattern of phosphoryl- ation sites associated with DDR to be regulated in equal measure by all radiation qualities. Moreover, phosphorylation sites responding differentially to X-ray and particle based treatments were confirmed. Herewith, we demonstrate the existence of radiation quality dependent signaling events in the acute cell response. X-ray dose to an alternative radiation quality dose which produces the same biological effect such as cell survival or other surrogates such as the number of double-strand breaks (DSBs). EXPERIMENTAL PROCEDURES Public Access to Mass Spectrometry Data—All LC-MS/MS data of the discovery and targeted spike-in experiments have been depos- ited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) (17) via the PRIDE partner repository (18) with the data set identifier PXD004817. Entire MaxQuant folders are in- cluded as well as Skyline files for re-evaluation and detailed inspec- tion of the results. In the present study, we aimed to systematically decipher acute signaling events induced by different radiation qualities using high-resolution mass spectrometry based proteomics. To this end, we irradiated SILAC-labeled human lung adeno- carcinoma cells (A549) with X-rays, protons and carbon ions in clinical-like setting. A549 cells have an intact DNA damage repair machinery (e.g. p53 wild-type) leading to a relatively low background of DNA damage foci compared with a panel of well-known cancer cell lines (6). Therefore, A549 cells are particularly suitable to study radiation induced DNA damage response. Proteomic and phosphoproteomic analyses per- formed 2 h post irradiation showed extensive alterations of the phosphorylation status whereas the protein expression itself remained largely unaffected. Phosphorylation events be- Materials—Triton X-100 (#A1388), Na-deoxycholate (#A1531) and dithiothreitol (DTT, #A2948) were purchased from AppliChem (Darmstadt, Germany). Water (H2O, #232141), acetonitrile (ACN, #12041G5), trifluoroacetic acid (TFA, #202341), methanol (MeOH, #13641G5) and formic acid (FA, #069141A8) were obtained from Biosolve (Valkenswaard, Netherlands). Ammonium bicarbonate (ABC, #09830) and trizma base (#93349) were obtained from Fluka (Stein- heim, Germany). Iron(III) chloride (FeCl3, #1.03943.0250), ammonia solution (NH3, #1.05428.0250) and acetic acid (AA, #1.00063.2511) were obtained from Merck (Darmstadt, Germany). L-proline (#P5607), iodoacetamide (IAA, #I1149), glycolic acid (#124737) and hexafluoro- 2-propanol (HFIP, #10522) were obtained from Sigma (St. Louis, MO). Further chemicals were purchased from different suppliers: SDS (#2326.2, Roth, Karlsruhe, Germany), sequencing grade trypsin (#9002-07-7, Promega, Madison, WI), chloroform (#22711.290, VWR, Radnor, PA), EDTA (#11278.01, SERVA, Heidelberg, Germany), tita- nium dioxide (TiO2, #5020–75000, GL Sciences, Tokyo, Japan). Cell Culture and Irradiation—Human lung adenocarcinoma cells (A549, #CCL-185, ATCC, Manassas, VA) were cultured at 37 °C with 5% CO2 in SILAC Dulbecco’s Modified Eagle medium (DMEM, SILANTES GmbH, Munich, Germany) containing 10% dialyzed fetal bovine serum (FBS, SILANTES GmbH) and supplemented with either 0.798 mM 12C6,14N2-lysine and 0.398 mM 12C6,14N4-arginine or 0.798 mM 13C6,15N2-lysine and 0.398 mM 13C6,15N4-arginine (SILANTES GmbH). In order to prevent arginine-to-proline conversion additional 200 mg/l proline were added to the cell culture medium (19). Martin Winter‡§¶, Ivana Dokic§¶, Julian Schlegel§¶, Uwe Warnken‡, Ju¨ rgen Debus§¶, Amir Abdollahi§¶§§, and Martina Schno¨ lzer‡ ‡‡§§ Although the RBE for protons is assumed to be com- parable to X-rays, carbon ions are more effective in inducing unrepairable DNA damage (6) and may therefore efficiently eradicate formerly radioresistant tumors (7–9). This is in part because of the fact, that carbon ions predominantly induce clustered and direct DNA damage, which is considered to be less dependent on cell cycle stage, oxygen level, genetic background and is less well repaired by DNA repair mecha- nisms (3). So far, limited studies exist comparing X-rays to particle based radiations and are mainly focused on investigating the alterations of single or panel genes (10, 11). However, cells are equipped with versatile signaling cascades mainly trans- duced by post-translational modifications (PTMs) in order to minimize adverse effects of DNA damage (12). Post-transla- tionally modified proteins are key targets especially to im- prove the understanding of acute signaling events. In DNA damage response (DDR), protein phosphorylation plays a cru- cial role to trigger a chain of events, starting with sensing DNA lesions, signaling cascade activation, cell cycle arrest and recruitment of DNA repair factors (13). Although phosphopro- teome data exist elucidating the cellular response to X-rays per se (13) as well as to various X-ray dose levels (14, 15), radiation quality dependent phosphoproteome studies are urgently needed. In summary, we established a robust and efficient workflow for the investigation and validation of phosphorylation changes in response to X-rays and particle based irradiations. We show for the first time, that differential acute signaling events are triggered by different radiation qualities. With this we provide valuable information to better understand molec- ular effects of radiation qualities and offer potential drug tar- gets for modulation and optimization of cancer radiotherapy. H2AX, histone H2AX; HFIP, hexafluoro-2-propanol; HIT, Heidelberg Ion Beam Therapy Center; KPNA2, importin subunit alpha-1; LET, linear energy transfer; MAP1B, microtubule-associated protein 1B; MKI67, antigen KI-67; MOPS, 3-(N-morpholino)propanesulfonic acid; NHEJ, nonhomologous end joining; NUMA1, nuclear mitotic apparatus protein 1; PRKDC, DNA-dependent protein kinase catalytic subunit; RAD50, DNA repair protein RAD50; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; RRBP1, ribosome-binding pro- tein 1; SF30, survival fraction 30%; SILAC, stable isotope labeling by amino acids in cell culture; SPE, solid phase extraction; SRC, proto- oncogene tyrosine-protein kinase Src; STMN1, stathmin; TP53BP1, tumor suppressor p53-binding protein 1; VIM, vimentin. H2AX, histone H2AX; HFIP, hexafluoro-2-propanol; HIT, Heidelberg Ion Beam Therapy Center; KPNA2, importin subunit alpha-1; LET, linear energy transfer; MAP1B, microtubule-associated protein 1B; MKI67, antigen KI-67; MOPS, 3-(N-morpholino)propanesulfonic acid; NHEJ, nonhomologous end joining; NUMA1, nuclear mitotic apparatus protein 1; PRKDC, DNA-dependent protein kinase catalytic subunit; RAD50, DNA repair protein RAD50; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; RRBP1, ribosome-binding pro- tein 1; SF30, survival fraction 30%; SILAC, stable isotope labeling by amino acids in cell culture; SPE, solid phase extraction; SRC, proto- oncogene tyrosine-protein kinase Src; STMN1, stathmin; TP53BP1, tumor suppressor p53-binding protein 1; VIM, vimentin. EXPERIMENTAL PROCEDURES The planned physical dose levels were 3.5 Gy for protons (5.5 keV/ m) and 2 Gy for carbon ions (95.2 keV/m) at dose rate 0.5 Gy/min. Irradiation with particles was performed at the Heidelberg Ion Beam Therapy Center (HIT). Detailed information on particle irradia- tion protocol and dosimetry are described in Dokic et al. (6). After treatment, all cell culture flasks were returned to the incubator for 2 h at 37 °C and 5% CO2. Control cells were sham irradiated and handled in the same way as irradiated cells. Finally, cells were washed twice with chilled PBS and harvested with a cell scrapper prior to cell lysis. After overnight digestion the supernatants were collected in PCR tubes while gel pieces were subjected to four further extraction steps. Gel pieces were sonicated for 5min in ACN/0.1% aqueous TFA 50:50 (v/v). Following centrifugation the supernatant was collected and gel pieces were sonicated for 5min in ACN. After collecting the superna- tant gel pieces were sonicated for 5min in 0.1% TFA followed by another extraction step with ACN. The combined supernatants were evaporated to dryness in a speed-vac concentrator at 37 °C. Pep- tides were dissolved in 5 l 0.1% TFA/2.5% HFIP by sonication for 5min and subsequently analyzed by nanoLC-ESI-MS/MS. Clonogenic Survival Experiments—To determine radiobiological equivalent doses cells were irradiated with dose series of X-rays, protons and carbon ions and the survival fraction measured by clono- genic survival assays as described (6). Briefly, cells were seeded as single cells in triplicates in six-well plates. After attachment, cells were irradiated at different doses or left untreated (sham) and incubated. After colonies were formed, they were fixed with 75% methanol and 25% acetic acid for 10 min at room temperature and labeled with 0.1% crystal violet for 15 min. Colonies with more than 50 cells were counted as survivors to determine isoeffective doses. StageTip and SPE Purification—10 l TipOne RTP tips (STARLAB GmbH, Hamburg, Germany) were blocked with C18 Empore plugs (Varian, Paolo Alto, CA) and filled with sufficient amount of Oligo R3 (Life Technologies, Carlsbad, CA) material. Reverse phase material was activated by adding 100% ACN and subsequently equilibrated with 2.5% FA. Sample was loaded by applying constant pressure using a 5 ml syringe. One wash step was performed using 2.5% FA. Finally, desalted peptides were eluted with 80% ACN/0.6% AA and evaporated to dryness in a speed-vac concentrator at 37 °C. EXPERIMENTAL PROCEDURES To ensure full incorporation of the SILAC labels, cells were cultured for at least six cell doublings and incorporation was checked using a re- cently published R script (20) to be 95%. Molecular & Cellular Proteomics 16.5 856 Radiation Proteomics Proteome Analysis - SDS-PAGE and Protein Digestion—For pro- teome analysis 15 g of each light and heavy labeled protein lysate were combined and separated on a NuPAGE 4–12% Bis-Tris gradi- ent gel (Invitrogen, Carlsbad, CA) using a MOPS-buffer system. The gel was stained with colloidal Coomassie Blue and each lane was cut into 27 slices. Gel slices were washed once with 100 l H2O and 100 l H2O/ACN 50/50 (v/v) and incubated for 5min at 37 °C on a thermal mixer at 600 rpm. For reduction of disulfide bonds, 100 l of 10 mM DTT in 40 mM ABC were added for 1 h at 56 °C followed by a washing step with 100 l H2O for 5 min at 37 °C. For alkylation of cysteine residues 100 l of 55 mM IAA in 40 mM ABC were added and gel slices were incubated for 30 min at 25 °C in the dark. Subsequently, slices were washed alternately 3 times with 150 l H2O and 150 l H2O/ACN 50/50 (v/v) at 37 °C. Before adding trypsin, slices were incubated for 1 min in 100% ACN for dehydration. ACN was removed and slices were dried for 10 min. Gel pieces were rehydrated with 10 l 40 mM ABC containing 150 ng trypsin and finally incubated overnight at 37 °C. Cells were irradiated with different radiation qualities at 65–75% confluence in T75 cell culture flasks (app. 8106 cells). Cells treated with X-rays were irradiated with 6 Gy at 320 keV and a dose rate of 110 cGy/min using the XRAD320 X-ray device (Precision X-Ray, North Branford, CT). Field size for the X-ray irradiation was 20 20 cm to fully cover the flasks and the source to surface distance was 50 cm. A filter unit comprised of 0.75 mm tin, 0.25 mm copper and 1.5 mm aluminum (half value layer 3.7 mm Cu) was used. Cells treated with particles were positioned in the middle of a 1 cm wide spread-out Bragg peak centered at about 3.5 cm water-equivalent depth in a field size adjusted to cover two T75 cell culture flasks next to each other. EXPERIMENTAL PROCEDURES Antibody Staining for Microscopy—Cells were plated onto 12-mm glass slides in 24-well plate format, irradiated 24 h later and incubated for desired time periods. Cells were fixed and stored in 70% ethanol at 20 °C for at least 24 h. For staining, cells on cover glasses were first permeabilized and blocked in PBS containing 0.1% (v/v) Triton- X100 and 3% (w/v) serum albumin for 20min. Primary antibody (TP53BP1, Cell Signaling Technologies (CST), Danvers, MA #4937) was applied at 1:200 in PBS containing 0.02% (v/v) Triton-X100 and 0.6% (w/v) serum albumin at 4 °C overnight. Cells were washed twice with PBS containing 0.02% (v/v) Triton-X100 and 0.6% (w/v) serum albumin. Secondary antibody (Alexa-555, Invitrogen, A-21428) was applied at 1:400 in PBS containing 0.02% (v/v) Triton-X100 and 0.6% (w/v) serum albumin at 4 °C for 4 h. Washing was performed as before, followed by nuclear counter-staining with 1 g/ml DAPI. Cells were washed once with PBS and then with H2O. Glass slides were fixed in Fluoromount G. Imaging was performed on an Olympus Cell Vivo microscope with a 20x objective. ImageJ (version 1.48) was used for image processing. TP53BP1-foci were quantified by performing background subtraction followed by global thresholding and water- shed. Foci larger than 3 pixels and with a minimum circularity of 0.5 were measured. For desalting prior to phosphopeptide enrichment SPE cartridges were manually prepared using Bond Elut empty SPE cartridges (Agi- lent Technologies, Santa Clara, CA), 20 m polypropylene frits and Oligo R3 material. Before sample loading the Oligo R3 material was activated by 100% ACN and subsequently equilibrated with 2.5% FA. After loading, five washing steps were performed using 2.5% FA. Finally, desalted peptides were eluted with 80% ACN/0.6% AA. Prior to phosphopeptide enrichment the samples were diluted with 0.6% AA to a final concentration of 60% ACN/0.6% AA. Phosphoproteome Analysis—Digestion and Enrichment—For phos- phoproteome analysis 300 g of the protein lysates (150 g of each light and heavy labeled sample combined) were used. First, proteins were precipitated with methanol/chloroform according to Wessel and Flu¨ gge (22). The obtained protein pellet was dissolved in 40 mM ABC and incubated for at least 30min at 25 °C on a thermal mixer at 600 rpm. Protein disulfide bonds were reduced by adding 1.5 mM DTT, followed by alkylation using 3 mM IAA. Afterward additional 1.5 mM DTT was added to quench remaining IAA. EXPERIMENTAL PROCEDURES To enrich for phosphorylated peptides we incubated the samples with the IMAC material in a sample/IMAC ratio of 1:10 for 1.5 h under constant rotation at room temperature. The supernatants were trans- ferred to new IMAC material and incubated again under the same conditions. The IMAC material of both steps was washed three times with 60% ACN/0.6% AA. Experimental Design and Statistical Rationale—To define the mo- lecular changes induced by ionizing radiation, we analyzed three biological replicates of SILAC labeled A549 cells. After treatment, SILAC states were mixed, processed and analyzed by LC-MS/MS. MaxQuant was used to identify proteins and phosphopeptides in the raw files with an FDR 1%. Additional filtering for quantitative values was applied to have at least two valid values in the three biological replicates of each sample group (X-rays, protons, carbon ions). To define significantly regulated proteins/phosphorylation sites between irradiated samples and control samples one-sample t test was per- formed to be different from 0 (p value: 0.01). In addition, proteins/ phosphorylation sites were only considered to be regulated if their abundance was changed more than 1.5-fold compared with the con- trol. In order to find proteins/phosphorylation sites being differentially regulated between radiation qualities we applied a two-sample t test (p value: 0.01). In addition, they were only considered to be signifi- cantly regulated if their abundance was changed more than 1.5-fold between the two radiation qualities considered. Peptides were eluted by adding 1 M glycolic acid solution in 80% ACN/5% TFA. Both supernatants were incubated separately with TiO2 (sample: TiO2 ratio of 1:8) for 15min under constant rotation. TiO2 material was washed with 1 M glycolic acid solution in 80% ACN/5% TFA, followed by 80% ACN/5% TFA and 20% ACN/0.2% TFA. Finally the phosphopeptides were eluted from the TiO2 material using 1% NH3. Prior to LC-MS/MS analysis, samples were desalted using Stage- Tips as described above and stored at 20 °C. p Liquid Chromatography—Mass spectrometry—Peptide mixtures were separated using a nanoAcquity UPLC system. For trapping we used a C18 precolumn (180 m 20 mm) with a particle size of 5 m (Waters GmbH, Eschborn, Germany). Liquid chromatography sepa- ration was performed on a BEH130 C18 main-column (100 m 100 mm) with a particle size of 1.7 m (Waters GmbH). EXPERIMENTAL PROCEDURES Peptide mixtures were loaded on the trap column at a flow rate of 5 l/min and were eluted with a gradient at a flow rate of 0.4 l/min. The proteome samples were separated by a 1 h gradient. This was set as follows: from 0 to 4% B in 1 min, from 4 to 40% B in 39 min, from 40 to 60% B in 5 min, from 60 to 85% B in 0.1 min, 6min at 85% B, from 85 to 0% B in 0.1 min, and 9min at 0% B. The phosphoproteome samples were separated by a 3 h gradient, which was set as follows: from 0 to 4% B in 1 min, from 4 to 25% B in 139 min, from 25 to 40% B in 15 min, from 40 to 85% B in 10 min, 5 min at 85% B, from 85 to 4% B in 2 min, and 15 min at 4% B. Solvent A contained 98.9% water, 1% ACN and 0.1% FA, solvent B contained 99.9% ACN and 0.1% FA. The nanoUPLC system was coupled online to an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany). The mass spectrometer was operated in data-dependent mode to automatically measure MS1 and MS2. Data were acquired by scan cycles of one FTMS scan with a resolution of 60.000 at m/z 400 and a range from 300 to 2000 m/z in parallel with six MS/MS scans in the linear ion trap of the most abundant precursor ions. Software Tools for Functional Analysis—Significant hits were iso- lated and transformed by z-scoring prior to hierarchical clustering. Principle component analysis was performed using default settings in Perseus. Remaining data visualizations were performed in R (http:// www.r-project.org/index.html, last accessed February 9, 2016). Ma- jority of the plots were created using the ggplot2 package (https:// cran.r-project.org/web/packages/ggplot2/index.html, last accessed February 9, 2016). Heat maps were created with heatmap.2 function from the gplots package (http://cran.r-project.org/web/packages/ gplots/index.html, last accessed February 9, 2016). For interpretation of the data GO term enrichment was performed with the Cytoscape 3.2.1 (26) plugin ClueGO (version 2.2.3) (27) and CluePedia (version 1.2.3) (28). Phosphoproteins significantly enriched in the data set were analyzed against the ontologies reference set. Significance level was set to p 0.01 as determined by right-sided hypergeometric test and Bonferroni step down correction. Enrich- ment was performed using the GeneOntology databases: GOBP, GOCC, and GOMF (29). EXPERIMENTAL PROCEDURES For digestion, trypsin was added to a final enzyme to sample ratio of 1:100 and incubated overnight at 37 °C. After digestion, samples were acidified to a final concentration of 2% FA and desalted with SPE cartridges using oligo R3 material (Life Technologies). Cell Lysis and Protein Quantification—Lysis was performed using RIPA buffer (50 mM Tris-HCl pH 7.5; 150 mM NaCl; 1% Triton X-100; 0.5% Na-deoxycholate; 0.1% SDS, 1% DTT) supplemented with miniComplete EDTA-free protease inhibitor mixture tablet and Phos- STOP phosphatase inhibitor mixture tablet (Roche Diagnostics, Mannheim, Germany) (21). Additionally 1% benzonase (Merck) was added to remove nucleic acids. After incubation on an orbital shaker for 1 h on ice the lysates were cleared by centrifugation at 15,000 rpm, 30 min at 4 °C. Protein concentration of supernatants was measured in triplicate using 2-D Quant Kit (GE Healthcare Bio-Sci- ences, Uppsala, Sweden). For phosphopeptide enrichment we used a combination of IMAC and TiO2 enrichment. First, IMAC material was prepared as previously described, with minor modifications (23). All solutions were prepared with ultrapure water (Biosolve). Ni-NTA material was extracted from Qiagen spin columns (Qiagen, Venlo, Netherland) and incubated for Molecular & Cellular Proteomics 16.5 857 Radiation Proteomics 20 min under constant rotation in 100 mM EDTA (pH 8, adjusted by NH3) followed by two washing steps with 100 mM EDTA. Subse- quently the material was washed two times with water followed by three times 0.6% AA. For loading with iron(III)-ions the material was incubated three times with 100 mM FeCl3 in 0.6% AA for 20 min under constant rotation. To remove excessive iron(III)-ions, the material was washed four times with 0.6% AA and stored in 0.6% AA at 4 °C. and “match between runs” features were implemented with default settings to increase the number of peptides which could be used for quantification. Data transformation and evaluation was performed with the freely available Perseus software (version 1.5.2.4), which includes all nec- essary functionalities. Contaminants as well as proteins identified by site modification and proteins derived from decoy database contain- ing reversed protein sequences were strictly excluded from further analysis. Protein and phosphopeptide ratios were normalized, so that the median of their logarithms is zero, according to (24). For confident protein identification at least two unique peptides were required. EXPERIMENTAL PROCEDURES In this case, the dose required to achieve 30% survival fraction (SF30) was deter- mined by clonogenic survival assay that is considered gold standard for determination of the RBE (Fig. 1A). This assay led to the following biological equivalent doses: 6 Gy X-rays, 3.5 Gy protons and 2 Gy carbon ions. In line with the survival data, similar level of initial and residual radiation induced nuclear TP53BP1 foci (RIF) representing DNA damage response (DDR) at radiation induced double-strand break (DSB) sites were found at depicted isoeffective doses (Fig. 1B). Especially residual DNA damage foci are considered to be predictive for lethal DNA damage (36) and show very good correlation be- tween the radiation qualities in our setup (Fig. 1C). Moreover, same level of DDR activation was confirmed at selected isoef- fective doses for each radiation quality by investigating a panel of central DDR components including H2AX (S139), pATM (S1981), pCHK2 (T68), pNUMA1 (S395), and pRAD50 (S635) (Fig. 1D, left panel) whereas basal protein expression remains unaffected (Fig. 1D, right panel). In Fig. 1E, results are illustrated by an interaction network including enriched bio- logical processes elucidating their affiliation to the DDR and equal upregulation of phosphorylation in response to applied radiation qualities. Because of these results, all subsequent studies were conducted at SF30 isoeffective RBE of 1.7 for protons and 3.0 for carbon ions to assure that alterations detected at proteome and phosphoproteome level are unbi- ased by differences of the prescribed dose across different radiation qualities. Synthetic Peptides—HPLC purified stable isotope-labeled phos- phopeptides were obtained from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). Peptide quality control included HPLC chromatograms and MALDI mass spectra. Stock solutions were pro- duced at a concentration of 1 mg/ml in 3% ACN/1% FA and stored at 80 °C until use. Cysteine residues were carbamidomethylated using IAA. Stable isotopes were introduced in each peptide using one of the following amino acids during synthesis: 13C9,15N1-phenylala- nine (10.0272), 13C6,15N1-isoleucine (7.0172), 13C6,15N1-leucine (7.0172) or 13C5,15N1-valine (6.0138). Targeted Phosphopeptide Validation—Parallel reaction monitoring was performed using a list of 28 synthetic phosphopeptides selected from the discovery data set. Peak integration was accomplished using Skyline (35) and all integrations were manually checked for correct peak detection and absence of interferences. Spectral librar- ies were created in Skyline by injecting a 50 fmol standard mixture containing all synthetic phosphopeptides. Top4 transitions were se- lected on the presence of high abundant y- or b-ions. EXPERIMENTAL PROCEDURES If necessary, additional transitions were selected that enable the differentiation between potential phosphorylation sites on the peptide. MS/MS spectra for all synthetic peptides with the selected transitions can be found in supplemental Fig. S2. Optimal spike-in amount for each peptide was obtained by setting up a standard curve between 0.1 fmol and 500 fmol and adjusting the spike-in level to the maximal intensity obtained in the discovery experiment. Information concern- ing the synthetic phosphopeptides can be found in supplemental Table S3. Corresponding spike-in amounts of each phosphopeptide were added to enriched phosphopeptide samples and analyzed with the LC-MS/MS method described above with minor modifications. Most important is the usage of a parent mass list for data dependent MS/MS acquisition. Six MS/MS scans of the most abundant precur- sors from this parent list were performed in parallel with one FTMS scan. The parent mass list contained three entries for each phospho- peptide: light (endogenous), medium (synthetic) and heavy (endoge- nous) peptide m/z. Confirmation of the phosphopeptides was performed by verifying coelution of the phosphopeptide precursors as well as MS/MS tran- sition profiles combined with the inspection of the relative abundance proportion of the fragment ions between endogenous and synthetic peptides. Information for each peptide can be found in supplemental Fig. S3. Study Design for Identification of Radiation Induced Pro- teome and Phosphoproteome Alterations—To study acute molecular response to different radiation qualities we per- formed a comparative proteomic as well as phosphopro- teomic study 2 h post irradiation. The experimental setup of the presented study is depicted in Fig. 2A. Cells were ex- posed to 6 Gy X-rays, 3.5 Gy protons, and 2 Gy carbon ions representing biological equivalent doses in terms of cell kill for this particular cell line. For accurate quantification, cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) (37, 38). Cells of the light SILAC state were sham irradiated and the ones of the heavy state were irradiated with the respective radiation qualities. Follow- ing a waiting period of 2 h, cells were harvested and equal protein amounts were mixed after cell lysis. The time point 2 h post ionizing radiation was chosen as a compromise between short-lived acute phosphorylation events and long-term events according to the results of Bennetzen et al. (13). For protein quantification, lysates were subjected to SDS-PAGE, gel slicing and subsequent in-gel digestion using trypsin. EXPERIMENTAL PROCEDURES Data validation and classification—LC-MS/MS raw data were pro- cessed with MaxQuant (version 1.5.3.8) (24). Peptide identification was performed using the Andromeda search engine integrated into the MaxQuant environment (25) against the human UniProt database (downloaded: 06/07/2015; 146.661 sequences). SILAC quantification was enabled using 13C6,15N4-arginine (10.0083) and 13C6,15N2- lysine (8.0142) for the heavy state and their 12C,14N-containing counterparts for the light state. Cysteine carbamidomethylation (57.0215) was used as fixed modification; methionine oxidation (15.9949), protein N-terminal acetylation (42.0106), deamidation of asparagine as well as glutamine (0.9840) and additionally for the phosphoproteome data set serine, threonine, and tyrosine phosphor- ylation (79.9663) as variable modifications. To gather information about the phosphorylation sites identified in the data set we consulted the PhosphoSitePlus database (PSP, http://www.phosphosite.org/homeAction.action, last accessed Feb- ruary 15, 2016) (30). For identification of over-represented kinase motifs phosphopeptide sequences being differentially regulated by ionizing radiation were submitted to the Motif-X software (31). Se- quence stretches were centered on the considered phosphosite and extended to 13 amino acids ( 6 residues). Entire set of phosphopep- tides identified in this study was used as background. Significance threshold was set to p 0.01 and the occurrence threshold of motifs in the data set was set to 20. Enriched consensus sequences were assigned to kinases with matching substrate motifs according to the Human Protein Reference Database (http://www.hprd.org/ serine_motifs, last accessed February 9, 2016). NetworKIN algorithm 3.0 in KinomeXplorer was used to predict kinases responsible for selected regulated phosphorylation sites (32, 33). Cutoff for the Net- A false discovery rate (FDR) of 0.01 for proteins, peptides and sites, a minimum peptide length of 7 amino acids, a mass tolerance of 7 ppm for precursor and 0.5 Da for fragment ions were required. En- zyme specificity was set to trypsin/p with an allowed maximum of two missed cleavages for the proteome analysis and four missed cleav- ages for the phosphoproteome analysis. Additionally, the “requantify” Molecular & Cellular Proteomics 16.5 858 Radiation Proteomics worKIN score was set to 3. To illustrate connections between kinases and their targets we applied the Cytoscape plugin PhosphoPath (34). X-rays, protons and carbon ions at the Heidelberg Ion Beam Therapy Center (HIT) in order to determine their RBE. Applying isoeffective doses enables direct comparison of cellular re- sponse to different radiation qualities, leading to the same biological effect for a given end point. EXPERIMENTAL PROCEDURES For phosphoproteome analysis lysates were tryptically digested in-solution and enriched using a combination of IMAC and Western Blot Analysis—Proteins from whole cell lysates were sep- arated under reducing conditions on a NuPAGE 4–12% Bis-Tris gradient gel (Invitrogen) using a MOPS-buffer system and transferred to 0.45 m nitrocellulose membranes (GE Healthcare). After blocking with 5% nonfat milk or 5% bovine serum albumin, the membranes were probed with primary antibodies against proteins or phosphoryl- ation sites of interest. Secondary antibodies with horseradish perox- idase conjugate were used for visualization with enhanced chemi- luminescence reagent (GE Healthcare). Commercial antibodies were purchased and used at the manufacturer’s recommended dilutions: ATM (CST, #2873), pATM (S1981) (CST, #4526), CHK2 (CST, #3440), pCHK2 (T68) (CST, #2661), RAD50 (CST, 3427), pRAD50 (S635) (CST, #14223), H2AX (CST, #2595), H2AX (S139) (CST, #5438), NUMA1 (CST, #3888), pNUMA1 (S395) (CST, #3429), SRC (CST, #2109), pSRC (S75) (Abcam, Cambridge, UK, ab79308) and GAPDH (Merck Milli- pore, CB1001). Molecular & Cellular Proteomics 16.5 RESULTS Identification of Relative Biological Effectiveness (RBE) of Different Radiation Qualities—We exposed well-established human lung adenocarcinoma cells (A549) to a dose series of Molecular & Cellular Proteomics 16.5 859 Radiation Proteomics FIG. 1. Determination and validation of radiobiological equivalent doses. A, Clonogenic survival assay for A549 cells exposed to do ies of X-rays, protons and carbon ions. Doses were set to achieve 30% survival fraction leading to the following doses used throughout dy: 6 Gy X-ray, 3.5 Gy protons and 2 Gy carbon ions. Data represents mean S.D. of three independent experiments. B, Representat ages of TP53BP1 foci (red) initial (1h) and residual (24h) after irradiation with the different radiation qualities. Cell nuclei were counterstain h DAPI (blue). Selected nuclei are magnified to emphasize the foci pattern following the individual treatments. C, Percentage of ce ntaining none, one or more residual TP53BP1 foci per nucleus 24h after irradiation. Foci were counted in two independent experime 2000 cell nuclei per condition and experiment) using ImageJ and mean S.D. is illustrated. D, Western blot analysis of phosphorylat es and their basal protein expression in response to different radiation qualities 2h after irradiation. Selected sites represent key player in A damage signaling and are known to respond to ionizing radiation dependent DNA damage. GAPDH was used as loading control. E, D mage response network, illustrating the quantitative Western blot results using Cytoscape plugin PhosphoPath (34). Additional proteins w ded to complement the network. Functional analysis of the network using the ClueGO plugin within Cytoscape revealed three predomin logical processes demonstrating their affiliation to the DNA damage response as well as equal regulation by the different radiation qualiti FIG. 1. Determination and validation of radiobiological equivalent doses. A, Clonogenic survival assay for A549 cells exposed to dose series of X-rays, protons and carbon ions. Doses were set to achieve 30% survival fraction leading to the following doses used throughout the study: 6 Gy X-ray, 3.5 Gy protons and 2 Gy carbon ions. Data represents mean S.D. of three independent experiments. B, Representative images of TP53BP1 foci (red) initial (1h) and residual (24h) after irradiation with the different radiation qualities. Cell nuclei were counterstained with DAPI (blue). Selected nuclei are magnified to emphasize the foci pattern following the individual treatments. C, Percentage of cells containing none, one or more residual TP53BP1 foci per nucleus 24h after irradiation. RESULTS Foci were counted in two independent experiments (n2000 cell nuclei per condition and experiment) using ImageJ and mean S.D. is illustrated. D, Western blot analysis of phosphorylation sites and their basal protein expression in response to different radiation qualities 2h after irradiation. Selected sites represent key player in the DNA damage signaling and are known to respond to ionizing radiation dependent DNA damage. GAPDH was used as loading control. E, DNA damage response network, illustrating the quantitative Western blot results using Cytoscape plugin PhosphoPath (34). Additional proteins were added to complement the network. Functional analysis of the network using the ClueGO plugin within Cytoscape revealed three predominant biological processes demonstrating their affiliation to the DNA damage response as well as equal regulation by the different radiation qualities. FIG. 1. Determination and validation of radiobiological equivalent doses. A, Clonogenic survival assay for A549 cells exposed to dose series of X-rays, protons and carbon ions. Doses were set to achieve 30% survival fraction leading to the following doses used throughout the study: 6 Gy X-ray, 3.5 Gy protons and 2 Gy carbon ions. Data represents mean S.D. of three independent experiments. B, Representative images of TP53BP1 foci (red) initial (1h) and residual (24h) after irradiation with the different radiation qualities. Cell nuclei were counterstained with DAPI (blue). Selected nuclei are magnified to emphasize the foci pattern following the individual treatments. C, Percentage of cells containing none, one or more residual TP53BP1 foci per nucleus 24h after irradiation. Foci were counted in two independent experiments (n2000 cell nuclei per condition and experiment) using ImageJ and mean S.D. is illustrated. D, Western blot analysis of phosphorylation sites and their basal protein expression in response to different radiation qualities 2h after irradiation. Selected sites represent key player in the DNA damage signaling and are known to respond to ionizing radiation dependent DNA damage. GAPDH was used as loading control. E, DNA damage response network, illustrating the quantitative Western blot results using Cytoscape plugin PhosphoPath (34). Additional proteins were added to complement the network. Functional analysis of the network using the ClueGO plugin within Cytoscape revealed three predominant biological processes demonstrating their affiliation to the DNA damage response as well as equal regulation by the different radiation qualities. FIG. 1. Determination and validation of radiobiological equivalent doses. A, Clonogenic survival assay for A549 cells exposed to dose series of X-rays, protons and carbon ions. RESULTS Doses were set to achieve 30% survival fraction leading to the following doses used throughout the study: 6 Gy X-ray, 3.5 Gy protons and 2 Gy carbon ions. Data represents mean S.D. of three independent experiments. B, Representative images of TP53BP1 foci (red) initial (1h) and residual (24h) after irradiation with the different radiation qualities. Cell nuclei were counterstained with DAPI (blue). Selected nuclei are magnified to emphasize the foci pattern following the individual treatments. C, Percentage of cells containing none, one or more residual TP53BP1 foci per nucleus 24h after irradiation. Foci were counted in two independent experiments (n2000 cell nuclei per condition and experiment) using ImageJ and mean S.D. is illustrated. D, Western blot analysis of phosphorylation sites and their basal protein expression in response to different radiation qualities 2h after irradiation. Selected sites represent key player in the DNA damage signaling and are known to respond to ionizing radiation dependent DNA damage. GAPDH was used as loading control. E, DNA damage response network, illustrating the quantitative Western blot results using Cytoscape plugin PhosphoPath (34). Additional proteins were added to complement the network. Functional analysis of the network using the ClueGO plugin within Cytoscape revealed three predominant biological processes demonstrating their affiliation to the DNA damage response as well as equal regulation by the different radiation qualities. TiO2. Subsequently, peptide mixtures were analyzed by high- resolution mass spectrometry and obtained raw data were processed using MaxQuant (24). peptide enrichment in order to prevent overloading of the affinity material and associated loss of endogenous phospho- peptides. As we used the synthetic phosphopeptides for iden- tity confirmation only and not for quantification, spike-in after the phosphopeptide enrichment was suitable. Usage of these synthetic phosphopeptides enabled to verify chromatographic coelution of the endogenous and the synthetic phosphopeptide and compare their fragmentation pattern, including fragments This discovery data set was used to select phosphorylation sites of interest to be confirmed in a targeted spike-in exper- iment. To this end, selected phosphopeptides were synthe- sized as medium isotope labeled counterparts. As indicated in Fig. 2A, they were added to the samples after the phospho- Molecular & Cellular Proteomics 16.5 860 Radiation Proteomics f t i t f th h h it l li ti it id tifi d ith f l di t (FDR) f l IG. 2. Experimental workflow for the analysis of radiation induced changes of proteome and phosphoproteome. RESULTS Moreover, the standard devi- ation is almost equal between the replicates as well as between the radiation qualities (X-rays: 0.48, Proton: 0.50, Carbon: 0.50) confirming the determined radiobiological equiv- alent doses used in this experimental setup. On the protein level, only eight features were significantly regulated by ionizing radiation in our data set (supplemental Table S1C). Thus, only negligible changes occur at protein expression level following this short period of time after irra- diation. Additionally, none of these eight regulated proteins contains phosphorylation sites in our data set. Because of these findings we focused on investigating phosphorylation changes in subsequent studies of acute radiation response. Kinase Motif Analysis of Regulated Phosphorylation Sites— One advantage of not using motif specific antibodies for phosphopeptide enrichment is independency of the consen- sus sequence surrounding the phosphorylation site. We ap- plied a combination of IMAC and TiO2 both being affinity- based techniques independent of the amino acids adjacent to the phosphorylation site. This led to an unbiased population of phosphopeptides and enabled the identification of enriched consensus sequence stretches in the data set. In order to identify kinase motifs we analyzed a 13-amino acid sequence stretch surrounding the central phosphorylated residue for all regulated phosphosites compared with nonregulated sites using Motif-X (31). Regulated Phosphorylation Sites in Response to Ionizing Radiation—In line with the fast protein phosphorylation kinet- ics of the cellular post translational modification machinery we identified many differentially regulated phosphorylation sites 2 h after irradiation. Overall, 181 sites were regulated on 141 proteins, whereas 2637 sites on 1269 proteins showed no regulation. The majority of identified phosphorylation sites (4702 out of 5107; 90%) were annotated in the Phospho- SitePlus database (30). This database contains 732 phosphor- ylation sites previously reported to be regulated by ionizing radiation which are predominantly derived from one compre- hensive study by Bennetzen et al. (13). However, only 30 of the 181 regulated phosphorylation sites in our study were already described in radiation context. Hence our study con- siderably increases the content of the database by 151 novel phosphorylation sites that were found to be associated with cellular response to ionizing radiation per se. Five consensus sequences were identified to be signifi- cantly enriched within the set of radiation regulated phosphor- ylation sites: p(S/T)P, pSPXXK, RXXpS, pSXXE, and pSQ as illustrated in Fig. 4. RESULTS A, SILAC labeled 49 cells were irradiated with radiobiological equivalent doses and harvested 2h after treatment. Heavy labeled samples irradiated with ays, protons and carbon ions respectively, were mixed with light labeled control samples for accurate quantification. SDS-PAGE with sequent in-gel tryptic digestion was performed for proteome analysis and a combination of IMAC and TiO2 phosphopeptide enrichment r in-solution digestion for phosphoproteome analysis. Resulting samples were analyzed by LC-MS/MS and data processing was conducted h MaxQuant. Selected phosphorylation sites were subsequently validated using synthetic medium isotope labeled phosphopeptides in a geted spike-in approach. B, Descriptive results of the proteome and phosphoproteome profiling of irradiated A549 cells (Regulation ween irradiated and control samples; §According to PhosphoSitePlus). C, Distribution of phosphorylated serine, threonine, and tyrosine dues among quantified phosphosites. D, Distribution of singly and doubly phosphorylated peptides among quantified phosphosites. Radiation Proteomics FIG. 2. Experimental workflow for the analysis of radiation induced changes of proteome and phosphoproteome. A, SILAC labeled A549 cells were irradiated with radiobiological equivalent doses and harvested 2h after treatment. Heavy labeled samples irradiated with X-rays, protons and carbon ions respectively, were mixed with light labeled control samples for accurate quantification. SDS-PAGE with subsequent in-gel tryptic digestion was performed for proteome analysis and a combination of IMAC and TiO2 phosphopeptide enrichment after in-solution digestion for phosphoproteome analysis. Resulting samples were analyzed by LC-MS/MS and data processing was conducted with MaxQuant. Selected phosphorylation sites were subsequently validated using synthetic medium isotope labeled phosphopeptides in a targeted spike-in approach. B, Descriptive results of the proteome and phosphoproteome profiling of irradiated A549 cells (Regulation between irradiated and control samples; §According to PhosphoSitePlus). C, Distribution of phosphorylated serine, threonine, and tyrosine residues among quantified phosphosites. D, Distribution of singly and doubly phosphorylated peptides among quantified phosphosites. necessary for exact assignment of the phosphosite localization. These parameters were verified comprehensively using Skyline as described (35). sites were identified with a false discovery rate (FDR) of less than 1% as depicted in Fig. 2B. Conservative thresholds were applied in order to use only high confident features during statistical analysis. Proteins and phosphorylation sites re- quired identification in at least two out of three replicates of Descriptive Results of the Proteome and Phosphoproteome Analyses—A total of 3851 proteins and 5107 phosphorylation Molecular & Cellular Proteomics 16.5 861 Radiation Proteomics each sample group (X-rays, protons, carbon ions). RESULTS Overall 2634 proteins and 2818 phosphorylation sites were subject of further statistical evaluations. Among these, 2724 (97%) phosphorylation sites were mapped with high confidence (class I (39)). Predominant phosphorylated amino acid was phosphoserine with 2536 phosphorylation sites (90%), fol- lowed by phosphothreonine represented by 269 sites (9.5%) and phosphotyrosine for which 13 sites (0.5%) were detected (Fig. 2C). Majority of these phosphopeptides were singly phosphorylated (2764, 98.1%) and only minority of peptides showed phosphorylation at two residues (54, 1.9%), whereas no higher phosphorylated peptides were quantified (Fig. 2D). indicates that the radiation qualities are mainly separated into two clusters, namely X-rays (X) and particle based irradiations (1H: proton and 12C: carbon). This is intriguing, considering that proton irradiation is believed to elicit similar biological effects compared with X-rays for planning patient treatment. Functional Analysis of Regulated Phosphorylation Sites—To better understand ongoing molecular processes triggered by ionizing radiation we used the Cytoscape (26) plugin ClueGO (27) to search for enriched GO terms (29) in the 141 proteins, containing regulated phosphorylation sites. Enriched biologi- cal processes in this data set contain mainly terms involved in the DDR: DNA repair, DNA metabolic process, DNA confor- mation change, DSB repair via NHEJ, base-excision repair and execution phase of apoptosis (Fig. 3B). These enriched biological functions are consistent with current concepts con- sidering DNA damage as hallmark of acute cellular response to ionizing radiation. Accordingly, most of these phosphopro- teins are located in or translocated into the nucleus. This is accompanied by their biological functions, to repair damaged DNA and prevent chromosomal instability. Enriched molecu- lar functions of the regulated phosphoproteins contain mainly nucleic acid binding motifs indicating that these phosphopro- teins bind to DNA damage sites or at regulatory transcription sites for their respective function. A key representative of these findings is TP53BP1, which is located in the nucleus, involved in DNA damage response by regulating repair path- way choice and directly recruited to damaged chromatin by recognition of a specific histone code (40). Normalized log2-transformed ratios were plotted for pro- teome and phosphoproteome in order to estimate reproduc- ibility and extent of regulation in the different sample groups (supplemental Fig. S1). These plots indicate minor changes at protein expression level. At phosphoproteome level each ex- periment showed similar spread of log2-ratios and good cor- relation between the replicates. RESULTS All five kinase motifs were previously characterized to be involved in different cellular signaling pro- cesses (41). To compare our findings with the 732 ionizing radiation dependent sites described in the PhosphoSitePlus database these sites were enriched in the same manner. All motifs identified by us were also present in the motifs en- riched in the database, implying consistency between our study and the previously described ionizing radiation depend- ent phosphorylation sites. Next, we matched the enriched motifs to potential kinases described in the Human Protein Hierarchical cluster analysis of the significantly regulated phosphorylation sites is displayed as heatmap in Fig. 3A. Similar numbers of phosphorylation sites were up- and down- regulated indicating that next to phosphorylation events also dephosphorylation plays an important role in the response to ionizing radiation. The dendrogram of the treatment arms Molecular & Cellular Proteomics 16.5 862 Radiation Proteomics FIG. 3. Clustering and GO enrichment analysis of radiation regulated phosphorylation sites. A, Hierarchical clustering of the 181 regulated phosphosites following treatment with different radiation qualities in three biological replicates each (X: X-rays, 1H: Proton, 12C:Carbon). Values represent the normalized log2-transformed ratios for the change in phosphorylation status between irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the data set of regulated phosphoproteins. Enriched GO terms were grouped according to GO hierarchy and displayed in pie charts representing the number of associated genes per group term. Detailed information about the enrichment analysis can be found in supplemental Table S4. FIG. 3. Clustering and GO enrichment analysis of radiation regulated phosphorylation sites. A, Hierarchical clustering of the 181 regulated phosphosites following treatment with different radiation qualities in three biological replicates each (X: X-rays, 1H: Proton, 12C:Carbon). Values represent the normalized log2-transformed ratios for the change in phosphorylation status between irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the data set of regulated phosphoproteins. Enriched GO terms were grouped according to GO hierarchy and displayed in pie charts representing the number of associated genes per group term. Detailed information about the enrichment analysis can be found in supplemental Table S4. proliferation (44–46). Casein kinase II represented by the SXXE motif also regulates important cellular functions as cell proliferation, differentiation and death (47). Reference database and assigned the radiation regulated phosphopeptides to each motif. RESULTS Thereby we were able to identify three kinase motifs being mainly associated with phosphorylation events and three mainly with dephosphory- lation. Highly upregulated are phosphorylation sites of the three phosphoinositide-3-like kinases (PIKK): ataxia telangi- ectasia mutated (ATM), ataxia telangiectasia and Rad3-re- lated protein (ATR) and DNA-dependent protein kinase cata- lytic subunit (PRKDC), with the joint sequence motif p(S/T)Q. This finding correlates well with their proposed involvement in the DDR following treatment with ionizing radiation (42, 43). In contrast, the motifs p(S/T)P and pSPXXK show mainly dephosphorylation in response to ionizing radiation. These represent consensus motifs of cyclin-dependent kinases (CDKs), glycogen synthase kinase 3 (GSK-3) and mitogen- activated protein kinases (MAPKs/ERKs) with common func- tionalities in cell cycle regulation, cell survival and motility. Sites of the RXXS and SXXE motifs cannot be clearly assigned to one direction of regulation however upregulation is slightly pronounced. RXXS is a target of three potential kinases: pro- tein kinase A (PKA), Ca2/calmodulin-dependent protein ki- nase II (CaMKII) and RAC-alpha serine/threonine-protein ki- nase (Akt) with joint functions including cell survival and Differentially Regulated Protein Phosphorylation by X-ray Versus Particle Irradiation—A central goal of our study was to decipher potential differences in acute radiation response to irradiation with X-rays, protons and carbon ions. Importantly, we aimed to exclude that these differences arise from distinct cellular fates resulting from variation in prescribed doses. In line with current standard approach to define biologically equivalent dose, the clonogenic survival was considered as the key readout to determine isoeffectiveness as described in Fig. 1. Based on current understanding of the radiobiology the two less densely ionizing radiations, X-ray and the low-LET protons were anticipated to show similar cellular responses versus the high-LET carbon irradiation. However, to our sur- prise, both unsupervised hierarchical clustering (Fig. 3A) and principle component analysis (Fig. 5A) predominantly discrim- inated between X-ray and particle irradiations (proton/car- bon). Based on this data we searched for phosphorylation sites being differentially regulation between radiation qualities and identified 55 corresponding sites listed in supplemental Table S2D. Differences between X-rays and particle radiations In contrast, the motifs p(S/T)P and pSPXXK show mainly dephosphorylation in response to ionizing radiation. These represent consensus motifs of cyclin-dependent kinases (CDKs), glycogen synthase kinase 3 (GSK-3) and mitogen- activated protein kinases (MAPKs/ERKs) with common func- tionalities in cell cycle regulation, cell survival and motility. RESULTS Sites of the RXXS and SXXE motifs cannot be clearly assigned to one direction of regulation however upregulation is slightly pronounced. RXXS is a target of three potential kinases: pro- tein kinase A (PKA), Ca2/calmodulin-dependent protein ki- nase II (CaMKII) and RAC-alpha serine/threonine-protein ki- nase (Akt) with joint functions including cell survival and Molecular & Cellular Proteomics 16.5 863 Radiation Proteomics FIG. 4. Phosphorylation consensus sequences and putative kinases of radiation regulated phosphorylation sites. Six consensus sequences were identified using the Motif-X software to be overrepresented in the set of 181 regulated phosphorylation sites compared with nonregulated sites during the response to ionizing radiation. Putative kinases were assigned and the heatmaps show the phosphorylation sites belonging to the different motifs (GSK-3: glycogen synthase kinase 3, ERK1/2: mitogen-activated protein kinases, CDK5: cyclin-dependent kinase 5, PKA: cAMP-dependent protein kinase, CaMKII: Ca2/calmodulin-dependent protein kinase II, Akt: RAC-alpha serine/threonine- protein kinase, ATM: ataxia telangiectasia mutated). FIG. 4. Phosphorylation consensus sequences and putative kinases of radiation regulated phosphorylation sites. Six consensus sequences were identified using the Motif-X software to be overrepresented in the set of 181 regulated phosphorylation sites compared with nonregulated sites during the response to ionizing radiation. Putative kinases were assigned and the heatmaps show the phosphorylation sites belonging to the different motifs (GSK-3: glycogen synthase kinase 3, ERK1/2: mitogen-activated protein kinases, CDK5: cyclin-dependent kinase 5, PKA: cAMP-dependent protein kinase, CaMKII: Ca2/calmodulin-dependent protein kinase II, Akt: RAC-alpha serine/threonine- protein kinase, ATM: ataxia telangiectasia mutated). were not yet described to be regulated by ionizing radiation in the PhosphoSitePlus database (30). Additionally, nine phos- phopeptides were selected with known regulation by ionizing radiation in order to confirm the experimental workflow. More- over, the selected candidates contain phosphorylation sites that are mainly responsible for the separation in the principle component analysis presented above between X-ray and the particle based irradiation. Because TP53BP1 was identified to play a central role in cellular response to radiation with sig- nificant enrichment of its phosphorylation sites across all radiation induced alterations (p value: 2.3e-6, Fisher’s exact test), we decided to particularly focus on validating radiation induced TP53BP1 regulation by tracking ten phosphorylation sites on this protein. were more pronounced whereas protons and carbon ions induce very similar phosphosite regulation as indicated in the volcano plots in Fig. 5B. RESULTS A second level separation is apparent between the low-LET protons versus high-LET carbon ions in the second PCA component although not as pronounced as the first level separation. These results indicate that differences in acute signaling events induced by different radiation qualities exist. Validation of mass spectrometry based proteomic results can be achieved by Western blotting. For three distinct phos- phorylation sites where phosphosite specific antibodies were commercially available we confirmed the SILAC based quan- tifications: DDR associated sites NUMA1 (S395) and RAD50 (S635) are shown in Fig. 1D and differentially phosphorylated site on SRC (S75) is shown in Fig. 5C. Comparison of the two orthogonal analyses revealed excellent correlation at phos- phorylation as well as basal protein level indicating the high impact of the presented discovery data set and the power of SILAC based quantitative proteomics (Fig. 5D). Initially, optimal spike-in amount was determined for each synthetic peptide. This was conducted by performing linear regression between 0.1 fmol and 500 fmol on column and comparing received intensities to the maximum intensity ob- tained in the discovery data set for the corresponding endog- enous phosphopeptide. This led to a mean spike-in level of 35.3 54.3 fmol on column (in detail listed in supplemental Table S3). The optimized peptide amounts were spiked into the enriched phosphopeptides and analyzed simultaneously by LC- MS/MS as indicated in the experimental workflow (Fig. 2A). Targeted Validation of Regulated Phosphorylation Sites Us- ing Synthetic Phosphopeptides—To further confirm our re- sults and circumvent the necessity of phosphosite specific antibodies we used synthetic isotope labeled phosphopep- tides in a targeted spike-in approach to validate selected candidates. A similar strategy was recently introduced by Kennedy et al. who characterized the response of DNA dam- age-responsive phosphorylation sites by using synthetic phosphopeptides (16). We selected 28 phosphopeptides listed in Table I using different criteria. 19 phosphopeptides This strategy enabled validation of 28 selected candidates as exemplarily shown in Fig. 6 for the phosphosite S56 on VIM. In Fig. 6A three parameters were checked to confirm the peptide characteristics: (i) quantity in the samples differen- tially treated, (ii) identification by comparing proportion of Molecular & Cellular Proteomics 16.5 864 Radiation Proteomics FIG. 5. Differential regulation of phosphorylation sites between radiation qualities. A, Unsupervised principle component analysis for confirmation of differences between the radiations’ mode of action. Of note, clear separation between X-rays and particle based radiations in the first component. RESULTS B, Volcano plots showing the phosphorylation sites being differentially regulated between the radiation qualities. Phosphosites with p value 0.01 and 0.67 fold change 1.5 are colored in blue; sites with p value 0.001 and 0.5 fold change 2 are colored in orange and labeled with their corresponding gene name. C, Western blot of the differentially regulated phosphorylation site S75 on SRC indicating strong dephosphorylation by both particle radiations whereas X-rays induce no significant regulation. Basal protein expression level shows no alterations. GAPDH was used as loading control. D, Bar charts illustrating quantitative Western blot results as well as SILAC based LC-MS/MS results, both at phosphorylation and basal protein expression level for three independent phosphorylation sites: RAD50 (S635), NUMA1 (S395) and SRC (S75) revealing excellent correlation between the two orthogonal analyses. FIG. 5. Differential regulation of phosphorylation sites between radiation qualities. A, Unsupervised principle component analysis for confirmation of differences between the radiations’ mode of action. Of note, clear separation between X-rays and particle based radiations in the first component. B, Volcano plots showing the phosphorylation sites being differentially regulated between the radiation qualities. Phosphosites with p value 0.01 and 0.67 fold change 1.5 are colored in blue; sites with p value 0.001 and 0.5 fold change 2 are colored in orange and labeled with their corresponding gene name. C, Western blot of the differentially regulated phosphorylation site S75 on SRC indicating strong dephosphorylation by both particle radiations whereas X-rays induce no significant regulation. Basal protein expression level shows no alterations. GAPDH was used as loading control. D, Bar charts illustrating quantitative Western blot results as well as SILAC based LC-MS/MS results, both at phosphorylation and basal protein expression level for three independent phosphorylation sites: RAD50 (S635), NUMA1 (S395) and SRC (S75) revealing excellent correlation between the two orthogonal analyses. selected transitions of the light (sham), medium (synthetic), and heavy (irradiated) peptides, and (iii) retention time com- parison between the phosphopeptide identified in the discov- ery experiment and the corresponding synthetic peptide. De- tailed results for each of the 28 selected phosphopeptides can be found in supplemental Fig. S3. For elucidation of the procedure additional information is given for VIM (S56) in Fig. 6B. Left, three mass spectra are shown, one for each of the three radiation treatments. RESULTS Several of these sites show differential Molecular & Cellular Proteomics 16.5 865 Radiation Proteomics TABLE I List of phosphopeptides validated in the spike-in experiment using synthetic isotope labeled peptides. Underlined amino acids are isotopically labeled: #heavy SILAC peptide-12C6,14N4-arginine or 13C6,15N2-lysine; $medium synthetic peptide-13C9,15N1-phenylalanine, 13C6,15N1-isoleucine, 13C6,15N1-leucine or 13C5,15N1-valine. §Phosphorylation site with known regulation by ionizing radiation according to PhosphoSitePlus List of phosphopeptides validated in the spike-in experiment using synthetic isotope labeled peptides. Underlined amino acids are isotopically labeled: #heavy SILAC peptide-12C6,14N4-arginine or 13C6,15N2-lysine; $medium synthetic peptide-13C9,15N1-phenylalanine, 13C6,15N1-isoleucine, 13C6,15N1-leucine or 13C5,15N1-valine. §Phosphorylation site with known regulation by ionizing radiation according to PhosphoSitePlus 6, 1 5, 1 p y g y g g p Gene symbol Modified peptide sequence Modified site Protein name Known IR response§ BRCA1 NYPS(ph)QEEL$IK# S1524 Breast cancer type 1 susceptibility protein CDK1 I$GEGTY(ph)GVVYK# Y15 Cyclin-dependent kinase 1 ELAVL1 NVALLSQL$YHS(ph)PAR# S202 ELAV-like protein 1 FLNB LDV$TILS(ph)PSR# S983 Filamin-B KPNA2 NVSSFPDDATS(ph)PL$QENR# S62 Importin subunit alpha-1 LMNA S(ph)VGGSGGGSFGDNL$VTR# S628 Lamin-A/C MAP1B SV$NFSLT(ph)PNEIK# T1282 Microtubule-associatedprotein 1B MCM6 (ac)MDLAAAAEPGAGS(ph)QHL$EVR# S13 DNA replication licensing factor MCM6 MCM6 EIESEIDS(ph)EEELI$NK# S762 DNA replication licensing factor MCM6 MKI67 ELF$QT(ph)PDHTEESTTDDK# T2203 Antigen KI-67 NUMA1 LSQLEEHLS(ph)QL$QDNPPQEK# S395 Nuclear mitoticapparatus protein 1 PLEC AQL$EPVAS(ph)PAK# S1435 Plectin PRKDC LTPLPEDNS(ph)MNV$DQDGDPSDR# S3205 DNA-dependent proteinkinase catalytic subunit RAD50 LFDVCGS(ph)QDFESDL$DR# S635 DNA repair protein RAD50 RRBP1 NTDV$AQS(ph)PEAPK#QEAPAK# S615 Ribosome-binding protein 1 SRC LFGGF$NSSDTVTS(ph)PQR# S75 Proto-oncogene tyrosine- proteinkinase Src STMN1 ESVPEFPL$S(ph)PPK# S38 Stathmin TOP2A TQMAEVLPS(ph)PR# S1213 DNA topoisomerase 2-alpha TP53BP1 S(ph)PEPEVLSTQEDLF$DQSNK# S294 Tumor suppressor p53-binding protein 1 TP53BP1 QDK#PM(ox)DTSVLS(ph)EEGGEPF$QK# S398 Tumor suppressor p53-binding protein 1 TP53BP1 FVPAENDSILMNPAQDGEVQL$S(ph)QNDDK# S580 Tumor suppressor p53-binding protein 1 TP53BP1 SGTAETEPVEQDS(ph)SQPSLPL$VR# S830 Tumor suppressor p53-binding protein 1 TP53BP1 SGTAETEPVEQDSS(ph)QPSLPL$VR# S831 Tumor suppressor p53-binding protein 1 TP53BP1 ADDPLRLDQEL$QQPQT(ph)QEK# T855 Tumor suppressor p53-binding protein 1 TP53BP1 GNLLHF$PSS(ph)QGEEEK#EK# S1068 Tumor suppressor p53-binding protein 1 TP53BP1 QSQQPMK#PI$S(ph)PVK#DPVS(ph)PASQK# S1101; S1094 Tumor suppressor p53-binding protein 1 TP53BP1 LPDGPTGSS(ph)EEEEEFLEIPPF$NK# S1759 Tumor suppressor p53-binding protein 1 VIM SLYASS(ph)PGGV$YATR# S56 Vimentin regulation between X-ray and the particle radiations. Potential reason for this may be deactivation of common upstream regulator leading to uniform dephosphorylation pattern of cor- responding target sites. Putative kinases for these sites are illustrated in Fig. 7C determined by the NetworKIN algorithm (33). Six sites have cyclin-dependent kinases (CDKs) as reg- ulatory kinase in common emphasizing their potential role as upstream regulator of this differential signaling. Enriched GO terms in cluster 2 (Fig. RESULTS It can be seen, that the light (§) and medium ($) peptide are comparable between the treatments but heavy (#) shows clear aberrations. For X-rays the heavy peptide has almost same abundance as the light peptide and for the two particle irradiations almost no heavy peptide can be detected which precisely confirms the quantification illus- trated in Fig. 6A. On the right of Fig. 6B representative frag- ment spectra are additionally shown for the three peptide species. Exemplarily, the zoom shows the y8-ion for each isotope label. Combination of the selected transitions con- firms identity and exact site of phosphorylation for each of the phosphopeptides. selected transitions of the light (sham), medium (synthetic), and heavy (irradiated) peptides, and (iii) retention time com- parison between the phosphopeptide identified in the discov- ery experiment and the corresponding synthetic peptide. De- tailed results for each of the 28 selected phosphopeptides can be found in supplemental Fig. S3. For elucidation of the procedure additional information is given for VIM (S56) in Fig. 6B. Left, three mass spectra are shown, one for each of the three radiation treatments. It can be seen, that the light (§) and medium ($) peptide are comparable between the treatments but heavy (#) shows clear aberrations. For X-rays the heavy peptide has almost same abundance as the light peptide and for the two particle irradiations almost no heavy peptide can be detected which precisely confirms the quantification illus- trated in Fig. 6A. On the right of Fig. 6B representative frag- ment spectra are additionally shown for the three peptide species. Exemplarily, the zoom shows the y8-ion for each A heatmap containing all 28 validated candidates is illus- trated in Fig. 7A. The heatmap is divided into two clusters containing phosphorylation sites mainly comprising two dis- tinct sequence motifs. Cluster 1 represents the sites of the p(S/T)Q-motif and few nonassigned sites which show intense upregulation and additionally uniform regulation across all radiation qualities. These sites are significantly enriched in the nuclear parts of the cell going along with their associated biological functions in the DDR (Fig. 7B). Thus, basic DDR functions maintaining genome integrity are similarly regulated by the different radiation qualities. Phosphorylation sites associated to cluster 2 comprise sites of the p(S/T)P-motif which are dephosphorylated in re- sponse to irradiation. RESULTS B, MS1 spectra of VIM S56 peptides for the three different treatments (X-ray, 1H: protons, 12C: carbon ions). C, MS2 spectra of VIM S56 peptides for the three different isotopic states (§light peptide (sham), $medium peptide (synthetic); #heavy peptide (irradiated)). Zoom demonstrates exemplarily the mass shift between the isotopic states for the y8-ion of the depicted peptide. RESULTS 7B) highlight their role in apoptosis and DNA metabolic processes as well as their localization in the outer parts of the cell (cytoplasm, cytoskeleton, cytosol). TP53BP1 with its crucial role in the DDR links the two clusters with several sites in both of them. Seven TP53BP1 residues show strong upregulation induced by the ATM/ ATR kinases (S398, S580, S831, T855, S1068) and PRKDC kinase (S830, S1759), respectively. In contrast, three TP53BP1 sites showed downregulation correlating with de- creased CDK1 activity (S294, S1094, S1101) (Fig. 7D). Here- with, we underline the versatile mediator role of TP53BP1 in response to ionizing radiation. Allocation of these regulated sites at TP53BP1 is depicted schematically in Fig. 7E. This illustrates an enrichment of regulated phosphorylation sites Molecular & Cellular Proteomics 16.5 866 Radiation Proteomics FIG. 6. Validation of phosphorylation sites using synthetic isotope labeled phosphopeptides. A, Validation of the differentially regulate hosphorylation sites exemplified by S56 on VIM. Quantification of the depicted phosphorylation site after irradiation shows differentia egulation between X-rays and the two particle treatments as seen in the left panel (mean S.E., n 3). Identification of the phosphosit verified by comparing selected transitions (central panel) and comparing the retention time in the discovery data set with the retentio me of the synthetic peptide (right panel). B, MS1 spectra of VIM S56 peptides for the three different treatments (X-ray, 1H: protons, 12C arbon ions). C, MS2 spectra of VIM S56 peptides for the three different isotopic states (§light peptide (sham), $medium peptide (synthetic heavy peptide (irradiated)). Zoom demonstrates exemplarily the mass shift between the isotopic states for the y8-ion of the depicte eptide. 6 V lid ti f h h l ti it i th ti i t l b l d h h tid A V lid ti f th diff ti ll FIG. 6. Validation of phosphorylation sites using synthetic isotope labeled phosphopeptides. A, Validation of the differentially regulated phosphorylation sites exemplified by S56 on VIM. Quantification of the depicted phosphorylation site after irradiation shows differential regulation between X-rays and the two particle treatments as seen in the left panel (mean S.E., n 3). Identification of the phosphosite is verified by comparing selected transitions (central panel) and comparing the retention time in the discovery data set with the retention time of the synthetic peptide (right panel). DISCUSSION A, Phosphosite regulation following irradiation with X-rays, protons (1H) an bon ions (12C) plotted as heatmap for 28 phosphopeptides validated using synthetic isotope labeled phosphopeptides in a spike-i FIG. 7. Validated response to different radiation qualities. A, Phosphosite regulation following irradiation with X-rays, protons (1H) and carbon ions (12C) plotted as heatmap for 28 phosphopeptides validated using synthetic isotope labeled phosphopeptides in a spike-in experiment. Phosphopeptides are grouped into two clusters. Cluster 1 contains peptides of the p(S/T)Q and nonassigned motifs. Cluster 2 contains the peptides of the p(S/T)P motif. Values represent the normalized log2-transformed ratios for the change in phosphorylation status between irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the set of validated phosphopeptides represented by their corresponding protein. Analysis was performed in the two illustrated clusters separately to uncover their particular characteristics concerning cellular localization and biological function. Illustrated are the five most significant terms in each cluster by their corresponding p value. C, NetworKIN algorithm in KinomeXplorer (32, 33) was used to predict associated kinases in order to estimate potential upstream regulators of the identified alterations. Kinase prediction for phosphorylation sites being differentially regulated between the radiation qualities. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin PhosphoPath (34) D, Sites on TP53BP1 show likewise up- and downregulation. Upregulations are conducted by the phosphoinositide-3-like kinases (PIKK); ATM, ATR and PRKDC. Concurrently, three phosphorylation sites are downregulated by decreased CDK1 activity. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin PhosphoPath (34) E, Schematic graph of TP53BP1 (1,972 amino acids) including two noteworthy structural features containing radiation regulated phosphorylation sites identified in this study: N-Terminal clusters of the p(S/T)-Q motif and two C-terminal BRCT domains. Domains not affected by our results are left out. The two depicted domains are responsible for TP53BP1 interaction with RIF1/PTIP and p53/EXPAND1 respectively (40). FIG. 7. Validated response to different radiation qualities. A, Phosphosite regulation following irradiation with X-rays, protons (1H) and carbon ions (12C) plotted as heatmap for 28 phosphopeptides validated using synthetic isotope labeled phosphopeptides in a spike-in experiment. Phosphopeptides are grouped into two clusters. Cluster 1 contains peptides of the p(S/T)Q and nonassigned motifs. Cluster 2 contains the peptides of the p(S/T)P motif. DISCUSSION in the N-terminal p(S/T)Q repeats responsible for interaction with telomere-associated protein RIF1 and PAX-interacting protein 1 (PTIP) as well as a single site in the BRCA1 C-Terminal domain (BRCT) associated with tumor anti- gen p53 and PWWP domain-containing protein MUM1 (EXPAND1) interaction mediating different cellular functions (40). Intriguingly, we identified a number of regulated phos- phorylation sites on TP53BP1 for which no functional infor- mation is currently available at the PhosphoSitePlus data- base (30). Precision radiotherapy such as particle radiation exhibits physical and biological advantages in comparison to conven- tional X-ray therapy (3). The application of particle radiation is especially of interest for difficult to reach tumors such as those in close proximity to organs at risk, where highly precise conformity of irradiation beams is needed and tumors being resistant to conventional X-ray therapy (i.e. hypoxic tumors) (48–50). Although particle radiation is already applied for pa- tient treatment, there is still only limited knowledge about the Molecular & Cellular Proteomics 16.5 867 Radiation Proteomics FIG. 7. Validated response to different radiation qualities. A, Phosphosite regulation following irradiation with X-rays, protons (1H) and rbon ions (12C) plotted as heatmap for 28 phosphopeptides validated using synthetic isotope labeled phosphopeptides in a spike-in periment. Phosphopeptides are grouped into two clusters. Cluster 1 contains peptides of the p(S/T)Q and nonassigned motifs. Cluster 2 ntains the peptides of the p(S/T)P motif. Values represent the normalized log2-transformed ratios for the change in phosphorylation status tween irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the t of validated phosphopeptides represented by their corresponding protein. Analysis was performed in the two illustrated clusters separately uncover their particular characteristics concerning cellular localization and biological function. Illustrated are the five most significant terms each cluster by their corresponding p value. C, NetworKIN algorithm in KinomeXplorer (32, 33) was used to predict associated kinases in der to estimate potential upstream regulators of the identified alterations. Kinase prediction for phosphorylation sites being differentially gulated between the radiation qualities. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin osphoPath (34) D, Sites on TP53BP1 show likewise up- and downregulation. Upregulations are conducted by the phosphoinositide-3-like nases (PIKK); ATM, ATR and PRKDC. Concurrently, three phosphorylation sites are downregulated by decreased CDK1 activity. Regulation IG. 7. Validated response to different radiation qualities. DISCUSSION These findings together emphasize, that radiation in- duced acute signaling pathways are dominated by DNA repair functions, cell cycle regulation and cell survival/death signals as previously reported (68). Our data enables the assignment of phosphorylation sites to distinct cellular functions and facilitate further investigations of exact phosphosite functionality. Applying SILAC based phosphoproteomics, we identified a total of 5107 phosphorylation sites, including 2818 quantified sites that were subject of statistical analysis because of strin- gent exclusion thresholds. A phosphopeptide enrichment protocol was adopted which required only small sample amount (150 g per SILAC state). Other enrichment strategies such as extensive sample fractionation would enhance the number of identifications and deepen the coverage of regu- lated signaling pathways (52–55), however this would require a larger amount of starting material. Moreover, our enrichment protocol was optimized for peptides containing one phosphor- ylation site only. Enhancement of multiple phosphorylated peptides would necessitate adjustments of the protocol such as reduction of IMAC material during enrichments (56). An additional extension by a single-step immunoaffinity purifica- tion would enlarge the proportion of phosphotyrosine resi- dues (57, 58). However, as irradiated sample material was very limited because of restricted time slots for researchers caused by privileged beam time for patient treatment we could not randomly extend sample amount to apply additional enrichment strategies. Despite employing depicted isoeffective doses, 55 phos- phorylation sites were identified to respond differentially be- tween the three radiations. We identified distinct p(S/T)P sites with radiation quality dependent regulation being mostly re- flected by minor downregulation of phosphorylation sites after X-ray treatment and pronounced downregulation following particle irradiation. This coherent pattern leads to the deduc- tion that a common upstream regulator is responsible for these regulations. Our data points to the CDK family with its versatile regulation of cellular processes including most im- portantly cell cycle and transcription regulation (69). We could show that particle irradiation leads to dephosphorylation of proteins involved in cell mitosis such as MKI67, MAP1B, To examine cellular signaling events differentially regulated by the radiation qualities, application of radiobiological equiv- alent doses is a prerequisite. Therefore, we determined the RBE utilizing the gold standard clonogenic survival assay. DISCUSSION Our goal was to characterize the triggered signaling events and decipher aberrant signaling following treatment with the applied radiation qualities. The depicted study represents a robust and efficient strat- egy to elucidate proteomic and phosphoproteomic changes in response to ionizing radiation. Alterations of protein abun- dance were observed at this acute time point for eight pro- teins only. In contrast, the prevailing pattern of cellular response was mediated by phosphorylation as a fast mech- anism of signal transduction. In line with our observation, 6 Gy X-ray irradiation of GM00130 cells was also found to elicit limited alterations in proteome abundance within a relatively short time frame (5min - 8h) as recently published by Bennet- zen et al. (13) confirming our decision to focus on changes at phosphorylation level. Single or fractionated high radiation doses as employed in the area of stereotactic ablative radio- therapy (SABR, radiosurgery) may result in higher number of differentially expressed proteins as suggested by Cho et al. (51). In contrast, in this study we focused on investigating dose ranges relevant to clinical routine applications. A consensus radiation quality independent phosphorylation pattern of p(S/T)Q sites was found mainly enriched for sites in the DDR, in line with current knowledge that DDR is a hallmark of ionizing radiation (59, 60), which is induced and anticipated by the usage of cell kill based isoeffective doses. Observed downregulation of p(S/T)P and pSPXXK sites representing motifs of the CDK, GSK-3 and MAP kinases have crucial role in the cell response to ionizing radiation per se. CDKs play a key role in maintaining genome integrity (61). Our finding that radiation induced DNA damage affect protein substrates of CDKs hence provides a plausible explanation for the phenom- enological observation of cell cycle arrest after irradiation. Indeed, radiation induced cell cycle arrest is important for proper DNA repair (62, 63). Recent findings indicate that CDKs additionally contribute to further upstream events such as checkpoint control and DNA repair (64). GSK-3 executes heterogeneous signaling functions including both cell death and survival signals dependent on the signaling context (65). The MAP kinases ERK1/ERK2 likewise conduct regulation of cell proliferation, migration and death (66, 67). The prevalent downregulation of phosphorylation sites belonging to these kinase motifs implies that cells enter a state of proliferative arrest to enable proper DNA repair and maintain genomic stability. DISCUSSION Values represent the normalized log2-transformed ratios for the change in phosphorylation status between irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the set of validated phosphopeptides represented by their corresponding protein. Analysis was performed in the two illustrated clusters separately to uncover their particular characteristics concerning cellular localization and biological function. Illustrated are the five most significant terms in each cluster by their corresponding p value. C, NetworKIN algorithm in KinomeXplorer (32, 33) was used to predict associated kinases in order to estimate potential upstream regulators of the identified alterations. Kinase prediction for phosphorylation sites being differentially regulated between the radiation qualities. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin PhosphoPath (34) D, Sites on TP53BP1 show likewise up- and downregulation. Upregulations are conducted by the phosphoinositide-3-like kinases (PIKK); ATM, ATR and PRKDC. Concurrently, three phosphorylation sites are downregulated by decreased CDK1 activity. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin PhosphoPath (34) E, Schematic graph of TP53BP1 (1,972 amino acids) including two noteworthy structural features containing radiation regulated phosphorylation sites identified in this study: N-Terminal clusters of the p(S/T)-Q motif and two C-terminal BRCT domains. Domains not affected by our results are left out. The two depicted domains are responsible for TP53BP1 interaction with RIF1/PTIP and p53/EXPAND1 respectively (40). Molecular & Cellular Proteomics 16.5 868 Radiation Proteomics bon). Statistical analyses were conducted to: (i) identify phos- phorylation sites with common regulation pattern across all radiation qualities and (ii) identify sites with differential phos- phorylation pattern between the three radiation qualities. A total of 181 phosphorylation sites are regulated in response to ionizing radiation. One hundred twenty-six sites thereof are regulated regardless of the radiation quality, whereas 55 show differential regulation between the qualities. We found enrich- ment of five different kinase sequence motifs in our data and could confirm similar enrichment of motif families by analysis of previously published radiation dependent phosphoproteome al- terations deposited in the PhosphoSitePlus database. molecular and cellular effects induced by the different radia- tion qualities. In our study, we compared the acute proteome and phosphoproteome response of human lung adenocarci- noma (A549) cells 2h after irradiation with X-rays, protons and carbon ions, respectively. Molecular & Cellular Proteomics 16.5 DISCUSSION At isoeffective doses of all three radiation qualities, resulting in 30% survival fraction, we were able to identify a large number of regulated phosphorylation sites and most importantly could show for the first time differential signaling induced by X-rays versus the raster-scanning particle irradiations (proton/car- Molecular & Cellular Proteomics 16.5 869 Radiation Proteomics ROS production and indirect damage, high-LET carbon ion irradiation induces direct complex DNA damages (6). Hence differences between these two radiation qualities were antic- ipated. In contrast, proton irradiation was so far thought to elicit similar biological effects to X-rays reflected by the cur- rently fixed proton RBE of 1.1 for patient treatment. Surpris- ingly, unsupervised clustering and principle component anal- ysis clearly separated X-ray irradiation from proton and carbon irradiation. Hence, the low-LET proton irradiation shared more common phosphoproteome characteristics with high-LET carbon than with conventional X-ray irradiation. This is an interesting unexpected finding, however in line with more recent data indicating potential less appreciated radiobiolog- ical differences attributed to protons beyond the classical cell killing effect (11). Further, in the field of radiotherapy the paradigm of fixed proton RBE of 1.1 is currently controver- sially debated and increasing body of data imply that im- proved radiobiological models are urgently needed to better characterize the exact effect of proton irradiation (77). Our observations at phosphoproteome level indicate that differ- ences in radiobiology between X-ray and proton irradiation exist even at doses that produce the same level of cell kill. RRBP1, and KPNA2 to a larger extent than X-ray irradiation. Sites on STMN1, FLNB, and VIM that are involved in cell migration and invasion show a similar pattern. These obser- vations support the previously published data on functional analysis of different cell lines after proton and carbon irradi- ation in comparison to X-rays, suggesting altered regulation of angiogenesis (10, 11, 70), cell migration (71, 72), and mitosis (73) by different radiation qualities. We identified phosphor- ylation sites corresponding to these processes that were not yet described in the context of ionizing radiation. However, their exact role within the context of radiation biology remains to be elucidated. For validation purposes, we selected phosphorylation sites of interest and confirmed their identity in an unbiased spike-in approach using synthetic isotope labeled phosphopeptides. DISCUSSION Compared with Western blotting, the use of synthetic pep- tides has tremendous advantage: (i) no phosphosite specific antibodies are needed, (ii) uncomplicated and inexpensive production of synthetic peptides, (iii) misinterpretation of Western blot signals because of cross-reactivity of the anti- bodies is avoided (74), and (iv) distinct multiplexing possibil- ities in a targeted spike-in approach (16, 75). Moreover, in future experiments the same pool of synthetic peptides can be used as internal standards in tissue samples or in vivo experiments (76). Easy handling of synthetic peptides makes a distribution between laboratories possible. The availability of synthetic isotope labeled phosphopeptide pools, similar to antibody arrays, would be a valuable tool to study complete signaling pathways with high confidence and accurate quan- tification in a fast and reproducible manner. When the amount of each synthetic peptide is adjusted to its ionization effi- ciency as conducted in our study and a list of suitable tran- sitions for the identification is included, it would enable a fast and easy usage of these synthetic phosphopeptide pools. In summary, we present a versatile workflow for identifica- tion, quantification and validation of phosphorylation sites in response to ionizing radiation that could be employed for a broad spectrum of applications in life sciences. Our data stimulate radiation research at novel frontiers of intracellular signaling and cell-cell communication. These are instrumental for a better understanding of discrepancies found on tissue level responses to different radiation qualities. Further explo- ration in this direction will supplement and adjust our view on the so far predominantly tumor cell kill centric modeling of radiobiological effects. Especially for the ongoing debate about proton RBE in patient treatment we offer exclusive data. Moreover, the presented phosphorylation sites, that are differentially regulated, offer attractive targets for modulation and improvement of radiotherapy in the clinical setting. Our pool contained 28 synthetic phosphopeptides covering a broad range of characteristic, in order to confirm multiple hypotheses in a single experiment. We confirmed that the DNA damage responsive phosphorylation sites of the p(S/T)Q motif show similar regulation pattern between the radiation qualities. Among these, sites on TP53BP1, PRKDC, and RAD50 with well-known DDR activity were similarly phosphor- ylated by the different radiation qualities. Moreover, our re- sults indicate TP53BP1 to be a key regulator in response to ionizing radiation, supporting the recently published data which highlighted TP53BP1 in a central position to maintain genome integrity (40). □ S This article contains supplemental material. 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DISCUSSION In this work, we validated nine phospho- peptides containing ten phosphorylation sites on TP53BP1, which are regulated by crucial kinases simultaneously (ATM, PRKDC, and CDK1), underlining the versatile role of TP53BP1 as one of the main genome guardian molecules (40). Acknowledgments—We thank Claudia Rittmu¨ ller for her excellent technical assistance with all cell experiments, Andrea Mairani and Stephan Brons for providing medical physics support at HIT for particle beam irradiation and Ramona Mayer for her excellent tech- nical assistance during sample preparations. * This work was supported by German Research Council (DFG- KFO214), Deutsche Krebshilfe (Max-Eder 108876) and intramural grants from National Center for Tumor diseases (NCT/DKFZ-DKTK, Heidelberg, Germany). M.W. was financially supported by the Helm- holtz International Graduate School for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. □ S This article contains supplemental material. ‡‡ To whom correspondence should be addressed: German Can- cer Research Center (DKFZ), Functional Proteome Analysis, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Tel.: 49 6221 42-2723; Fax: 49 6221 42 4562; E-mail: m.schnoelzer@dkfz.de. §§ Shared senior authors. ‡‡ To whom correspondence should be addressed: German Can- cer Research Center (DKFZ), Functional Proteome Analysis, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Tel.: 49 6221 42-2723; Fax: 49 6221 42 4562; E-mail: m.schnoelzer@dkfz.de. §§ Shared senior authors. Moreover we confirmed ten phosphorylation sites with dif- ferential regulation pattern between the radiation qualities which is because of the radiations mode of action. 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https://openalex.org/W4392878576	https://e-journal.uniflor.ac.id/index.php/JPE/article/download/638/648	Indonesian	null	Penggunaan Model Pembelajaran Kooperatif Tipe Group Investigation Untuk Meningkatkan Hasil Belajar Bahasa Indonesia Pada Siswa Kelas III Mis Ar-Rahman IPI	Ekspektasi	2,020	cc-by-sa	3,403	Keywords: group investigation, learning outcomes Penggunaan Model Pembelajaran Kooperatif Tipe Group Investigation Untuk Meningkatkan Hasil Belajar Bahasa Indonesia Pada Siswa Kelas III Mis Ar-Rahman IPI enggunaan Model Pembelajaran Kooperatif Tipe Group Investigation Untuk Meningkatkan Hasil Belajar Bahasa Indonesia Pada Siswa Kelas III Mis Ar-Rahman IPI Nur Hasna e-mail: nurhasnasharail@gmail.com Guru Mis Ar-Rahman IPI Ende PENDAHULUAN merubah perilaku siswa itu sendiri baik dari ranah kognitif, afektif dan psikomotor. Bahasa memiliki peranan penting dalam perkembangan berbagai ilmu pengetahuan dan kemajuan daya pikir manusia. Pada hakikatnya, pembelajaran bahasa Indonesia diarahkan untuk meningkatkan kemampuan siswa dalam berkomunikasi menggunakan bahasa Indonesia baik secara lisan maupun tulisan (Susanto, 2013). Dalam proses pembelajaran, penguasaan bahasa yang tidak baik atau kurang sempurna akan mempengaruhi tujuan pembelajaran itu sendiri. Pembelajaran bahasa Indonesia di SD/MI bertujuan agar siswa memiliki keterampilan berbahasa Indonesia yang baik dan benar, serta dapat menghayati bahasa. Keterampilan berbahasa terdiri dari empat komponen yaitu keterampilan menyimak, keterampilan berbicara, keterampilan membaca, dan keterampilan menulis (Nurhidayat, 2019). Salah satu cara umtuk mengukur keterampilan bahasa adalah melalui test hasil belajar. Slameto (2010) menggolongkan faktor hasil belajar menjadi dua yaitu faktor intern dan faktor ekstern. Faktor intern adalah faktor yang berasal dari dalam diri individu yang sedang belajar yaitu faktor jasmani, faktor psikologis, dan faktor kelelahan. Sedangkan faktor ekstern adalah faktor yang ada di luar individu, yaitu faktor keluarga, faktor sekolah dan faktor masyarakat. Sedangkan menurut Suryabrata (2010) faktor yang mempengaruhi hasil belajar adalah faktor internal dan faktor eksternal. Faktor internal digolongkan menjadi dua yaitu faktor fisiologi dan faktor psikologi. Sedangkan faktor eksternal digolongkan menjadi faktor nonsosial dan faktor sosial. Berdasarkan pendapat tersebut maka dapat diambil kesimpulan bahwa baik tidaknya hasil belajar siswa dipengaruhi oleh banyak faktor. Berdasarkan pengamatan selama proses pembelajaran dan data hasil belajar ditemukan bahwa hasil belajar bahasa Indonesia siswa kelas III masih belum maksimal, hasil yang diperoleh belum bisa mencapai tujuan pembelajaran yang diharapkan. Rendahnya hasil belajar ini disebabkan karena dua faktor yaitu faktor dari dalam diri siswa itu sendiri dan faktor dari luar siswa. Faktor pertama adalah faktor yang berasal dari dalam diri siswa yaitu minat dan motivasi yang masih rendah. Hal ini terlihat pada proses pembelajaran, dimana sebagian besar siswa masih kurang memperhatikan apa yang diajarkan guru di depan kelas. Beberapa siswa ada yang mengganggu temannya yang sedang belajar dan ada beberapa anak yang sibuk melakukan aktivitas yang tidak berkaitan dengan apa yang sedang diajarkan guru seperti mengambar dan mencoret-coret kertas. j Susanto (2013) menyatakan bahwa hasil belajar adalah kemampuan yang diperoleh anak setelah melalui kegiatan belajar. Belajar merupakan proses dari seorang yang berusaha untuk memperoleh suatu bentuk perubahan perilaku yang relatif menetap. Hasil belajar juga diartikan sebagai perubahan-perubahan yang terjadi pada diri siswa. Guru Mis Ar-Rahman IPI Ende ABSTRAK: Penelitian ini bertujuan untuk meningkatkan hasil belajar bahasa Indonesia melalui penggunaan model pembelajaran kooperatif tipe Group Investigation pada siswa kelas III Mis Ar-rahman Ipi. Penelitian ini adalah penelitian tindakan kelas yang menggunakan model Kemmis & Taggart. Teknik pengumpulan data dilaksanakan dengan cara observasi, tes dan dokumentasi. Data dianalisis dengan cara deskriptif kualitatif dan deskriptif kuantitatif. Hasil penelitian menunjukkan bahwa hasil belajar bahasa Indonesia melalui penggunaan model pembelajaran kooperatif tipe Group Investigation mengalami peningkatan. Pada siklus I menunjukkan bahwa dari 21 orang siswa, hanya 11 siswa saja yang memenuhi kriteria ketuntasan klasikal dengan persentase 52,38% dan nilai rata-rata sebesar 67,61, dan pada siklus II, nilai rata-rata mengalami peningkatan menjadi 81,42 dengan ketuntasan klasikal mencapai 100%. Berdasarkan hasil tersebut dapat disimpulkan bahwa penggunaa model pembelajaran kooperatif tipe Group Investigation dapat meningkatkan hasil belajar bahasa Indonesia. Kata kunci: group investigation, hasil belajar ABSTRACT: This research aims to improve the results of Indonesian language learning through the use of Group Investigation Cooperative learning model in grade III students at Mis Ar- Rahman IPI. This research is a class action research that uses the Kemmis & Taggart model. Data collection techniques are performed by observation, test and documentation. Data is analyzed in a qualitative and quantitative descriptive way. The results showed that the results of Indonesian language learning through the use of Group Investigation Cooperative learning models have increased. In cycle I showed that out of 21 students, only 11 students meet the classifications criterion with a percentage of 52.38% and an average value of 67.61, and in cycle II, the average value has increased to 81.42 by The classical dictancy reaches 100%. Based on the results it can be concluded that the use of Group Investigation Cooperative learning model can improve Indonesian language learning outcomes. 33 Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 PENDAHULUAN Perubahan tersebut berkaitan dengan perubahan pengetahuan (kognitif), sikap (afektif), dan keterampilan (psikomotorik) sebagai hasil dari proses kegiatan belajar. Sejalan dengan Susanto, Sudjana (2013) menyatakan bahwa hasil belajar merupakan kemampuan-kemampuan yang dimiliki siswa setelah ia menerima pengalaman belajarnya. Sehingga dapat disimpulkan bahwa hasil belajar adalah proses yang dilakukan siswa selama pembelajaran yang menghasilkan pengalaman belajar yang 34 Faktor kedua adalah faktor yang berasal dari luar siswa. Berdasarkan pengamatan yang dilakukan selama pembelajaran di sekolah berlangsung, ditemukan bahwa pembelajaran yang dilakukan belum terlaksana dengan baik. Pemilihan model pembelajaran yang digunakan guru kurang tepat. Kurang tepatnya model yang digunakan mempengaruhi minat dan motivasi belajar bahasa Indonesia pada siswa kelas III, sehingga hal ini berpengaruh pula pada hasil belajar yang diperoleh menjadi kurang maksimal. Ada banyak model pembelajaran yang bisa diterapkan pada pembelajaran bahasa Indonesia. Salah satu nya adalah model pembelajaran kooperatif tipe Group Investigation 2) Merencanakan Tugas yang akan dipelajari Pada tahap ini, peserta didik merencanakan bersama mengenai apa yang akan dipelajari? Bagaimana cara mempelajarinya? Siapa yang melakukannya? (pembagian tugas) untuk tujuan atau kepentingan apa menginvestigasi topik ini? 3) 3) Melaksanakan Investigasi Tahap ini para peseta didik mengumpulkan informasi, mengnalisis data, dan membuat kesimpulan. Tiap kelompok berkontribusi untiuk usaha- usaha yang dilakukan kelompoknya.para peserta didik saling bertukar, berdiskusi, mengklarifikasi, dan mensintesis semua gagasan. 4) Slavin (2005) mengemukakan bahwa model pembelajaran kooperatif tipe Group Investigation, yaitu kelompok investigasi. Group investigation yang dikembangkan oleh Shlomo dan Yael Sharan di Universitas Tel aviv, merupakan perencanaan pengaturan kelas yang umum dimana para siswa bekerja dalam kelompok kecil menggunaan pertanyaan kooperatif, diskusi kelompok, serta perencanaan dan proyek kooperatif. Menurut Slavin (2005) ada beberapa langkah-langkah model pembelajaran kooperatif tipe group investigation yaitu: 4) Menyiapkan Laporan Akhir Kegiatan yang dilakukan pada tahap ini adalah setiap anggota kelompok menentukan pesan-pesan esensial dan proyek mereka. Anggota kelompok merencanakan apa yang akan mereka laporkan dan bagaimana mereka secara kelompok akan membuat presentasi mereka. Setiap wakil-wakil kelompok membentuk sebuah panitia acara untuk mengkoordinasikan rencana-rencana presentasi. 5) Mempresentasikan Laporan Akhir Presentasi yang dibuat untuk seluruh kelas dalam berbagai macam bentuk. Bagian presentasi tersebut harus dapat melibatkan pendengaran secara aktif. Para pendengar tersebut mengevaluasi kerjasama dan penampilan presentasi berdasarkan kriteria yang telah ditentukan sebelumnya oleh seluruh anggota kelas. E l i y 1) Mengidentifikaskan Topik dan Mengatur Siswa dalam Kelompok Pada tahap ini peserta didik meneliti beberapa sumber, mengusulkan sejumlah topik, dan mengkategorikan saran-saran. Peserta didik bergabung dengan kelompoknya untuk mempelajari topik yang telah mereka pilih. PENDAHULUAN Adapun komposisi kelompok didasarkan pada ketertarikan peserta didik dan harus bersifat heterogen. Pada tahap ini pula guru membantu pengumpulan informasi/ memfasilitasi pengaturan. 1) Mengidentifikaskan Topik dan Mengatur Siswa dalam Kelompok Pada tahap ini peserta didik meneliti beberapa sumber, mengusulkan sejumlah topik, dan mengkategorikan saran-saran. Peserta didik bergabung dengan kelompoknya untuk mempelajari topik yang telah mereka pilih. Adapun komposisi kelompok didasarkan pada ketertarikan peserta didik dan harus bersifat heterogen. Pada tahap ini pula guru membantu pengumpulan informasi/ memfasilitasi pengaturan. 6) Evaluasi Para peserta didik saling memberikan umpan balik mengenai topik tersebut, mengenai tugas yang 35 pektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 telah mereka kerjakan, mengenai keefektifan pengalaman-pengalaman mereka. Guru dan peserta didik berkolaborasi dalam mengevaluasi pembelajaran. Hasil Belajar Mata Pelajaran Sosiologi Pada Siswa Kelas X 3 SMA Negeri Colomadu Tahun Pelajaran 2011/2012’’. Hasil penelitian menunjukkan bahwa keaktifan dan hasil belajar sosiologi siswa setelah diterapkan pembelajaran model kooperatif tipe group investigation pada materi perilaku penyimpangan dan pengendalian sosial mengalami peningkatan . Hasil Belajar Mata Pelajaran Sosiologi Pada Siswa Kelas X 3 SMA Negeri Colomadu Tahun Pelajaran 2011/2012’’. Hasil penelitian menunjukkan bahwa keaktifan dan hasil belajar sosiologi siswa setelah diterapkan pembelajaran model kooperatif tipe group investigation pada materi perilaku penyimpangan dan pengendalian sosial mengalami peningkatan . Rusman (2014) mengemukakan beberapa kelebihan pembelajaran kooperatif tipe group investigation yaitu sebagai berikut: Penelitian kedua dilakukan oleh Ayuwati (2016) yang berjudul “Meningkatkan Aktivitas dan Hasil Belajar Matematika Menggunakan Model Pembelajaran Kooperatif Tipe Group Investigation di SMK Tuma’nnah Yasin Metro”. Hasil penelitian menunjukkan bahwa pembelajaran dengan menggunakan cooperative learning model group investigation dapat meningkatkan aktivitas belajar matematika siswa kelas X SMK Tuma’ninah Yasin Metro. Hasil belajar menggunakan model group investigation mengalami peningkatan dari 27,5% pada siklus I menjadi 54,54% pada siklus II dan meningkat menjadi 81,8% pada siklus III. 1) Dirancang untuk membantu terjadinya pembagian tanggung jawab ketika siswa mengikuti pembelajaran. 2) Berorientasi menuju pembentukan siswa menjadi manusia social 3) Dapat mengembangkan kreativitas siswa, baik secara individu maupun secara kelompok. 4) Membentuk kesempatan berkolaborasi dengan teman sebaya dalam bentuk diskusi kelompok untuk memecahkan suatu masalah 5) Mengaktifkan siswa dalam proses pembelajaran yang diberikan guru sehingga membangun pengetahuan siswa. Berdasarkan hasil penelitian yang dilakukan oleh peneliti terdahulu, ditemukan bahwa penerapan model pembelajaran kooperatif tipe group investigation efektif meningkatkan aktivitas dan hasil belajar. Melihat banyaknya manfaat dan efektivitas dari model pembelajaran kooperatif tipe group investigation ini, maka peneliti tertarik untuk menerapkan model pembelajaran kooperatif tipe group investigation untuk mengatasi permasalahan yang dihadapi di kelas. Adapun perbedaan penelitian ini dengan penelitian terdahulu adalah penelitian ini dilakukan untuk mengatasi permasalahan hasil belajar pada pembelajaran bahasa Indonesia di tingkat SD/MIS. Penelitian yang dilakukan peneliti adalah untuk mengatasi permasalahan yang ditemukan selama proses pembelajaran. (Trianto,2011: .63) Sedangkan untuk menghitung persentase ketuntasan belajar klasikal, digunakan rumus sebagai berikut : ∑ ∑ 100% (Daryanto, 2011: 192) Pembelajaran dengan menggunakan model pembelajaran kooperatif tipe group investigation pada siswa kelas III Mis Ar- Rahman Ipi dikatakan efektif meningkatkan hasil belajar bahasa Indonesia, apabila hasil belajar siswa ≥ 72, dengan persentase ketuntasan klasikal sekurang-kurangnya 85 %. KB = 100 METODE PENELITIAN Metode penelitian yang dilakukan dalam penelitian ini adalah metode penelitian tindakan kelas (PTK). PTK yang dilakukan peneliti merujuk pada prosedur penelitian dari Kemmis dan Mc Taggart. Penelitian ini terdiri dalam 4 tahapan yaitu tahap perencanaan, pelaksanaan, observasi dan refleksi. Subjek dalam penelitianini adalahsiswa kelas III Mis Ar-Rahman Ipi yang berjumlah 21 orang, 12 orang laki-laki dan 9 orang perempuan. Teknik pengumpulan data dilakukan melalui observasi dan tes. Data dianalisis secara deskriptif kualitatif dan kuantitatif. Bentuk analisis persentase hasil belajar adalah sebagai berikut: siswa kelas III Mis Ar-Rahman Ipi. Keterangan: KB: Ketuntasa Belajar T: Skor yang Diperoleh Siswa Tt: Skor Maksimal Keterangan: KB: Ketuntasa Belajar T: Skor yang Diperoleh Siswa Tt: Skor Maksimal Evaluasi Adapun tujuan dari penelitian ini adalah untuk meningkatkan hasil belajar bahasa Dari penjelasan tersebut diatas maka dapat diambil kesimpulan bahwa pembelajaran kooperatif tipe group investigation memiliki kelebihan, dimana dalam pembelajaran ini siswa dapat memiliki rasa tanggung jawab baik secara individu maupun kelompok, melalui kegiatan diskusi siswa menjadi lebih aktif dan mampu berinteraksi dengan teman sebaya serta mampu memecahkan permasalahan yang ada. Ada beberapa penelitian yang pernah dilakukan dengan menggunakan pembelajaran kooperatif tipe group investigation. Yang pertama penelitian yang dilakukan oleh Rahmawati (2012) yang berjudul “Penerapan Model Pembelajaran Kooperatif Tipe Group Investigation (IG) untuk Meningkatkan Keaktifan Belajar dan Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 36 Indonesia melalui model pembelajaran kooperatif tipe group investigation pada PEMBAHASAN HASIL penggunaan model pembelajaran kooperatif tipe group investigation pada siswa kelas III Mis Ar-Rahman Ipi. Hasil penelitian dapat dilihat pada tabel 1 berikut ini: penggunaan model pembelajaran kooperatif tipe group investigation pada siswa kelas III Mis Ar-Rahman Ipi. Hasil penelitian dapat dilihat pada tabel 1 berikut ini: penelitian ini bertujuan untuk meningkatkan hasil belajar pada pembelajaran bahasa Indonesia melalui penelitian ini bertujuan untuk meningkatkan hasil belajar pada pembelajaran bahasa Indonesia melalui Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 37 Tabel 1. Nilai hasil belajar Bahasa Indonesia No Nilai hasil belajar Siklus I Siklus II 1 Rata-rata 67,61 81,42 2 Tuntas 52,38% 100% 3 Tidak Tuntas 47,61%% - Berdasarkan data pada tabel 1, diketahui bahwa persentase ketuntasan belajar Pada siklus I adalah 52,38% dengan nilai rata-rata sebesar 67,61. Mengalami peningkatan pada siklus II, dimana nilai rata-rata kelas meningkat menjadi 81,42 dengan persentase ketuntasan sebesar 100%. Hasil pada siklus II sudah memenuhi target yang ingin dicapai. Dengan demikian penelitian dengan menggunakan model pembelajaran kooperatif tipe group investigation telah Tabel 1. Nilai hasil belajar Bahasa Indonesia Tabel 1. Nilai hasil belajar Bahasa Indonesia Berdasarkan data pada tabel 1, diketahui bahwa persentase ketuntasan belajar Pada siklus I adalah 52,38% dengan nilai rata-rata sebesar 67,61. Mengalami peningkatan pada siklus II, dimana nilai rata-rata kelas meningkat menjadi 81,42 dengan persentase 37 berhasil meningkatkan hasil belajar siswa kelas III Mis Ar-Rahman Ipi. kelompoknya. Anggota kelompok dipilih berdasarkan keterampilan dan keheterogenan, mengidentifikasikan topik, memotivasi peserta didik untuk terlibat dalam investigasi guna mencapai tujuan pembelajaran. kelompoknya. Anggota kelompok dipilih berdasarkan keterampilan dan keheterogenan, mengidentifikasikan topik, memotivasi peserta didik untuk terlibat dalam investigasi guna mencapai tujuan pembelajaran. Penelitian tindakan kelas yang dilakukan dalam penelitian ini melalui empat tahapan yaitu tahap perencanaan, tahap pelaksanaan, tahap observasi dan tahap refleksi. Pada tahap perencanaan, peneliti menyiapkan perangkat pembelajaran seperti menentukan materi yang akan diajarkan, membuat RPP yang menggunakan model pembelajaran kooperatif tipe group investigation, menyiapkan alat dan bahan sesuai dengan materi yang akan diajarkan, membuat lembar kerja siswa, dan menyusun evaluasi pembelajaran. 2) Merencanakan tugas yang akan dipelajari Kegiatan yang dilakukan guru pada langkah kedua ini adalah guru meminta peserta didik untuk merencanakan tugas yang diberikan, membagi tugas, menggali informasi, bekerjasama dan berdiskusi. Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 PEMBAHASAN HASIL 2) Merencanakan tugas yang akan dipelajari 3) Melaksanakan Investigasi Pada langkah ketiga ini guru meminta peserta didik melakukan investigasi secara berkelompok, mengumpulkan informasi yang didapat untuk menyelesaikan topic yang dipilihnya, kemudian meminta peseta didik untuk menganalisis data dan membuat kesimpulan terkait dengan permasalahan yang diselidiki Tahap yang kedua adalah tahap pelaksanaan tindakan. Penelitian ini dilaksanakan pada bulan September 2019. Penelitian ini dilakukan di Mis Ar-Rahman Ipi dengan menggunakan model pembelajaran kooperatif tipe group investigation dengan berpedoman pada rencana pelaksanaan pembelajaran (RPP) yang telah disiapkan. Adapun langka- langkah pembelajaran menggunakan model pembelajaran kooperatif tipe group investigation adalah sebagai berikut: 4) Menyiapkan laporan akhir Kegiatan yang dilakukan guru pada tahap ini ameminta anggota kelompok menentukan pesan-pesan penting dari topic dalam kelompoknya masing-masing, meminta anggota kelompok merencanakan apa yang akan mereka laporkan, format laporan dan bagaimana mereka akan mempresentasikan laporannya. 1) Mengidentifikasikan topik dan mengatur peserta didik ke dalam kelompok Pada langkah pertama ini kegiatan yang dilakukan guru adalah menyampaikan tujuan pembelajaran yang akan dilakukan, menjelaskan model pembelajaran yang akan digunakan dalam pembelajaran, mengelompokkan peserta didik menjadi 4 kelompok yang terdiri dari 5-6 orang dalam setiap 5) Mempresentasikan laporan akhir Pada langkah kelima ini, guru meminta peserta didik yang bertugas untuk mewakili kelompok menyajikan hasil atau simpulan dari ktasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 38 dengan pembelajaran yang menggunakan model kooperatif tipe group investigation. (2) kurangnya pemahaman akan pembelajaran yang menggunakan model ini menyebabkan pembelajaran tidak terlaksana secara runtut yang mengakibatkan penggunaan waktu dalam pemebelajaran tidak cukup atau kurang (3) pembimbingan dan pemberian motivasi yang diberikan guru pada siswa belum merata sehingga berpengaruh terahadap hasil belajar. Berdasarkan hasil refleksi tersebut maka penelitian dilanjutkan ke siklus berikutnya. investigasi yang telah dilaksanakan, meminta peserta didik yang bukan sebagai penyaji, mengajukan pertanyaan, saran tentang topik yang disajikan, meminta peserta didik mencatat topik yang di paparkan oleh pemateri. investigasi yang telah dilaksanakan, meminta peserta didik yang bukan sebagai penyaji, mengajukan pertanyaan, saran tentang topik yang disajikan, meminta peserta didik mencatat topik yang di paparkan oleh pemateri. 6) Evaluasi Kegiatan yang dilakukan guru pada tahap evaluasi ini adalah mengajak peseta didik untuk bersama-sama mengevaluasi pembelajaran, menyimpulkan dan menggabungkan semua topik yang ada, meminta peserta didik merangkum dan mencata topik yang disajikan. Langkah-langkah pada siklus II dilakukan sama seperti pada siklus I, mulai dari tahap perencanaan, pelaksanaan, observasi dan refleksi. Daftar Pustaka Ayuwanti, Irma. 2016. Meningkatkan Aktivitas dan Hasil Belajar Matematika Menggunakan Model Pembelajaran Kooperatif Tipe Group Investigation di SMK Tuma’ninah Yasin Metro (versi elektronik). Jurnal SAP Vol 1 No 2, 105-114. Nurhidayat, Muhamad. 2019. Peningkatan hasil belajar Bahasa Indonesia Materi Paragraf Terpisah Menggunakan Model Pembelajaran Kooperatif tipe Group Investigation siswa kelas 3 MI Darussalam Kecamatan Beringin Kabupaten SemarangSemester I Tahun Pelajaran 2018/2019. http://e- repository.perpus.iainsalatiga.ac.id/ 5539/. Diakses tanggal 13 maret 2020. Rahmawati, Endah Dwi. 2012. Penerapan Model Pembelajaran Kooperatif Tipe Group Investigation (IG) untuk Meningkatkan Keaktifan Belajar dan Hasil Belajar Mata Pelajaran Sosiologi Pada Siswa Kelas X 3 SMA Negeri Colomadu Tahun Pelajaran 2011/2012. Jurnal Sosialitas Vol 2 No 1. http://www.jurnal.fkip.uns.ac.id/ind ex.php/sosant/article/view/394/195. Diakses tanggal 14 maret 2020. PEMBAHASAN HASIL Akan tetapi kegiatan pada siklus II ini dilakukan berdasarkan hasil refleksi pada siklus I. Dalam pelaksanaan pembelajaran pada siklus II ini, peneliti sudah memberikan pemahaman akan pembelajaran yang akan dipelajari dengan menggunakan model kooperatif tipe group investigation dengan maksimal. Pemahaman yang diberikan guru pada peserta didik membuat pembelajaran melalui model ini terlaksana dengan baik dan runtut sesuai dengan alokasi waktu yang ditentukan. Dalam proses pembelajaran guru sudah membimbing dan memberikan motivasi dengan maksimal secara merata pada semua peserta didik. Hasil penelitian pada siklus II menunjukkan adanya perubahan hasil belajar. Dimana pada siklus ini hasil belajar meningkat dengan nilai rata- rata sebesar 81,42 dengan persentase ketuntasan sebesar 100%. Tahap ketiga, yaitu tahap observasi, pada tahap ini guru melakukan kegiatan pengamatan sesuai dengan lembar observasi yang sudah disiapkan pada tahap perencanaan yaitu tentang keterlaksanaan pembelajaran melalui model pembelajaran kooperatif tipe group investigation. Tahap berikutnya adalah tahap refleksi pada tahap ini kegiatan refleksi dilakukan berdasarkan hasil evaluasi dan dari hasil observasi. Berdasarkan hasil evaluasi, diketahui bahwa pada siklus I, hasil belajar dari sebagian besar siswa belum mencapai KKM 72. Dari 21 orang siswa hanya 11 orang yang tuntas, sedangkan 10 orang lainnya tidak tuntas. Hal ini menunjukan bahwa sebagian peserta didik belum terlalu memahami pembelajaran menggunakan model ini dengan baik Pada siklus I peneliti menemukan bahwa ada beberapa kelemahan-kelemahan yang harus diperbaiki yaitu: (1) sebagian besar peserta didik belum terlalu paham Dari hasil penelitian diketahui bahwa hasil belajar siswa di Mis Ar-Rahman Ipi sudah mencapai keberhasilan sesuai yang Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 39 diharapkan. Hal ini dapat dilihat dari rata- rata kemampuan siswa pada siklus I dan siklus II. Persentase ketuntasan belajar pada siklus I sebesar 52,38% meningkat menjadai 100% pada siklus II dengan Nilai rata-rata sebesar 81,42. Dari data yang diperoleh tersebut, menunjukan bahwa dalam penggunaan model kooperatif tipe group investigation dapat meningkatkan hasil belajar bahasa Indonesia siswa kelas III di Mis Ar-Rahman Ipi. Berdasarkan hasil yang dicapai pada siklus II, maka penelitian tentang penggunaan model pembelajaran kooperatif tipe group investigation untuk meningkatkan hasil belajar bahasa Indonesia pada siswa kelas III di Mis Ar-Rahman Ipi, tidak dilanjutkan ke siklus berikutnya, karena hasil yang di dapat sudah mencapai target yang diharapkan. Hasil belajar siswa sudah memenuhi KKM ≥72 dengan ketuntasan klasikal 100%. pembelajaran yang ingin dicapai dapat terwujud. KESIMPULAN Dari hasil penelitian dan pembahasan, dapat disimpulkan bahwa penggunaan model pembelajaran kooperatif tipe group investigation dapat meningkatkan hasil belajar bahasa Indonesia pada siswa kelas III Mis Ar-Rahman Ipi. Hal ini dapat dilihat dari hasil belajar siswa pada siklus I dengan rata-rata 67,61 dengan persentase ketuntasan sebesar 52,38% meningkat pada siklus II dengan nilai rata-rata 81,42 dan persentase ketuntasan mencapai 100%. Rusman. 2014. Model-Model Pembelajaran Mengembangkan Profesionalisme Guru. Edisi Ke-2. Jakarta: PT Raja Grafindo Persada. Berdasarkan hasil tersebut, maka saran peneliti bagi guru di tingkat sekolah dasar adalah agar selalu menyiapkan perangkat pembelajaran dan memilih model pembelajaran yang bervariasi sesuai dengan materi yang akan diajarkan agar tujuan Slameto. 2010. Belajar dan faktor-faktor yang Mempengaruhinya. Jakarta: PT. Rineka Cipta Slavin, Robert E. 2005. Cooperatif Learning: theory, Research and practice (Terjemahan N. Yusron.). pektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 40 London: Allymand Bacon. (Buku asli diterbitkan tahun 2005). Sudjana, Nana. 2013. Dasar-dasar Proses Belajar Mengajar. Bandung: Sinar Baru Algesindo. Suryabrata. 2010. Metodologi Penelitian. Jakarta: Rajawali Pers Susanto, Ahmad. 2013. Teori Belajar dan Pembelajaran di Sekolah. Jakarta: Kencana London: Allymand Bacon. (Buku asli diterbitkan tahun 2005). Suryabrata. 2010. Metodologi Penelitian. Jakarta: Rajawali Pers Susanto, Ahmad. 2013. Teori Belajar dan Pembelajaran di Sekolah. Jakarta: Kencana Sudjana, Nana. 2013. Dasar-dasar Proses Belajar Mengajar. Bandung: Sinar Baru Algesindo. Ekspektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 1, Juni 2020 E-ISSN 2722-3353 41
https://openalex.org/W4387098757	https://link.springer.com/content/pdf/10.1007/978-3-031-37663-4_4.pdf	English	null	Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps and the Teacher	Springer eBooks	2,023	cc-by	5,111	Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps and the Teacher Silje Fyllingsnes Christiansen 1 Kindergarten teachers in Norway have a bachelor’s degree in kindergarten teacher education, and make up 42.3% of the staff in kindergarten (UDIR, 2022). S. F. Christiansen () Western Norway University of Applied Sciences, Bergen, Norway e-mail: sifc@hvl.no © The Author(s) 2024 H. Palmér et al. (eds.), Teaching Mathematics as to be Meaningful – Foregrounding Play and Children’s Perspectives, https://doi.org/10.1007/978-3-031-37663-4_4 39 S. F. Christiansen () Western Norway University of Applied Sciences, Bergen, Norway e-mail: sifc@hvl.no © The Author(s) 2024 H. Palmér et al. (eds.), Teaching Mathematics as to be Meaningful – Foregrounding Play and Children’s Perspectives, https://doi.org/10.1007/978-3-031-37663-4_4 Introduction Digital tools are becoming increasingly more common all over the globe, with young children encountering them at home as well as in early childhood education (Otterborn et al., 2019). Although there is some research on older children using digital tools, little is known about the youngest children, especially when it comes to mathematics. Yet digital tools receive a lot of political attention, with an OECD report stating that “linking the way children interact with ICT inside of school to the way they already use it outside of school can be a key to unlocking technology’s potential for learning” (Schleicher, 2019, p. 10). Similarly, a link between play and learning is commonly made in Early Childhood Education and Care (ECEC) prac tice and research (Nilsson et al., 2018). For example, Lundtofte’s (2020) literature review of children’s tablet play found that children’s use of tablets is often con nected to digital literacy and learning. 1 2018 2017 2018 1 Kindergarten teachers in Norway have a bachelor’s degree in kindergarten teacher education, and make up 42.3% of the staff in kindergarten (UDIR, 2022). 39 39 40 S. F. Christiansen S. F. Christiansen S. F. Christiansen 2022 Teaching in Digital Environments Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps… 41 The Mathematical Activity of Locating More than 30 years ago Bishop (1988) identified locating as one of six mathemati cal activities that are present in all cultures. In Norway, the curriculum known as the Framework Plan for Kindergartens (Ministry of Education, 2017) includes the learning area of quantities, spaces and shapes, which is based on Bishop’s (1988) activities (Reikerås, 2008). Researchers have also found that situations involving Bishop’s (1988) activities are present in children’s culture (for example, Helenius et al., 2016; Johansson et al., 2014). Bishop (1988) described locating as more or less sophisticated activities con nected to navigating and communicating about the environment which are closely linked to language as language develops through taking part in these situations. Lowrie (2015) found that video games for 5-year-old children required visuospatial skills similar to those needed in real life-situations, suggesting that digital games can provide opportunities to engage children in locating activities. Although verbal language can both make locating ideas easier to spot and focus the children’s atten tion on the mathematical aspects of a situation, children are also able to engage in the different mathematical activities without using verbal language (Flottorp, 2010; Meaney, 2016). Children’s engagement with locating ideas does not have to be done through verbal language. What remains unknown is how apps might mediate the kind of support, including the development of verbal mathematical language, that teachers could provide to increase young children’s opportunities to engage in locating tasks. Theoretical Framework This article is part of a wider study where I have used Bishop’s (1988) six funda mental mathematical activities to describe children’s mathematics. In this article, I focus on the mathematical activity of locating as this is mostly what these very young children engaged with when using digital apps. To investigate how digital apps mediate kindergarten teachers’ support, I use Ladel and Kortenkamp’s (2011, 2014) artefact-centric activity theory (ACAT) which has been developed to capture the complexity of children’s use of digital tools. Teaching in Digital Environments Although digital tools such as mobile technologies have been around for some time, their entry into early childhood education remains contested, with opinions often divided into two groups: those who see digital tools as a threat, and those who think they can make the world better (Danby et al., 2018; Palaiologou, 2016). Given this contested space, it can be challenging for teachers to implement digital tools in their teaching. Vangsnes et al. (2012) suggested that teachers rarely bring the Nordic pedagogy of valuing play into digital game-playing, indicating that the digital tools’ entry into kindergarten might entail a risk of changing the children’s pedagogical environment to one with less focus on children’s play (Christiansen & Meaney, 2020). Fleer (2014) argued that that there is a need for further research to recognize new opportunities for play in digital settings. Tablets with multitouch capabilities are often considered an intuitive tool suit able for children’s developing coordination skills. For example, Geist (2012) found that when toddlers use digital touch screens, they showed a high level of skills in using the screens independently, and explored options in similar ways to how they used other play materials such as building blocks. Nevertheless, digital apps for children are often given a strong learning focus, being marketed as “fun learning” (Kvåle, 2021). In apps with a linear build-up of tasks, children can often guess their way to the correct answer and seem to be motivated by the digital stardust or con fetti they get for finding the right answer, rather than by learning ideas or concepts (Nilsen, 2018). Children can also change the purpose of digital activities from what was planned by teachers. Lafton (2019) found that children playing a digital mem ory game ignored the intended rules of the game, and instead created their own game with different rules. Digital apps with strong framing, those with clear control over the communication and the pace of the app, can sometimes limit children’s participation (Palmér, 2015). Lembrér and Meaney (2016) suggested that children’s agency could be strengthened by giving them control over the game, which could lead to them needing or wanting to explain what they were doing. This need could support children’s reflective mathematical thinking. Playful apps with a weak fram ing, where the app provides the children with control, could engage children in mathematical language and thinking when they played by themselves (Christiansen, 2022). Methodology As part of a wider project, video recordings were made of children engaging with digital tools at a Norwegian kindergarten over a 2-month period. For this article, I analyse 20 min of video, where two one-and-a-half-year-old children, Ole and Trine, played with three digital apps with a teacher. Artefact-Centric Activity Theory 2011 2014 2015 42 S. F. Christiansen S. F. Christiansen S. F. Christiansen 2014 1 The ACAT model consists of two triangles. In the first triangle, the artefact (the digital app) is connected to the nodes for rules (how the app is designed) and the object (the mathematical content). The triangle describes how the mathematical object is internalized in the app and then externalized through the tablet’s design. The rules (the design principles of the app) describe how the mathematical object is presented in the app (what kind of activities and feedback are available). In the second triangle, the focus is on how the artefact (the digital app) is used by the subject (the child who is playing) and the relationship to the group (the social group around the child). The focus is on what the subject internalizes from engaging with the app, and how the subject externalizes that engagement through their words 2011 2011 Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps… 43 and actions. The group influences the child’s engagement because it can facilitate or hinder, or be facilitated or hindered, by the way that the app presents the mathemati cal content (as a basis for what happens in the first triangle). and actions. The group influences the child’s engagement because it can facilitate or hinder, or be facilitated or hindered, by the way that the app presents the mathemati cal content (as a basis for what happens in the first triangle). Informants and the App The teacher was informed about the wider project in a series of staff meetings before filming began. She gave her informed consent to be part of the project. The parents also gave written consent for their children’s participation after a parents’ evening was held to inform about the project. The children were given time to get to know the researcher before the video recordings began. The video recordings were carried out in the rooms the children usually used in the kindergarten, and they were always with one of their permanent teachers. There was an agreement to turn off the camera if the children seemed uncomfortable with being filmed. The two children were learning Norwegian as their first language. In the videos, the children engage with three different apps which they chose from what the kindergarten staff made avail able on the tablets. These apps were: My PlayHome, an app designed to excite and captivate children (My PlayHome, 2022); Toca Kitchen, an app designed for play (TOCA BOCA, 2022); and Crocro’s Friends Village, an app designed to “both entertain and encourage them [children] to learn, develop, and flourish” (Samsung, 2022). Both the children had previously tried all three apps. Analysis 2014 1 I then organized the analyses into two parts, each part focused on one of the tri angles in ACAT. Everything related to how the children used the app to engage with S. F. Christiansen 44 Table 1 Transcript of datamaterial Verbal engagement Bodily engagement Screenshot from the video Teacher: Oh [pauses] You need to press the dog. Then he will move. Can you see him walking along? Do you see that? The teacher traces her finger along the tablet, from the dog and towards the end of the screen. Table 1 Transcript of datamaterial p Verbal engagement Bodily engagement Screenshot from the video Teacher: Oh [pauses] You need to press the dog. Then he will move. Can you see him walking along? Do you see that? The teacher traces her finger along the tablet, from the dog and towards the end of the screen. Screenshot from the video Screenshot from the video the mathematical activity of locating (actions and vocabulary linked to locating and navigating in the virtual space of the app) was coded as part of the rules–object– artefact triangle. Following earlier research on children engaging with apps (Christiansen & Meaney, 2020; Geist, 2012; Kvåle, 2021; Nilsen, 2018). I wanted to examine apps which did not have (mathematics) learning as a stated aim of the app. Therefore, the chosen video recordings did not include the children using apps that were described by their developers as educational. After watching the children’s engagement, I identified locating as a mathemati cal object that was made available to the children through playing with the app. It may be that the teacher did not think that she was focusing on mathematics or locat ing when engaging with the apps with the children. However, this way of engaging in mathematics through playful participation is in line with the Norwegian frame work plan which states that children should learn through play (Ministry of Education, 2017). In the video, the teacher appeared to focus on communicating about the children’s navigating, and communicating about the environment, which is in line with Bishop’s (1988) understanding of locating. Everything related to how the child and the group around the child interacted with the app was coded as part of the subject–group–artefact triangle. In ACAT, the role of the teacher is not explicit, but the teacher is part of the “group” in the subject- group-artefact triangle. Analysis In the recordings, the children each had a tablet, but they mostly focussed on Ole’s screen. Therefore, I have made Ole the subject of the analysis. Trine and the teacher are part of the social group around Ole. The choices for this coding are discussed in more detail in the findings and discussion section. Findings and Discussion To answer the research question, I describe how the three digital apps supported the kindergarten teacher in facilitating the youngest children’s locating. I found that the pace of the apps appeared to have an impact on what kind of conversation the teacher and the children engaged in. If an app operated at a slow pace, there was an increase in opportunities for the teacher to facilitate children’s engagement with aspects of locating. Lembrér and Meaney (2016) had noted a similar result with preschool children using an interactive table and a balancing app. Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps… 45 Crocro’s Friends Village 2 2 2 S. F. Christiansen S. F. Christiansen 46 The second triangle focused on how the app (the artefact) was used by Ole (the subject) and his interactions with Trine and the teacher when playing with the app (the group). When the teacher suggested that “maybe he wants to have it inside his stomach” Ole was guided towards trying something new, and the teacher was able to focus on locating by asking, “Should he (the sloth) put it in his mouth?” Trine then put her finger inside her mouth and the teacher held her hand in front of her mouth. Trine did the same and the teacher said “Now we will be full up. In our stomachs” and rubbed her stomach. This exchange would have helped the children to understand about different parts of their own bodies, such as their mouth where food goes in, and their stomach where food ends up. Locating different parts of their bodies and knowing their names, such as stomach, are new and relevant aspects for many young children to learn. In Crocro’s Friends Village, there was a lot of quickly-moving objects. Ole did not have the fine motor skills needed to navigate the app, and consequently he did not always get feedback from the app that supported his engagement with aspects of locating, apart from watching the items move from left to right and tapping on the fish on the screen. The teacher’s communication, even if it was not explicitly about aspects of locating, appeared to support Ole to keep exploring. However, it seemed as if the somewhat fast-moving items, and a large number of small items on the screen, made it challenging for the teacher to engage in a discussion about this with Ole. Toca Kitchen 3 Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps… 47 3 3 However, the teacher focused Ole and Trine’s attention on locating by adding words to the onion going into the monster’s mouth. Using language to discuss envi ronments is, according to Bishop (1988), part of locating. The teachers’ support is also connected to supporting Ole and Trine in exploring the options available within the app. When the onion fell out of the fridge, the teacher opened the cooking menu and suggested that the children use it by asking “what do we have to do now”? However, when neither of the children responded to her invitation to explore the cooking options, she closed the menu. In Toca Kitchen, opportunities for engaging in locating were externalized through the opportunities to move the objects, and because moving the object towards the monster’s mouth would result in the monster eating the food. Although this app moved at a slower pace than Crocro’s Friends Village, it appeared to be difficult for the children to navigate; for instance when one wrong tap would close the fridge. The children were interested in, and managed to give the monster food to eat, and the teacher and the children discussed how the food disappeared and went into the stomach, also by using gestures such as rubbing their stomach. S. F. Christiansen 48 My PlayHome The slow pace gave the children the opportunity to explore the virtual space at a slower pace than in the two previous apps, and this also provided space for the teacher to mediate the locating through active engagement around the app. The teacher not only voices what the children are doing, but also uses gestures to underline the locating, such as showing the children that they are moving the mir ror and tracing the dog’s movement on the screen. The tracing of the dog’s move ment appears to encourage Ole to try to drag items instead of just tapping them. My PlayHome 3 3 3 4 When using My PlayHome, the children appeared to be interested in making things move, and both Ole and the teacher externalized language related to locating. The teacher’s language focused the children’s attention on locating, through using verbal language such as the baby being in the bed; the dog moving, jumping, walk ing along; the baby was gone and there. My PlayHome has the option of undoing actions. Unlike Crocro’s Friends Village and Toca Kitchen, where Ole and Trine Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps… 49 only had the option of doing the same thing over again, in My PlayHome they could move items back and forth. Ole explored this option when he covered and uncov ered the baby with the blanket, encouraged by his own reaction when the baby ‘disappeared’, the teacher’s question “Where is he?” and Trine’s excitement when the baby was still in the bed. Even if Ole navigated between rooms without doing so intentionally, this is a relatively slow-moving app where the children actively have to make something move by dragging it, although they sometimes make them move further than planned. The slow pace gave the children the opportunity to explore the virtual space at a slower pace than in the two previous apps, and this also provided space for the teacher to mediate the locating through active engagement around the app. The teacher not only voices what the children are doing, but also uses gestures to underline the locating, such as showing the children that they are moving the mir ror and tracing the dog’s movement on the screen. The tracing of the dog’s move ment appears to encourage Ole to try to drag items instead of just tapping them. only had the option of doing the same thing over again, in My PlayHome they could move items back and forth. Ole explored this option when he covered and uncov ered the baby with the blanket, encouraged by his own reaction when the baby ‘disappeared’, the teacher’s question “Where is he?” and Trine’s excitement when the baby was still in the bed. Even if Ole navigated between rooms without doing so intentionally, this is a relatively slow-moving app where the children actively have to make something move by dragging it, although they sometimes make them move further than planned. Conclusion The findings show that the apps provide opportunities for engaging in locating. This is because they provide opportunities for users, such as Ole and Trine using their fingers to make things move in the app, and such as opening cupboard doors in Toca 50 S. F. Christiansen S. F. Christiansen S. F. Christiansen Kitchen. When Ole and Trine interact with the apps, they do so mostly through body movements, and sometimes using verbal language to express what they are doing or want to do. The locating in the virtual word can sometimes lead to discussing locat ing in the actual world, for instance when Trine put her finger in her mouth, showing where the food went. With the very young children, it seems that the teacher has a key role in mediating locating. Although Ole did explored the apps to some degree, as Geist (2012) found tod dlers can do, moving small objects in the virtual space required good fine motor skills and this has an impact on the aspects of locating that the children could engage with. In Crocro’s Friends Village, the need for fine motor skills was combined with fast-moving objects. In Toca Kitchen, many things were happening on the screen at the same time. These design features of the apps appeared to make it harder for the children to engage with the opportunities for exploring aspects of locating by play ing with the app. When the children use My PlayHome, the pace is much slower. Although there is no built-in need for explanation such as Lembrér and Meaney (2016) suggest there should be when (older) children take part in the game, My PlayHome provides opportunities to discuss what is happening, especially when Ole pulls the blanket over the baby, and he can pull it back and forth many times to both see and discuss what is happening. Although playful apps have been shown to engage older children in mathemati cal language and thinking when they play by themselves (Christiansen, 2022), these younger children did not use a lot of verbal language when they engaged with any of the three apps. As the digital apps did not provide any verbal input, the teacher was the only one providing models of mathematical language. It was the teacher who voiced what the children did in relationship to aspects of locating in the virtual environments. Conclusion In fast-moving apps, it was harder for the teacher to focus the chil dren’s attention on these opportunities, as something new is happening all the time. The teacher had a key role as the mediator of the mathematics; the mathematical object (locating) is externalized to the children through what is seen on the screen and how they use their fingers to interact with it, but also through the teacher’s ver balization. The differences in how the three apps provided opportunities for chil dren’s interactions on the screen. The teacher focused on the aspects of locating that the children were interested in and allowed the children to decide what to do and how to do it. For example, with My PlayHome, the teacher followed what the children did, rather than limiting their exploration so that they were guided towards solving tasks. The teacher appeared to value the play, both in that she did not insist that the children engage with apps with what Kvåle (2021) call ‘fun learning’, and because she let the children explore freely while she highlighted aspects of locating. The kindergarten teacher seizes the space for action that arises when the child faces digital challenges and use it as an opportunity to discuss the mathematical object of locating. The teacher has an important role when young children engage with apps designed for play as the teacher becomes a mediator of the mathematical content in the app, just like the app itself. The teacher both highlights the mathematics in the app (in this case locating) and underlines the way the app externalizes the feedback, Supporting One-Year-Olds’ Digital Locating: The Mediating Role of the Apps… 51 such as when she says to Ole that the dog is moving when he taps it. As the teacher has this key role as a mediator of the mathematical object, I suggest that the model of ACAT should be expanded by adding a third triangle where the connection between the group (in this case Trine and the teacher) is highlighted. 5 In this study, I have shown that although digital apps have the potential to engage even very young children in locating and the teacher might have a key role as a mediator of the mathematical object. Conclusion The design of apps which provide an open virtual environment for children to explore at their own pace can provide spaces where the teacher can focus the children’s attention on locating by adding words to what the children are engaged in. Of the apps that have been investigated in this study, the non-linear and slow-moving apps without verbal language appeared to provide the best opportunities for such spaces for verbal language contributions from the teacher, who provided a supportive environment for the children’s own explorations. Although ACAT was useful in helping me make sense of the data, it was clear that the teacher’s role was not sufficiently highlighted in supporting the children in engaging with the mathematical objects of learning. Theoretical models 2011 2011 S. F. Christiansen 52 S. F. Christiansen used to analyse children’s digital engagements need to recognize the importance of the teacher. For future research, I would suggest looking into how digital apps can support other mathematical activities and whether the design elements in other apps can support engagement with all of Bishop’s (1988) mathematical activities. References Bishop, A. J. (1988). 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Christiansen Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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 chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter'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.
https://openalex.org/W3206350548	http://www.scielo.cl/pdf/rmc/v144n1/art04.pdf	Spanish; Castilian	null	[Long-term outcomes with expanded criteria donors in kidney transplantation].	null	2,016	cc-by	4,987	Long-term outcomes with expanded criteria donors in kidney transplantation Background: Kidney transplantation of expanded criteria deceased donors (DCE) has become a common clinical practice. However, DCE outcomes are inferior compared to kidney transplants from standard criteria donors (DCS). Aim: To evaluate intermediate and long-term outcomes of DCE transplanted patients. Material and Methods: Cadaveric kidney transplants were evaluated using a retrospective cohort of eight consecutive years. Complications and long- term function of the transplant were assessed in DCE and DCS kidney recipients. Results: Of 213 patients analyzed, 34 (16%) underwent DCE transplantation. DCS recipients spent more time on the waiting list for transplantation (p = 0.04). DCE recipients showed higher frequency of surgical complications (p = 0.04), vascular complications (p = 0.02), acute transplant rejection (p = 0.05), and hospitalizations (p = 0.01). Creatinine (mg/dL) in DCE and DCS recipients was 2.3 and 1.5 respectively at year one (p < 0.01) and 2.6 and 1.6 respectively at year five (p < 0.01). Graft survival in the DCE group was significantly lower at 5 years (61 and 89% respectively, p < 0.01). Conclusions: DCE grafts are associated with lower survival, higher hospitalization rate and commonly develop surgical complications and rejections. (Rev Med Chile 2016; 144: 22-29) Key words: Aged; Donor selection; Graft rejection; Graft survival; Kidney transplantation. Key words: Aged; Donor selection; Graft rejection; Graft survival; Kidney transplantation. E l donante cadavérico ideal es considerado un individuo joven, previamente sano, que fallece producto de una lesión traumática del encéfalo, manteniendo intacta la funciona- lidad de órganos torácicos y abdominales. El aumento en la prevalencia de la enfermedad renal crónica avanzada ha provocado, a nivel mundial, un crecimiento sustancial en las listas de espera para trasplante renal cadavérico, que supera por mucho el número de riñones procurados. Esta crisis global en el suministro de órganos ha lleva- do a maximizar el uso de órganos obtenidos de donantes fallecidos, es así como en la actualidad donantes añosos y con condiciones antiguamente E consideradas no aptas para la donación, están siendo aceptados. Estos donantes “no ideales” son actualmente denominados donantes con criterios expandidos (DCE) y se caracterizan por poseer órganos con menor reserva funcional y capacidad regenerativa, lo que se ha vinculado, posterior al trasplante, con menor sobrevida a largo plazo y con mayor probabilidad de insuficiencia primaria, función retardada del injerto y episodios de recha- zo agudo, cuando son comparados con órganos de donantes con criterios estándar (DCS)1. Correspondencia a: Dr. Ricardo Valjalo Servicio de Medicina Interna, Hospital del Salvador. Avenida Salvador 364, Providencia. Santiago, Chile. Teléfono: (2) 25753532 rvaljalo@hotmail.com Resultados a largo plazo en trasplante renal de donantes con criterios expandidos 1Sección de Nefrología, Servicio de Medicina Interna, Hospital del Salvador. Santiago, Chile. 2Departamento de Medicina, Facultad de Medicina, Universidad de Chile. Santiago, Chile. Ricardo Valjalo1,2, Enrique Reynolds1,2, Patricia Herrera1,2, Óscar Espinoza1,2, Fernando González1,2 Recibido el 29 de abril de 2015, aceptado el 9 de octubre de 2015. Recibido el 29 de abril de 2015, aceptado el 9 de octubre de 2015. ARTÍCULOS DE INVESTIGACIÓN ARTÍCULOS DE INVESTIGACIÓN Rev Med Chile 2016; 144: 22-29 Long-term outcomes with expanded criteria donors in kidney transplantation Múltiples factores han sido implicados en estos resultados, incluyendo una masa nefronal reducida, presencia de daño renal previo, menor capacidad tisular 22 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al regenerativa, mayor susceptibilidad a la injuria isquemia-reperfusión y elevada inmunogenicidad del injerto2,3. reactividad de anticuerpos al panel de linfocitos (PRA) mayor a 20%, y el esquema inmunosu- presor inicial constó de terapia triasociada con glucocorticoides, ciclosporina y azatioprina en los pacientes con bajo nivel de sensibilización previa al trasplante y de glucocorticoides, tacrolimus y micofenolato en los pacientes con mayores niveles de sensibilización. Se utilizó ketoconazol como fármaco ahorrador de anticalcineurínicos. j Si bien los riñones provenientes de DCE pre- sentan una menor sobrevida a largo plazo que los provenientes de DCS, Ojo y cols. demostraron que aun así, los receptores de DCE se benefician en términos de sobrevida, cuando son comparados a los pacientes en diálisis que se encuentran en lista de espera y que no logran ser trasplantados4. A pesar de esto, la calidad limitada de dichos órganos hace que sigan siendo descartados con relativa frecuencia y muchos centros de trasplante prefieren no hacer uso de ellos5. Nuestro objetivo fue evaluar los resultados a mediano y largo plazo de los receptores sometidos a trasplante renal con DCE, en nuestro centro. Durante el seguimiento evaluamos la ocu- rrencia de rechazo agudo, disfunción primaria del trasplante, retraso de función del injerto (RFI), hospitalizaciones, complicaciones vascu- lares post-trasplante y complicaciones urológicas que necesitaron de re-intervención quirúrgica. Además, fueron evaluados a largo plazo la fun- ción renal, sobrevida del injerto y sobrevida de los receptores. Se consideró como RFI a la necesidad de diálisis durante la primera semana posterior al trasplante. La ausencia permanente de función renal posterior al trasplante, inde- pendientemente del motivo, fue considerada como disfunción primaria del trasplante. Se consideraron complicaciones quirúrgicas a la ocurrencia de complicaciones post-operatorias que requirieron de re-intervención quirúrgica (linfoceles, fístulas urinarias, colecciones, infec- ción de herida operatoria, etc.). Fueron incluidas como complicaciones vasculares la ocurrencia de trombosis arterial o venosa del injerto, el sangra- do post-operatorio desde el pedículo o a la apari- ción posterior de estenosis en la arteria renal del injerto. Todos los rechazos fueron confirmados histológicamente, de acuerdo a la clasificación de Banff vigente en el período en que se realizó la biopsia y tratados según el tipo de rechazo demos- trado. Long-term outcomes with expanded criteria donors in kidney transplantation Se contabilizaron las hospitalizaciones por cualquier motivo ocurridas durante el período de seguimiento, considerándose la razón entre las hospitalizaciones y los años de seguimiento. La velocidad de filtración glomerular (VFG) fue calculada en base a la fórmula MDRD-2. Las variables categóricas fueron evaluadas usando el test exacto de Fisher y las variables continuas utilizando la prueba t de Student, ANCOVA y U de Mann-Whitney. Utilizando curvas de Kaplan- Meier y análisis comparativo con log-rank, fueron analizadas las curvas de sobrevida de pacientes y de injertos, esta última censurada por muerte con injerto funcionante. Se utilizó el software Rev Med Chile 2016; 144: 22-29 Resultados fue de 4,5 años. Las características de los donantes, receptores y del trasplante están resumidas en la Tabla 1. Como era de esperarse por su definición, los DCE presentaron mayor edad (61 vs 38 años, p < 0,01), mayor prevalencia de HTA (p < 0,01) y fallecieron con mayor frecuencia por ACV (p < 0,01) que los DCS. El tiempo de espera en lista de trasplante fue significativamente menor en los re- ceptores de DCE (p = 0,04). Los receptores de DCE tuvieron mayor edad (58 vs 44 años, p < 0,01) y discordancias HLA que el grupo de comparación. En los ocho años fueron efectuados 215 tras- plantes con donante cadavérico. Se excluyó a dos trasplantados, uno, receptor de DCS, por conti- nuar seguimiento en otro centro y, el otro, por extravío de datos (n = 213). De estos 213 pacientes, 34 pacientes (16%) recibieron trasplante de DCE y 179 (84%) de DCS. El 47% de trasplantes de DCE fueron efectuados en los últimos 2 años del período considerado. El promedio de seguimiento Tabla 1. Pacientes y Método Se llevó a cabo un estudio de cohorte no concurrente, considerando todos los pacientes mayores de 18 años sometidos a trasplante renal exclusivo de donante cadavérico efectuados en el Hospital del Salvador (Santiago, Chile), entre el 01 de enero de 2005 y el 31 de diciembre de 2012. Fueron considerados como DCE los donantes que cumplían los siguientes criterios sugeridos por la United Network for Organ Sharing (UNOS): Mayores de 60 años, o los fallecidos de entre 50 y 59 años con al menos 2 de las siguientes condi- ciones médicas: Historia de hipertensión arterial (HTA), accidente cerebrovascular como causa de defunción o creatinina sérica previa a la procura mayor a 1,5 mg/dL6. Fueron considerados como DCS todos los donantes que no reuniesen los requisitos de DCE. El criterio de asignación de receptores para los DCE fue “old for old”, asignándoseles por tanto, a receptores de una edad similar7. La velocidad de filtración glomerular (VFG) fue calculada en base a la fórmula MDRD-2. Las variables categóricas fueron evaluadas usando el test exacto de Fisher y las variables continuas utilizando la prueba t de Student, ANCOVA y U de Mann-Whitney. Utilizando curvas de Kaplan- Meier y análisis comparativo con log-rank, fueron analizadas las curvas de sobrevida de pacientes y de injertos, esta última censurada por muerte con injerto funcionante. Se utilizó el software estadístico SPSS v22. Todos los pacientes fueron instruidos acerca de las expectativas, riesgos y beneficios relacio- nados al trasplante, y firmaron consentimiento informado. En algunas circunstancias, y en caso de no existir un receptor para el segundo riñón donado, los receptores de DCE recibieron un doble trasplante renal. Los riñones donados fueron conservados hasta el trasplante utilizando soluciones de preservación. El uso de inducción con anticuerpos se reservó para los pacientes con 23 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al Resultados La VFG calculada en base a la fórmula MDRD registrada al mes, al año y al quinto año, fue de 33 vs 40, 32 vs 51 y 29 vs 54 mL/min en receptores de DCE y DCS respectivamente (Figura 2). El análisis de ANCOVA, considerando la creatinina y VFG al mes de trasplante como covariable, también mostró que la función renal de ambos grupos, du- rante los años sucesivos, fueron estadísticamente diferentes, favoreciendo a los receptores de DCS. La función renal evidenciada en el subgrupo de DCE que recibió trasplante renal doble no mostró Los receptores de DCE presentaron (Tabla 2) mayor ocurrencia de complicaciones quirúrgicas (p = 0,04), complicaciones vasculares (p = 0,02) y de episodios de rechazo agudo confirmados por biopsia (p = 0,05). La tasa observada de hospitali- zaciones fue significativamente mayor (p = 0,01). No hubo diferencias entre ambos grupos en cuanto a desarrollo de RFI, disfunción primaria del injerto, neoplasias, diabetes post-trasplante y enfermedad por citomegalovirus (CMV). p g La función renal en ambos grupos evidenció diferencias a partir del primer mes post-trasplante, la que se hizo significativa desde el primer año y se Tabla 2. Resultados DCE (n = 34) DCS (n = 179) p Retraso de función del injerto 16 (47%) 90 (50%) 0,44 Disfunción primaria del injerto 4 (12%) 10 (6%) 0,17 Complicación quirúrgica 11 (32%) 30 (17%) 0,04 Complicación vascular 7 (21%) 13 (7%) 0,02 Rechazo agudo en primer año 11 (32%) 32 (18%) 0,05 Diabetes post-trasplante 3 (9%) 15 (8%) 0,57 Enfermedad por CMV 2 (6%) 12 (7%) 0,61 Nefropatía por BK 2 (6%) 4 (2%) 0,25 Desarrollo de neoplasias 1 (3%) 8 (5%) 0,56 Hospitalizaciones/año 1,5 ± 2,1 0,6 ± 0,8 0,01 Abreviaturas. CMV: Citomegalovirus. Figura 1. Creatininemia a los 6 años post-trasplante. Seguimiento de función renal medida con creatinina sérica, a los 6 años post-trasplante (p < 0,05). DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Tabla 2. Resultados Características de donantes, receptores y del trasplante DCE (n = 34) DCS (n = 179) p Edad del donante (años) 61,0 ± 5,7 38,3 ± 12,3 < 0,01 Muerte del donante: ACV 19 (56%) 36 (20%) < 0,01 Donante: HTA 20 (59%) 19 (11%) < 0,01 Donante: DM-2 1 (3%) 4 (2%) 0,59 Creatinina del donante (mg/dL) 0,86 ± 0,26 0,89 ± 0,36 0,85 Tiempo de isquemia fría (h) 16,6 ± 4,1 20,5 ± 6,4 < 0,01 Tiempo de isquemia caliente (min) 42,7 ± 10,8 39,7 ± 9,9 0,11 Edad del receptor (años) 57,7 ± 8,5 43,6 ± 11,6 < 0,01 Receptor: Sexo masculino 22 (65%) 104 (58%) 0,30 Tiempo en lista de espera (meses) 32,4 ± 28,1 41,9 ± 31,6 0,04 Trasplante renal previo 2 (6%) 9 (5%) 0,55 Tiempo en diálisis (meses) 66,7 ± 53,2 73,6 ± 46,4 0,24 PRA 0% 15 (44%) 52 (29%) 0,08 PRA > 20% 5 (15%) 55 (26%) 0,11 HLA-mismatch 3,7 ± 1,3 2,7 ± 1,2 < 0,01 Trasplante renal doble 7 (21%) 0 < 0,01 Receptor: HTA 22 (65%) 114 (64%) 0,54 Receptor: DM-2 3 (9%) 11 (6%) 0,39 Receptor: IgG CMV (+) 30 (88%) 162 (92%) 0,33 Inducción con Basiliximab 5 (15%) 35 (20%) 0,35 Inducción con rATG 1 (3%) 12 (7%) 0,35 Inmunosupresión: Ciclosporina 33 (97%) 167 (93%) 0,35 Inmunosupresión: Tacrolimus 1 (3%) 11 (6%) 0,40 Inmunosupresión: Azatioprina 31 (91%) 163 (91%) 0,64 Inmunosupresión: Micofenolato 3 (9%) 16 (9%) 0,64 Abreviaturas. ACV: Accidente cerebrovascular; HTA: Hipertensión arterial; DM-2: Diabetes mellitus tipo 2; PRA: Reactividad de anticuerpos al panel de linfocitos; HLA: Antígeno leucocitario humano; CMV: Citomegalovirus; rATG: Timoglobulina de conejo anti-timocitos. Tabla 1. Características de donantes, receptores y del trasplante Rev Med Chile 2016; 144: 22-29 24 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al No hubo diferencias significativas en cuanto a los esquemas inmunosupresores de inducción o de mantenimiento empleados. mantuvo durante el período de seguimiento. Los niveles de creatinina sérica al mes, al año y a los 5 años en los receptores de DCE versus DCS fueron de 2,9 vs 2,2 (p = 0,08); 2,3 vs 1,5 (p < 0,01) y 2,6 vs 1,6 (p < 0,01) mg/dL, respectivamente (Figura 1). Resultados Resultados DCE (n = 34) DCS (n = 179) p Retraso de función del injerto 16 (47%) 90 (50%) 0,44 Disfunción primaria del injerto 4 (12%) 10 (6%) 0,17 Complicación quirúrgica 11 (32%) 30 (17%) 0,04 Complicación vascular 7 (21%) 13 (7%) 0,02 Rechazo agudo en primer año 11 (32%) 32 (18%) 0,05 Diabetes post-trasplante 3 (9%) 15 (8%) 0,57 Enfermedad por CMV 2 (6%) 12 (7%) 0,61 Nefropatía por BK 2 (6%) 4 (2%) 0,25 Desarrollo de neoplasias 1 (3%) 8 (5%) 0,56 Hospitalizaciones/año 1,5 ± 2,1 0,6 ± 0,8 0,01 Abreviaturas. CMV: Citomegalovirus. Tabla 2. Resultados Tabla 2. Resultados Figura 1. Creatininemia a los 6 años post-trasplante. Seguimiento de función renal medida con creatinina sérica, a los 6 años post-trasplante (p < 0,05). DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Rev Med Chile 2016; 144: 22-29 25 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al Figura 2. VFG a 6 años post-trasplante. Seguimiento de función renal con VFG calculada por fórmula MDRD, a los 6 años post-trasplante (p < 0,05). VFG: Velocidad de filtración glomerular. DCS: Donantes de criterio estándar. DCE: Do- nantes de criterios expandidos. Figura 3. Tasa de supervivencia ac- tuarial del injerto, en receptores de trasplante renal de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Figura 2. VFG a 6 años post-trasplante. Seguimiento de función renal con VFG calculada por fórmula MDRD, a los 6 años post-trasplante (p < 0,05). VFG: Velocidad de filtración glomerular. DCS: Donantes de criterio estándar. DCE: Do- nantes de criterios expandidos. Figura 2. VFG a 6 años post-trasplante. Seguimiento de función renal con VFG calculada por fórmula MDRD, a los 6 años post-trasplante (*p < 0,05). VFG: Velocidad de filtración glomerular. DCS: Donantes de criterio estándar. DCE: Do- nantes de criterios expandidos. Figura 3. Tasa de supervivencia ac- tuarial del injerto, en receptores de trasplante renal de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Figura 3. Tasa de supervivencia ac- tuarial del injerto, en receptores de trasplante renal de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Figura 3. Tasa de supervivencia ac- tuarial del injerto, en receptores de trasplante renal de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Figura 3. Discusión Santiago, Chile. 2014). Esta amplia brecha entre candidatos a trasplante y disponibilidad de ór- ganos ha llevado a maximizar el uso de donantes vivos y cadavéricos antes considerados descarta- bles, trasplantando a modo de ejemplo riñones de donantes con edades extremas, con diferentes morbilidades, o con incompatibilidad de grupo ABO o HLA. El trasplante renal con DCE ha sido cuestionado por sus peores resultados: Mayor frecuencia de disfunción primaria, RFI, rechazo agudo y menor sobrevida del injerto (aproxi- madamente 4 años menor) en comparación a los receptores de DCS. Tales resultados podrían ser explicados por la menor masa nefronal funcionante, mayor frecuencia de alteraciones histológicas como glomeruloesclerosis, fibrosis intersticial, atrofia tubular y arterioesclerosis, una elevada antigenicidad del injerto y una mayor susceptibilidad a nefrotoxicidad y daño inmunológico2,3,13. Santiago, Chile. 2014). Esta amplia brecha entre candidatos a trasplante y disponibilidad de ór- ganos ha llevado a maximizar el uso de donantes vivos y cadavéricos antes considerados descarta- bles, trasplantando a modo de ejemplo riñones de donantes con edades extremas, con diferentes morbilidades, o con incompatibilidad de grupo ABO o HLA. El trasplante renal con DCE ha sido cuestionado por sus peores resultados: Mayor frecuencia de disfunción primaria, RFI, rechazo agudo y menor sobrevida del injerto (aproxi- madamente 4 años menor) en comparación a los receptores de DCS. Tales resultados podrían ser explicados por la menor masa nefronal funcionante, mayor frecuencia de alteraciones histológicas como glomeruloesclerosis, fibrosis intersticial, atrofia tubular y arterioesclerosis, una elevada antigenicidad del injerto y una mayor susceptibilidad a nefrotoxicidad y daño inmunológico2,3,13. El aumento sostenido de la enfermedad renal crónica en Chile es responsable de que al año 2014 existan 18.160 pacientes en programa de hemodiálisis crónica, estimándose una prevalen- cia de 1.019 pacientes por millón de habitantes, cifra considerada elevada en comparación a países de Latinoamérica y resto del mundo8,9. Entre las diferentes modalidades de sustitución renal, el trasplante ha sido considerada la terapia de elección por brindar una mejor expectativa y calidad de vida, y por ser la opción con perfil de costo-efectividad más favorable10-12. En nuestro país, semejante al resto del mun- do, hemos observado un progresivo incremento de los pacientes en lista de espera para trasplante renal, existiendo al año 2014 una tasa de 88 por millón de habitantes, cifra marcadamente supe- rior que la tasa de trasplantes cadavéricos a la misma fecha (12 por millón de habitantes). Resultados Tasa de supervivencia ac- tuarial del injerto, en receptores de trasplante renal de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. diferencias significativas, durante el período de seguimiento, respecto a los receptores de DCE que recibieron trasplante simple. actuarial del injerto al año, 3 años y 5 años fue de 88% vs 93%, 75% vs 91% y 61 vs 89% en el grupo de DCE y DCS, respectivamente, siendo estas dife- rencias estadísticamente significativas (Figura 3). actuarial del injerto al año, 3 años y 5 años fue de 88% vs 93%, 75% vs 91% y 61 vs 89% en el grupo de DCE y DCS, respectivamente, siendo estas dife- rencias estadísticamente significativas (Figura 3). La sobrevida de los pacientes a 5 años fue de 91% en los receptores de DCS y 74% en los recep- tores de DCE, diferencia que resultó significativa (Figura 4). La muerte con injerto funcionante ocurrió en 4 casos (12%) en el grupo de receptores de DCE, y en 14 casos (8%) en el grupo de receptores de DCS (p = 0,45). Tras censurar los casos de muerte con injerto funcionante, la tasa de supervivencia La sobrevida de los pacientes a 5 años fue de 91% en los receptores de DCS y 74% en los recep- tores de DCE, diferencia que resultó significativa (Figura 4). Rev Med Chile 2016; 144: 22-29 26 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al Figura 4. Tasa de supervivencia actua- rial de los pacientes receptores de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Figura 4. Tasa de supervivencia actua- rial de los pacientes receptores de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Figura 4. Tasa de supervivencia actua- rial de los pacientes receptores de DCE versus DCS. DCS: Donantes de criterio estándar. DCE: Donantes de criterios expandidos. Rev Med Chile 2016; 144: 22-29 Discusión El problema se ve incrementado si consideramos que sólo 8,2% de pacientes en hemodiálisis cró- nica se encuentra en lista de espera, recuento con- siderado bajo con respecto a otros países, en las que esta cifra bordea el 20% (Elgueta S. Registro nacional de trasplante renal 2014. XII Congreso latinoamericano de nefrología e hipertensión. En esta serie, y al igual que en la mayoría de las series publicadas, los resultados obtenidos en receptores de DCE son inferiores en términos de sobrevida del injerto, incidencia de rechazo y función renal a largo plazo. Las diferencias en función renal de ambos grupos se manifiestan precozmente desde el primer mes y se hacen sig- nificativas a partir del año en forma sostenida. Al Rev Med Chile 2016; 144: 22-29 27 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al aplicar un análisis de covarianza, pudimos deter- minar que las diferencias de creatininemia y VFG entre ambos grupos, durante el seguimiento, son independientes de la función renal obtenida al mes post-trasplante, por lo tanto, un buen resultado inicial no sería necesariamente un buen predictor de resultados satisfactorios a largo plazo. Debemos considerar que la incompatibilidad HLA en los re- ceptores de DCE fue mayor, y el uso de inducción fue infrecuente, lo que podría haber influido en la mayor ocurrencia de eventos inmunológicos en este grupo de pacientes particularmente sus- ceptibles. Del mismo modo, pareciere que los trasplantes de DCE no serían los candidatos más idóneos a recibir esquemas inmunosupresores libres de agentes anticalcineurínicos. La sobre- vida de los receptores de DCE fue más reducida, sin embargo, no puede dejar de considerarse la gran diferencia etaria entre ambos grupos, y la probabilidad implícita de presentar con mayor frecuencia eventos desfavorables en el grupo de edad más avanzada. El tiempo en lista de espera de los receptores de DCE fue considerablemente menor, sin embargo, creemos que este tiempo es aún más bajo tras observar que muchos de los receptores de DCE ingresaron a la lista de espera de DCE tras permanecer un largo tiempo en la lista estándar. var un mayor número de hospitalizaciones y de complicaciones post-quirúrgicas en los recepto- res de DCE. El mayor número de hospitalizacio- nes en este grupo fue explicado principalmente por la mayor ocurrencia de rechazos, de com- plicaciones quirúrgicas tardías y de infecciones respiratorias. Discusión Es importante destacar que, si bien el 21% de ellos recibió un doble trasplante renal, este subgrupo de pacientes presentó similar nú- mero de complicaciones peri-operatorias que los receptores de riñón único, sin poder demostrarse influencia de la doble intervención en la mayor ocurrencia de complicaciones quirúrgicas del grupo receptor de DCE. Son pocos los ensayos que han evaluado adecuadamente, y a largo plazo, la ocurrencia de dichos eventos en este grupo de pacientes, y los resultados reportados son dispa- res. Algunos autores han descrito un incremento en la frecuencia de linfoceles, mayor necesidad de re intervención quirúrgica y un riesgo aumentado de complicaciones urológicas y vasculares a corto y largo plazo19,20. Es muy probable que la alta pre- valencia de arterioesclerosis, tanto del donante como del receptor, juegue un rol protagónico en la génesis de las complicaciones vasculares en los sujetos más añosos21. Además, la mayor fragilidad linfática renal, la abundancia del tejido graso perihiliar que dificulta la disección quirúrgica, y una menor vascularización a nivel ureteral, po- drían ser los factores contribuyentes a la mayor ocurrencia de linfoceles y de complicaciones de la vía urinaria22. Numerosas han sido las estrategias para mejorar los resultados de los injertos de DCE, como seleccionar receptores con bajo riesgo inmunológico y HLA compatibles, reducir los tiempos de isquemia y mejorar la criopreser- vación de los injertos14,15. Remuzzi y cols. han obtenidos buenos resultados utilizando criterios histológicos para la aceptación de los injertos, pudiendo de esta manera descartar o aceptar el injerto donado según la puntuación histológica obtenida; sin embargo, esta práctica requiere un gran despliegue de recursos y es irrealizable en nuestro medio16. Una serie de otras medidas han demostrado ser efectivas en optimizar la utiliza- ción de este tipo de trasplantes, sugiriéndose así llevarlos a cabo con receptores mayores de 60 años o mayores de 40 años con nefropatía dia- bética en los que se prevé una larga permanencia en lista de espera. En este grupo se ha mostrado una mejor sobrevida recibiendo trasplante renal de DCE que permaneciendo en hemodiálisis y lista de espera17,18. Referencias 1. Carter JT, Lee CM, Weinstein RJ, Lu AD, Dafoe DC, Alfrey EJ. Evaluation of the older cadaveric kidney do- nor: the impact of donor hypertension and creatinine clearance on graft performance and survival. Transplan- tation 2000; 70 (5): 765-71. 13. Randhawa PS, Minervini MI, Lombardero M, Duques- noy R, Fung J, Demetris A, et al. Biopsy of marginal donor kidneys: correlation of histologic findings with graft dysfunction. Transplantation 2000; 69 (7): 1352- 7. 2. De Fijter J, Mallat M, Doxiadis I, Ringers J, Rosendaal F, Claas F, et al. Increased immunogenicity and cause of graft loss of old donor kidneys. J Am Soc Nephrol 2001; 12 (7): 1538-46. 14. Moers C, Smits JM, Maathuis MH, Treckmann J, van Gelder F, Ploeq RJ, et al. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 2009; 360 (1): 7-19. 3. Bajwa M, Cho YW, Pham PT, Shah T, Danovitch G, Wilkinson A, et al. Donor biopsy and kidney transplant outcomes: an analysis using the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database. Transplantation 2007; 84 (11): 1399-405. 15. Port FK, Bragg-Gresham JL, Metzger RA, Dykstra DM, Gillespie BW, Held PJ, et al. Donor characteristics associated with reduced graft survival: an approach to expanding the pool of kidney donors. Transplantation 2002; 74 (9): 1281-6. 4. Ojo A, Hanson J, Meier-Kriesche H, Okechuwku C, Wolfe R, Leichtman A, et al. Survival in recipients of marginal cadaveric donor kidneys compared with other recipients and wait-listed transplant candidates. J Am Soc Nephrol 2001; 12 (3): 589-97. 16. Remuzzi G, Cravedi P, Perna A, Dimitrov B, Turturro M, Ruggenenti P, et al. Long-Term Outcome of Renal Transplantation from Older Donors. N Engl J Med 2006; 354 (4): 343-52. 17. Pascual J, Zamora J, Pirsch J. A systematic review of kidney transplantation from expanded criteria donors. Am J Kidney Dis 2008; 52 (3): 553-86. 5. Lee C, Scandling J, Pavlakis M, Markezich A, Dafoe D, Alfrey E. A review of kidneys that nobody wanted: de- terminants of optimal outcome. Transplantation 1998; 65 (2): 213-9. 18. Rao P, Merion R, Ashby V, Port F, Wolfe R, Kayler L. Renal transplantation in elderly patients older than 70 years of age: results from the Scientific Registry of Transplant Recipients. Transplantation 2007; 83 (8): 1069-74. 6. Rosengard B, Feng S, Alfrey E, Zaroff J, Emond J, Del- monico F, et al. Discusión Una de las fortalezas de nuestro estudio es el largo tiempo de seguimiento promedio (4,5 años), que permite evaluar los desenlaces a largo plazo; además la buena calidad y tabulación de los registros clínicos, junto a la revisión acuciosa de las fichas clínicas lograron que fueran analizados el 99% de los trasplantados en el período consi- derado y permitieron obtener prácticamente la totalidad de eventos ocurridos. Finalmente, concluimos que la reducida su- pervivencia del injerto y mayor ocurrencia de complicaciones no significa una falta de benefi- cio terapéutico. Los trasplantes de DCE son una alternativa válida frente a los excesivos tiempos en lista de espera, pero deben realizarse esfuerzos adicionales y exhaustivos enfocados en seleccio- nar receptores adecuados, para así maximizar la utilidad del procedimiento y reducir la tasa de complicaciones. En nuestro análisis, además, pudimos obser- Rev Med Chile 2016; 144: 22-29 28 ARTÍCULOS DE INVESTIGACIÓN Trasplante renal de donantes con criterios expandidos - R. Valjalo et al Trasplante renal de donantes con criterios expandidos - R. Valjalo et al 12. Laupacis A, Keown P, Pus N, Krueger H, Ferguson B, Muirhead N, et al. A study of the quality of life and cost-utility of renal transplantation. Kidney int 1996; 50 (1): 235-42. Referencias Report of the Crystal City meeting to maximize the use of organs recovered from the cadaver donor. Am J Transplant 2002; 2 (8): 701-11. 19. Bentas W, Jones J, Karaoguz A, Tilp U, Probst M, Goss- mann J, et al. Renal transplantation in the elderly: sur- gical complications and outcome with special emphasis on the Eurotransplant Senior Programme. Nephrol Dial Transplant 2008; 23 (6): 2043-51. 7. Arns W, Citterio F, Campistol JM. ‘Old-for-old’-new strategies for renal transplantation. Nephrol Dial Trans- plant 2007; 22 (2): 336-41. 8. Poblete H. XXXIV Cuenta de hemodiálisis crónica en Chile. Obtenido de http://fmc-ag.cl/_file/file_54_cuen- ta%20hemodialisis%202015.pdf [Consultado el 27 de abril de 2015]. 20. Barba J, Zudaire JJ, Robles JE, Rosell D, Berian JM, Pascual I. Complications of kidney transplantation with grafts from expanded criteria donors. World J Urol 2013; 31 (4): 893-900. 9. Jha V, García-García G, Iseki K, Li Z, Naicker S, Yang CW, et al. Chronic kidney disease: global dimension and perspectives. Lancet 2013; 382 (9888): 260-72. 21. Giessing M, Budde K, Fritsche L, Slowinski T, Tuerk I, Loening SA, et al. “Old-for-old” cadaveric renal trans- plantation: surgical findings, perioperative complica- tions and outcome. Eur Urol 2003; 44 (6): 701-8. 10. Wolfe R, Ashby V, Milford E, Ojo A, Ettenger R, Port F, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and reci- pients of a first cadaveric transplant. N Engl J Med 1999; 341 (23): 1725-30. 22. Hernández D, Rufino M, Armas S, González A, Gutié- rrez P, Torres A, et al. Retrospective analysis of surgical complications following cadaveric kidney transplanta- tion in the modern transplant era. Nephrol Dial Trans- plant 2006; 21 (10): 2908-15. 11. Jofré R, López-Gómez J, Moreno F, Sanz-Guajardo D, Valderrábano F. Changes in quality of life after renal transplantation. Am J Kidney Dis 1998; 32 (1): 93-100. Rev Med Chile 2016; 144: 22-29 29
https://openalex.org/W2955005727	https://figshare.com/ndownloader/files/17801810	English	null	Linkage disequilibrium and haplotype block patterns in popcorn populations	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	460	S1 Table. Gene name, annotation, and chromosome localization, and the number of intragenic SNPs in each population. Gene Annotation Chr. SNPs Population Zm00001d002654 nkd1; naked endosperm1: double mutants have multiple (2-5) layers of 2 12 Biparental peripheral endosperm cells that lack starch granules or other features of 7 Synthetic starchy endosperm 7 BFc4 Zm00001d004817 Fatty acid amide hydrolase 2 14 Biparental 11 Synthetic 3 BFc4 Zm00001d005451 Cellulose synthase A catalytic subunit 5 [UDP-forming] 2 5 Biparental 8 Synthetic 6 BFc4 Zm00001d041972 Cellulose synthase-like protein G3 3 15 Biparental 9 Synthetic 10 BFc4 Zm00001d052263 Starch synthase 2 chloroplastic/amyloplastic 4 6 Biparental 10 Synthetic 6 BFc4 Zm00001d018033 Starch synthase IIb-2 5 19 Biparental 6 Synthetic 5 BFc4 Zm00001d035760 zp15; zein protein, 15kDa15: high methionine; genomic blot indicates one 6 5 Biparental or two copies 2 Synthetic 2 BFc4 Zm00001d036900 Cellulose synthase A catalytic subunit 7 [UDP-forming] 6 19 Biparental 15 Synthetic 8 BFc4 Zm00001d021731 Cellulose synthase-like protein D3 7 7 Biparental 9 Synthetic 9 BFc4 Zm00001d023810 Putative cellulose synthase-like family protein 10 5 Biparental 12 Synthetic 16 BFc4 Zm00001d025201 fab1; fatty acid biosynthesis1: endosperm cDNA 2C01H08 (uaz99) similar 10 9 Biparental to fatty acid biosynthesis enzyme 5 Synthetic 21 BFc4 Zm00001d026113 nkd2; naked endosperm2: double mutants have multiple (2-5) layers of 10 20 Biparental peripheral endosperm cells that lack starch granules or other features of 8 Synthetic starchy endosperm 11 BFc4 S1 Table. Gene name, annotation, and chromosome localization, and the number of intragenic SNPs in each population. Gene Annotation Chr. SNPs Population Zm00001d002654 nkd1; naked endosperm1: double mutants have multiple (2-5) layers of 2 12 Biparental peripheral endosperm cells that lack starch granules or other features of 7 Synthetic starchy endosperm 7 BFc4 Zm00001d004817 Fatty acid amide hydrolase 2 14 Biparental 11 Synthetic 3 BFc4 Zm00001d005451 Cellulose synthase A catalytic subunit 5 [UDP-forming] 2 5 Biparental 8 Synthetic 6 BFc4 Zm00001d041972 Cellulose synthase-like protein G3 3 15 Biparental 9 Synthetic 10 BFc4 Zm00001d052263 Starch synthase 2 chloroplastic/amyloplastic 4 6 Biparental 10 Synthetic 6 BFc4 Zm00001d018033 Starch synthase IIb-2 5 19 Biparental 6 Synthetic 5 BFc4 Zm00001d035760 zp15; zein protein, 15kDa15: high methionine; genomic blot indicates one 6 5 Biparental or two copies 2 Synthetic 2 BFc4 Zm00001d036900 Cellulose synthase A catalytic subunit 7 [UDP-forming] 6 19 Biparental 15 Synthetic 8 BFc4 Zm00001d021731 Cellulose synthase-like protein D3 7 7 Biparental 9 Synthetic 9 BFc4 Zm00001d023810 Putative cellulose synthase-like family protein 10 5 Biparental 12 Synthetic 16 BFc4 Zm00001d025201 fab1; fatty acid biosynthesis1: endosperm cDNA 2C01H08 (uaz99) similar 10 9 Biparental to fatty acid biosynthesis enzyme 5 Synthetic 21 BFc4 Zm00001d026113 nkd2; naked endosperm2: double mutants have multiple (2-5) layers of 10 20 Biparental peripheral endosperm cells that lack starch granules or other features of 8 Synthetic starchy endosperm 11 BFc4 1 1
https://openalex.org/W3010825428	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0230319&type=printable	English	null	Description and classification of echolocation clicks of Indian Ocean humpback (Sousa plumbea) and Indo-Pacific bottlenose (Tursiops aduncus) dolphins from Menai Bay, Zanzibar, East Africa	PloS one	2,020	cc-by	8,138	PLOS ONE RESEARCH ARTICLE Abstract Citation: Yang L, Sharpe M, Temple AJ, Jiddawi N, Xu X, Berggren P (2020) Description and classification of echolocation clicks of Indian Ocean humpback (Sousa plumbea) and Indo-Pacific bottlenose (Tursiops aduncus) dolphins from Menai Bay, Zanzibar, East Africa. PLoS ONE 15(3): e0230319. https://doi.org/10.1371/journal. pone.0230319 Passive acoustic monitoring (PAM) is a powerful method to study the occurrence, movement and behavior of echolocating odontocetes (toothed whales) in the wild. However, in areas occupied by more than one species, echolocation clicks need to be classified into species. The present study investigated whether the echolocation clicks produced by small, at-risk, resident sympatric populations of Indian Ocean humpback dolphin (Sousa plumbea) and Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Menai Bay, Zanzibar, East Africa, could be classified to allow species specific monitoring. Underwater sounds of S. plumbea and T. aduncus groups were recorded using a SoundTrap 202HF in January and June-August 2015. Eight acoustic parameters, i.e. -10 dB duration, peak, centroid, lower -3 and lower -10 dB frequencies, and -3 dB, -10 dB and root-mean-squared bandwidth, were used to describe and compare the two species’ echolocation clicks. Statistical analyses showed that S. plumbea clicks had signifi- cantly higher peak, centroid, lower -3 and lower -10 dB frequencies compared to T. aduncus, whereas duration and bandwidth parameters were similar for the two species. Random Forest (RF) classifiers were applied to determine parameters that could be used to classify the two species from echolocation clicks and achieved 28.6% and 90.2% correct species classification rates for S. plumbea and T. aduncus, respectively. Both species were classified at a higher rate than expected at random, however the identified classifiers would only be useful for T. aduncus monitoring. The frequency and bandwidth parameters provided most power for spe- cies classification. Further study is necessary to identify useful classifiers for S. plumbea. This study represents a first step in acoustic description and classification of S. plumbea and T. aduncus in the western Indian Ocean region, with potential application for future acoustic moni- toring of species-specific temporal and spatial occurrence in these sympatric species. Editor: William David Halliday, Wildlife Conservation Society Canada, CANADA Editor: William David Halliday, Wildlife Conservation Society Canada, CANADA Received: September 18, 2019 Accepted: February 26, 2020 Published: March 13, 2020 Copyright: © 2020 Yang et al. Description and classification of echolocation clicks of Indian Ocean humpback (Sousa plumbea) and Indo-Pacific bottlenose (Tursiops aduncus) dolphins from Menai Bay, Zanzibar, East Africa Liangliang YangID1,2, Matt Sharpe1, Andrew J. TempleID1, Narriman Jiddawi3, Xiaomei Xu2, Per Berggren1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, England, United Kingdom, 2 Key Laboratory of Underwater Acoustic Communication and Marine Information Technology of the Ministry of Education, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China, 3 Institute of Fisheries Research Zanzibar, Ministry of Agriculture, Natural Resources, Livestock and Fisheries, Zanzibar, Tanzania * xmxu@xmu.edu.cn (XX); per.berggren@newcastle.ac.uk (PB) * xmxu@xmu.edu.cn (XX); per.berggren@newcastle.ac.uk (PB) Abstract This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: William David Halliday, Wildlife Conservation Society Canada, CANADA Received: September 18, 2019 Accepted: February 26, 2020 Published: March 13, 2020 Copyright: © 2020 Yang 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. * xmxu@xmu.edu.cn (XX); per.berggren@newcastle.ac.uk (PB) PLOS ONE PLOS ONE Introduction Passive acoustic monitoring (PAM) is a powerful technique to study the occurrence, move- ment and behavior of odontocetes (toothed whales) in the wild [1–4]. Compared to traditional visual surveys for odontocetes, PAM detections are not affected by visibility, weather condi- tions, or human observer bias and may be used for long-term monitoring with minimal distur- bance to the study animals [5]. Odontocetes predominantly use two types of sounds; tonal frequency-modulated sounds for communication, e.g. whistles, [6]; and high frequency pulsed clicks, e.g. echolocation clicks for navigation, orientation and prey detection [7], and burst pulses for communication [8]. Whistles are highly variable at an individual level [9] whereas echolocation clicks (here on referred to as “clicks”), are more consistent and can be used for species classification [10–13]. However, some sympatric species of odontocetes produce simi- lar clicks which can limit the effectiveness of PAM for species-specific studies, as acoustic spe- cies classification can be challenging [14]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Acoustic parameters of odontocete clicks vary depending on their sound production mor- phology [14], with some species having the ability to optimize their clicks within the context of the specific habitat [15]. These parameters are further influenced by sound propagation [11], off-axis effects [16] and differences in recording systems [14]. Previous research has success- fully classified clicks for some species groups, such as narrowband high frequency clicks of phocoenids [17] and non-whistling delphinids [10], and at species-specific level e.g. sperm whales (Physeter microcephalus) [18], pygmy sperm whales (Kogia breviceps) [19], Cuvier’s beaked whales (Ziphius cavirostris) [20], Blainville’s beaked whales (Mesoplodon densirostris) [21] and Franciscana river dolphins (Pontoporia blainvillei) [22]. However, classification of clicks for delphinids, especially sympatric species, has proved difficult due to overlaps in some acoustic parameters among species [14, 23, 24]. If the species identity is known when collecting acoustic data, then supervised classification techniques can be developed and employed to attempt species classification using only click data. Supervised machine learning techniques, such as logic-based techniques (e.g. decision tree and rule-based classifiers), perception-based techniques (e.g. neural networks) and statisti- cal learning algorithms (e.g. Bayesian networks and instance-based learning), perform well in processing complex input tasks and may improve decision-making and prediction of unla- beled samples [25]. 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 the National Natural Science Foundation of China (41976178), the National Key Research and Development Program of China (Grant Number 2018YFC1406305), the Wildlife Conservation Society (WCS) and the Western Indian Ocean 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins Marine Science Association (WIOMSA) for funding project BYCAM (Grant Number MASMA/CP/2014/ 01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Marine Science Association (WIOMSA) for funding project BYCAM (Grant Number MASMA/CP/2014/ 01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Considerable efforts have been devoted to analyzing the species-specific aspects of sympatric delphinid clicks using various supervised machine learning methods [26, 27]. For example, clicks of melon-headed whales (Peponocephala electra), common bottlenose (Tursiops truncatus) and Gray’s spinner (Stenellla longirostris longirostris) dolphins were sepa- rated using spectral parameters and discriminant function analysis providing 93%, 75% and 54% correct classification rates for the three delphinid species, respectively [14]. Furthermore, clicks of seven delphinid species, striped dolphin (Stenella coeruleoalba), long-beaked common dolphin (Delphinus capensis), short-beaked common dolphin (Delphinus delphis), Risso’s dol- phin (Grampus griseus), Pacific white-sided dolphin (Lagenorhynchus obliquidens), pilot whale (Globicephala macrorhynchus) and killer whale (Orcinus orca), off the coasts of Washington, Oregon and California were classified using the Random Forest classification model with over- all correct classification score of 49%, which was significantly greater than that expected by chance for the seven species (14%) [12]. Eight species of delphinids have been identified around Zanzibar, East Africa [28, 29], but only the Indian Ocean humpback (Sousa plumbea) and the Indo-Pacific bottlenose dolphin (T. aduncus) are resident in Menai Bay off the southwest coast (Fig 1). Boat surveys using pho- tographic identification and capture-recapture analyses have estimated population sizes of 19 (95% CI 14–25) S. plumbea and 136 (95% CI 124–172) T. aduncus in the southern portion of 2 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins the Menai Bay [30]. Both species are usually observed in small groups of 5–10 individuals, but social and foraging groups may be larger [30, 31]. S. plumbea is generally distributed closer to shore than T. aduncus in areas where the two species distributions overlap [32]. This is also true for Menai Bay, however the two species distributions overlap and are frequently encoun- tered together in mixed-species groups [30]. Both species in Menai Bay are threatened by unsustainable fisheries bycatch [33, 34] and dolphin ecotourism [35–37]. The vocal repertoire of both S. plumbea and T. aduncus includes whistles and clicks, although to date only whistles of T. aduncus from Zanzibar have been described in detail [38]. A recent PAM study conducted in Menai Bay demonstrated broad scale spatio-temporal occurrence patterns for the delphinids [39]. However, that study was unable to acoustically classify the two species and therefore unable to assess species-specific spatio-temporal patterns, thus limiting the applications of the results. Data collection Data were collected on 19 and 20 January, and between 28 June and 19 August 2015 in the Menai Bay Conservation Area, off the southwest coast of Unguja Island, Zanzibar (Fig 1). The study area was surveyed for dolphins using an outboard powered 8 m boat during daytime in Beaufort Sea state <4. The water depth at the recording locations was between 10 m and 15 m depending on the tide. The seabed sediment was sand with scattered small coralline rocks. The time, date, location, species, group size and surface behavior were recorded for all encountered dolphin groups. The boat motored slowly ahead of the dolphin group, deployed the recording equipment, turned off the engine and drifted to reduce background noise. All acoustic recordings were made using a single SoundTrap (ST) 202HF (Ocean Instruments, New Zealand) with a flat frequency response from 20 Hz to 150 kHz (±3 dB). Full-scale responses and sampling rates were set as 173 dB re 1 μPa and 576 kHz, respectively. The ST has an anti-aliasing filter at 150 kHz, resulting in a– 6 dB roll-off per additional octave in fre- quency. The ST was deployed approximately 3 m below the sea surface, attached to a surface buoy and a small weight and tethered to the boat by a 50 m floating line. During recordings, dolphins passed or milled within 5–50 m from the boat. There was no apparent reaction by either species to the presence of the boat and the ST. Although some mixed species groups of S. plumbea and T. aduncus were encountered and recorded, for the purpose of this study only recordings from single species were used in the analyses. The temporal, spatial and species information for each recording session is given in Table 1. Introduction Yet, there is potential for species separation, with small but significant differences in some broadband click parameters demonstrated between T. aduncus and Australian humpback dolphins (S. sahulensis) [23], a sister taxon of S. plumbea. The main objectives of this study are (1) to describe and quantify the source parameters of clicks produced by S. plumbea and T. aduncus in Menai Bay, Zanzibar, and (2) to investigate whether there are sufficient differences in the acoustic click parameters of S. plumbea and T. aduncus to classify recorded clicks to species. Data analyses Click train selection. Recordings were first visually and aurally inspected using wave- forms and spectrograms [Hanning window, fast Fourier transform (FFT) size: 1024 points, 50% frequency overlap] produced in Adobe Audition (version 3.0, Adobe Systems Incorpo- rated, CA). All sound files were digitally filtered with a 4-order Butterworth band pass-filter (10–200 kHz) in Audition to minimize the influence of whistles and ambient noise. Only PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 3 / 13 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins Fig 1. Map of study area. Menai Bay study area (6˚310S to 6˚170S, 39˚110E to 39˚330E) off the southwest coast of Zanzibar, East Africa, where recordings of S. plumbea and T. aduncus echolocation clicks were conducted in 2015. https://doi.org/10.1371/journal.pone.0230319.g001 Fig 1. Map of study area. Menai Bay study area (6˚310S to 6˚170S, 39˚110E to 39˚330E) off the southwest coast of Zanzibar, East Africa, where recordings of S. plumbea and T. aduncus echolocation clicks were conducted in 2015. Fig 1. Map of study area. Menai Bay study area (6˚310S to 6˚170S, 39˚110E to 39˚330E) off the southwest coast of Zanzibar, East Africa, where recordings of S. plumbea and T. aduncus echolocation clicks were conducted in 2015. https://doi.org/10.1371/journal.pone.0230319.g001 https://doi.org/10.1371/journal.pone.0230319.g001 Table 1. Temporal, spatial and species information of acoustic recordings made during 2015 in Menai Bay, Zanzibar, East Africa during single species group encounters of S. plumbea and T. aduncus. Number of click trains, selected trains and individual clicks are presented for each recording session and species. tion of acoustic recordings made during 2015 in Menai Bay, Zanzibar, East Africa during single species group ber of click trains, selected trains and individual clicks are presented for each recording session and species. Table 1. Temporal, spatial and species information of acoustic recordings made during 2015 in Menai Bay, Zanzibar, East Africa during single species group encounters of S. plumbea and T. aduncus. Number of click trains, selected trains and individual clicks are presented for each recording session and species. Table 1. Temporal, spatial and species information of acoustic recordings made during 2015 in Menai Bay, Zanzibar, East Africa during single species group encounters of S. plumbea and T. aduncus. Number of click trains, selected trains and individual clicks are presented for each recording session and species. Species Recording session Date Start time Latitude (S), Longitude (E) Group size Click trains Selected trains selected clicks S. Data analyses plumbea 1 19 Jan 2:12 PM 6˚27’0", 39˚27036" 5 61 10 10 2 28 Jun 6:46 AM 6˚28’48", 39˚29024" 5 9 9 9 3 1 Jul 8:21 AM 6˚26’24", 39˚27036" 4 3 3 3 4 20 Jul 12:06 AM 6˚27’36", 39˚28012" 8 13 13 13 T. aduncus 1 20 Jan 7:09 AM 6˚27’36", 39˚28012" 25 40 40 40 2 28 Jun 9:47 AM 6˚27’0", 39˚27036" 14 1 1 1 3 4 Jul 9:26 AM 6˚28’48", 39˚29024" 27 4 4 4 4 17 Jul 7:03 AM 6˚28’48", 39˚30036" 5 24 10 10 5 1 Aug 7:28 AM 6˚28’12", 39˚32060" 7 2 2 2 6 9 Aug 9:07 AM 6˚28’12", 39˚28012" 17 2 2 2 7 11 Aug 8:59 AM n. a. 36 30 30 30 8 19 Aug 11:54 AM 6˚28’48", 39˚28048" 14 3 3 3 mporal, spatial and species information of acoustic recordings made during 2015 in Menai Bay, Zanzibar, East Africa du of S. plumbea and T. aduncus. Number of click trains, selected trains and individual clicks are presented for each recording s PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 4 / 13 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins sound files with “loud and clear” click trains were extracted, labeled by hand, and used in sub- sequent analyses. Click trains were considered to be “loud and clear”, if they were at least 10 dB re 1 μPa louder than background noise [40] and had no overlap with other strong pulsed sound. The click trains were inspected and potentially confounding sounds (e.g. snapping shrimp and bubble/sediment entrainment noise) were removed by hand. Furthermore, the chosen click trains were required to contain at least eight clicks per train with average inter- click interval (ICI) of > 10 ms and < 0.1 s using a playback rate of 0.01 [41, 42], thus excluding echolocation buzzes (click trains with high repetition rates used during prey capture) and burst pulses, to avoid introducing additional variance in the dataset. The total number of click trains used per recording session was limited to twice the estimated group size to reduce over- presentation of a single recording session [13]. Click trains were randomly selected until all available trains were selected or the limit was reached (Table 1). Click detection. Clicks were automatically detected from each chosen click train using an energy detector to identify impulse signals [43]. Data analyses Click trains were first divided into several 5 ms segments. Clicks were detected in the spectra domain (frequency vs spectral power, Han- ning window, FFT size: 576 points, 50% overlap) and calculated from each segment. When 13% or more of the frequency bins between 15 kHz and 95 kHz had signal-to-noise ratios over 15 dB [13], the segments were considered to contain a click candidate. An automated algorithm was used to remove false positive detections, including vessel noise and clipped clicks. Specifically, click candidates with a peak frequency less than 20 kHz [44] and with a maximum amplitude more than 80% of the maximum system capability [14] were considered as false positive detections. Given that only a single hydrophone was used during the field recordings, it was impossible to determine whether a click was recorded on the acoustic axis [45]. To mitigate against the impact of off-axis click use, only the highest amplitude click from each click train was extracted, following the methods for on-axis click analysis [46]. The aforementioned click detections and false positive removal were performed using customized routines in MATLAB (version R2016a, Mathworks, Natick, MA). Acoustic parameters. A 32-point rectangular window around the peak of the signal envelop was utilized for all selected clicks in order to minimize the risk of reflected clicks and background noise being included in the analysis. To assess potential differences in clicks between S. plumbea and T. aduncus, eight acoustic parameters, i.e. -10 dB duration, peak fre- quency, centroid frequency, lower -3 dB frequency, lower –10 dB frequency, -3 dB bandwidth, -10 dB bandwidth and RMS bandwidth, were calculated using custom written scripts in MATLAB R2016a (Mathworks, Natick, MA). The chosen parameters (Table 2) have been used Table 2. Description of eight acoustic parameters for echolocation clicks of S. plumbea and T. aduncus recorded in Menai Bay, Zanzibar, East Africa. Abbreviation used are shown in the parenthesis. Acoustic parameters Description -10 dB duration (D-10dB) Click duration in 10 dB below the peak of the envelope of the waveform [46]. Peak frequency (FP) Frequency value of maximum energy in the spectrum [7]. Centroid frequency (FC) Average power distributed across the frequency bins in the spectrum [48]. Lower -3dB frequency (FL3) Lower cut-off frequency of -3dB bandwidth [14]. Lower -10dB frequency (FL10) Lower cut-off frequency of -10dB bandwidth [14]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 Statistical analyses The click train selection criteria used identified 35 S. plumbea (S1 Audio) and 92 T. aduncus (S2 Audio) click trains as “loud and clear”, and thus suitable for analysis. All data of the highest amplitude clicks selected from each click train used for statistical analysis are available in sup- porting information (S1 Table). None of the analyzed parameters conformed to a normal dis- tribution (Kolmogorov-Smirnov tests, α = 0.05), had equal variance (Levene’s tests, α = 0.05) or could be successfully log-transformed. Median with 5th and 95th percentile values were used as the descriptive statistics for each parameter. Non-parametric Mann-Whitney U-tests were used to statistically compare the acoustic parameters between S. plumbea and T. aduncus. Significance level was set at α = 0.05. All parameters were used in subsequent classification analyses, with parameters showing significant differences between species expected to provide greatest classification power. Random Forest (RF) was used to separate echolocation clicks between S. plumbea and T. aduncus using all acoustic parameters. RF, as a supervised classification method, has demon- strated excellent performance in bio-acoustic studies [49]. The RF is an ensemble classifier, developed by Breiman [50], consisting of many independent classification trees [51], where each tree is generated by a randomly selected subset of the original training data (e.g. 92 T. aduncus clicks and 35 S. plumbea clicks used here) with replacement [50, 52]. At each split approximately 37% of the training data, named as “out of bag” (OOB) samples, are not selected when constructing each tree but used to assess the performance of the RF [50, 53]. The remain- ing samples, named as “in-bag” samples, are used to construct each tree using a random subset of all features (e.g. the eight acoustic parameters here) to split the node [50, 53]. Once the forest is built, individual trees are combined through a majority voting process to assign new candi- dates to a class [50]. The OOB error rate (OOBerror rate) was calculated as the median of the error rates from the constructed trees in the RF using OBB samples [50]. The percentage, i.e. 1- OOBerror rate, and its 5th and 95th quantiles are reported as a measure of the correct classification rate [54]. There are two significant parameters in the RF [53]: (1) the number of trees to construct (ntree), and (2) the number of randomly chosen variables (e.g. Data analyses -3dB bandwidth (BW-3dB) Frequency width between the 1/p2 of amplitude points of the spectrum on the linear scale [46]. -10dB bandwidth (BW-10dB) Frequency width between the 1/10 of amplitude points of the spectrum on the linear scale [46]. Root-mean-squared bandwidth (BWRMS) Spectral standard deviation around the centroid frequency of the spectrum [48]. https://doi.org/10.1371/journal.pone.0230319.t002 Table 2. Description of eight acoustic parameters for echolocation clicks of S. plumbea and T. aduncus recorded in Menai Bay, Zanzibar, East Africa. Abbreviation used are shown in the parenthesis. able 2. Description of eight acoustic parameters for echolocation clicks of S. plumbea and T. aduncus recorded Menai Bay, Zanzibar, East Africa. Abbreviation used are shown in the parenthesis. 5 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins by several other studies to characterize dolphin clicks [7, 14, 46]. The -10 dB duration was deter- mined from the interpolated (10 times linear interpolation) waveform for an individual click. The remaining seven frequency and bandwidth parameters were computed from the power spectra. The power spectrum of each detected click was calculated based on Welch method [47] using 32-point fast Fourier transform with a Hanning window, and interpolated with a factor of 10 using low-pass interpolation, resulting in a spectral resolution of 1.8 kHz. These settings allow direct comparison to previous published acoustic parameter measurements for the two species [23]. Inter-click interval (ICI) and Q (quality factor) parameters were not deemed appropriate for use in the analyses and so were not considered. ICIs are adjusted by the echolo- cating animal and shortened when approaching a target to facilitate close distance tracking and capture [14]. The Q parameter has been used to describe the relative bandwidth of click signals in previous research [23]. However, the Q parameter does not provide useful information for classification, as it is defined as the ratio of centroid frequency to RMS bandwidth. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 Ethics statement The research was undertaken with research permits obtained through the Zanzibar Ministry of Agriculture, Natural Resources, Livestock and Fisheries as well as ethical approval from Newcastle University, UK. PLOS ONE PLOS ONE Table 3. The median with the 5th and 95th percentile values of eight acoustic parameters for echolocation clicks of S. plumbea and T. aduncus in Menai Bay, Zanzi- bar, East Africa. Species comparisons of click parameters were conducted using Mann-Whitney U tests. Note: = p<0.05. Acoustic parameters S. plumbea (n = 35) T. aduncus (n = 92) p-value median (5–95%) median (5–95%) -10 dB duration (μs) 13 (11–22) 14 (11–23) 0.514 Peak frequency (kHz) 97 (52–119) 73 (47–115) 0.003 Centroid frequency (kHz) 87 (64–107) 81 (57–98) 0.002 Lower -3 dB frequency (kHz) 43 (29–95) 36 (27–59) 0.003 Lower -10 dB frequency (kHz) 22 (16–35) 18 (13–29) 0.017 -3 dB bandwidth (kHz) 81 (49–107) 85 (34–95) 0.223 -10 dB bandwidth (kHz) 126 (103–148) 130 (94–146) 0.445 RMS bandwidth (kHz) 23 (18–31) 25 (16–35) 0.365 https://doi.org/10.1371/journal.pone.0230319.t003 entile values of eight acoustic parameters for echolocation clicks of S. plumbea and T. aduncus in Menai Bay, Zanzi- ameters were conducted using Mann-Whitney U tests. Note: = p<0.05. Statistical analyses acoustic parameters in this case) used to split each node (mtry), which can be optimized via the OOB error estimation. As a result, the ntree and mtry were set to 3000 and pM based on the OOB error, where M repre- sents the total number of input variables. The above parameter comparison and RF model construction were all implemented in R (version 3.3.3, R Core Team, 2018) using the asht and randomForest packages, respectively. 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 Description and classification of echolocation clicks of humpback and bottlenose dolphins Results The values of the eight acoustic parameters of S. plumbea and T. aduncus echolocation clicks are summarized in Table 3 and their respective typical waveforms and the power spectra are presented in Fig 2. Although the clicks of S. plumbea and T. aduncus were similar, the four fre- quency parameters (peak frequency, centroid frequency, lower -3 dB frequency and lower -10 dB frequency) had significantly higher median values for S. plumbea compared to T. aduncus Fig 2. Echolocation click examples for S. plumbea and T. aduncus. Waveform and power spectrum [Sampling rate: 576 kHz, 32-point rectangular window around the peak of the envelope, and interpolated with a factor of 10 for a spectral resolution of 1.8 kHz] examples of echolocation clicks of S. plumbea [(a) and (b)] and T. aduncus [(c) and (d)] from Menai Bay, Zanzibar, East Africa. https://doi.org/10.1371/journal.pone.0230319.g002 Fig 2. Echolocation click examples for S. plumbea and T. aduncus. Waveform and power spectrum [Sampling rate: 576 kHz, 32-point rectangular window around the peak of the envelope, and interpolated with a factor of 10 for a spectral resolution of 1.8 kHz] examples of echolocation clicks of S. plumbea [(a) and (b)] and T. aduncus [(c) and (d)] from Menai Bay, Zanzibar, East Africa. https://doi org/10 1371/journal pone 0230319 g002 https://doi.org/10.1371/journal.pone.0230319.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 7 / 13 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins (Mann-Whitney U Tests, α = 0.05, Table 3). Neither -10 dB duration nor the three bandwidth parameters were significantly different between S. plumbea and T. aduncus. Overall, the RF model resulted in a 73.2% (5th and 95th quantiles 72.4%-74.0%) correct classification rate for S. plumbea and T. aduncus echolocation clicks submitted to the model, compared to 50% expected by random chance alone. Similarly, the RF model resulted in a cor- rect classification rate of 28.6% (5th and 95th quantiles 25.7%-31.4%) for S. plumbea, com- pared to an expected rate of 27.6%, and 90.2% (5th and 95th quantiles 90.2%-90.2%) for T. aduncus, compared to an expected rate of 72.4%. The RF model demonstrated the following order of importance for the acoustic parameters using mean decreasing accuracy measures for species classification: lower -10 dB frequency, -3 dB bandwidth, -10 dB bandwidth, lower -3 dB frequency, centroid frequency, RMS bandwidth, peak frequency and -10 dB duration. Discussion To our knowledge, this study represents the first report of echolocation click parameters for T. aduncus in the Western Indian Ocean and for S. plumbea globally. The study found differences in peak, centroid, lower -3 dB and lower -10 dB frequencies of clicks between species. This study further classified the echolocation clicks using a RF model and achieved an overall 73.2% correct species classification rate i.e. a better performance than what was expected by chance alone (50%). Click source parameters measured in this study differed substantively in some instances from those previously reported for T. aduncus and Sousa species in other geographic regions (Table 4). Both species had lower peak and centroid frequencies than previously reported in the published literature [23, 41, 55]. These differences may result from morphological species differences [14] and/or optimization of clicks for the specific environmental context of differ- ent habitats primarily occupied by the species [17]. Indeed, S. plumbea has a relatively longer and narrower skull compared to S. chinensis [56]. Additionally, differences among study meth- odologies may contribute to click parameter differences. The present study used a single hydrophone and selected only the highest amplitude click in each click train for inclusion in the analyses, in order to minimise the likelihood of off-axis click selection. Other studies were able to use hydrophone arrays thus having a higher likelihood of selecting only on-axis clicks [23, 55]. Statistically, all click frequency parameters (peak, centroid, lower -3 dB and lower -10 dB frequencies) measured for S. plumbea were higher than those of T. aduncus. Differences in Table 4. Comparisons of the mean ± standard deviation of source parameters of echolocation clicks of wild T. aduncus from different areas and different species of Sousa. Species -10 dB duration (μs) Peak frequency (kHz) Centroid frequency (kHz) -3 dB bandwidth (kHz) -10 dB bandwidth (kHz) RMS bandwidth (kHz) Reference T. aduncus (n = 54) 14±2 124±13 112±9 62±17 140±17 34±3 [23] T. aduncus (n = 89) 18±6 n.a 91±13 n. a. n. a. 35±3 [41] T. aduncus (n = 92) 16±4 77±24 80±13 81±19 127±15 24±4 This study S. chinensis (n = 77) 19±4 109±4 95±6 50±13 102±11 29±3 [55] S. sahulensis (n = 42) 15±2 114±12 106±11 59±18 116±20 29±4 [23] S. Discussion plumbea (n = 35) 16±3 91±22 87±12 79±15 125±11 24±3 This study htt //d i /10 1371/j l 0230319 t004 mparisons of the mean ± standard deviation of source parameters of echolocation clicks of wild T. aduncus from differen ons of the mean ± standard deviation of source parameters of echolocation clicks of wild T. aduncus from different areas Table 4. Comparisons of the mean ± standard deviation of source parameters of echolocation clicks of wild T. adunc Sousa. eviation of source parameters of echolocation clicks of wild T. aduncus from different areas and different species of eviation of source parameters of echolocation clicks of wild T. aduncus from different areas and different species of PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 8 / 13 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins preferred habitat and cranial morphology likely explain the interspecific click differences observed in these parameters. T. aduncus and S. plumbea in Menai Bay have overlapping dis- tributions. However, S. plumbea is only found in shallow waters, close to shore whereas T. aduncus occurs across the bay including offshore areas [30]. The significantly lower echoloca- tion click frequencies recorded for T. aduncus compared to S. plumbea would facilitate longer range echolocation which would be beneficial in the deeper and more open water habitats occupied by T. aduncus [57]. Furthermore, differences in skull morphology between species, with S. plumbea featuring “a small left posterior branch of the melon”, which may be an adap- tation that provide improved directionality when using high frequency sounds [57]. The parameters used for species classification in the present study were similar to those applied in other similar research [11, 14, 23]. Specifically, frequency parameters appear to be powerful for classification of delphinids from clicks. Bandwidth parameters appear, generally, weaker for classification, which is consistent with findings of no or limited differences in these parameters among species [14, 23]. Despite this, bandwidth parameters were found to be rela- tively important contributors to species classification in this study, suggesting that researchers should continue to consider these parameters in future. Interestingly, whilst none of the band- width parameters showed significant differences between the two species, both the -3 and -10 dB bandwidth parameters were important in the RF classifier. This reflects the multivariate nature of click signals and indicates likely interactions among parameters. Discussion It re-enforces the importance of retaining parameters for classification despite not showing significant differ- ences across all clicks. Conversely, click duration contributed little to click classification. This is congruent with the limited variability in this parameter for sympatric Tursiops and Sousa species observed in this study and other studies [23]. However, it is notable that click duration has been identified as a valuable parameter for classification of other delphinid species [12, 58] and thus cannot be assumed irrelevant in future works. Understanding drivers behind the interspecific variability in parameter utility for classification may be an important step in shap- ing future classifier development and ultimately its field application. Regarding classification of T. aducus and S. plumbea in the current study, both species were classified successfully above the expected rate. However, the improvement above the expected rate for S. plumbea was minimal. The sample size for S. plumbea was relatively small (n = 35) in comparison to T. aduncus (n = 92) and several of the parameters showed a high level of vari- ability. The relatively small sample size for S. plumbea makes it vulnerable to influence from unusual click trains or accidental inclusion of off-axis trains. Therefore, greater sampling effort is required to confirm and improve on the findings in the current study. The relatively high classification rate for T. aduncus demonstrates the potential to use PAM of clicks to monitor occurrence of this species. Thus, continued effort to further improve the classifiers and to develop algorithms to be incorporated into PAM monitoring systems for T. aduncus is strongly encouraged. In this study, eight acoustic parameters were considered in the analysis. Future efforts to classify species using clicks may be assisted by expanding the range of parameters considered [59, 60]. For example, measurements of intensity at different frequencies in the spectrum have been shown to facilitate identification of “peak and notch” patterns which have been used to classify both G. griseus and L. obliquidens [13]. Furthermore, comparing intensity at different frequency bands in the spectrum has improved classification results for M. densirostris, G. macrorhynchus and G. griseus [61, 62]. Previous research have also demonstrated that coeffi- cients in cepstral analysis (inverse Fourier transform of the logarithm of the estimated spec- trum of a signal) had good classification performance for some species (e.g. G. griesus, M. densirostris and G. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 S2 Audio. Echolocation click trains of Indo-Pacific bottlenose dolphin (Tursiops aduncus). (ZIP) S1 Table. Information for each click analyzed. For each click, information is provide indicat- ing species, file name, -10 dB duration (μs), peak, centroid, lower -3 and lower -10 dB frequen- cies (kHz), and -3 dB, -10 dB and root-mean-squared bandwidth (kHz). (XLSX) Acknowledgments We thank Juma Kifana, Mwalim Rajab, Foum Ramadhan and Khamis Ramadhan for assis- tance during the field work. We also thank the academic editor Dr William David Halliday and three reviewers for their constructive feedback that significantly improved this manuscript. We thank Juma Kifana, Mwalim Rajab, Foum Ramadhan and Khamis Ramadhan for assis- tance during the field work. We also thank the academic editor Dr William David Halliday and three reviewers for their constructive feedback that significantly improved this manuscript. Discussion macrorhynchus), with little influence of sound propagation and variation resulting from different recording platforms [15, 63]. Additionally, energy amplitude, off-axis PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 9 / 13 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins click distortion and transmission beam-widths may contain some species-specific information. However, these characteristics are dependent on knowing the exact location and orientation of echolocating individuals which requires a multiple hydrophone array which was not available in the present study. In conclusion, this study presents new information on echolocation click parameters recorded from wild S. plumbea and T. aduncus resident in Menai Bay, Zanzibar. We further explored whether variation in acoustic parameters of echolocation clicks may be used to iden- tify and classify sympatric living S. plumbea and T. aduncus. An overall 73.2% species click classification rate was achieved, indicating the potential to separate these two species using PAM. However, the identified classifiers were only at a sufficiently high rate (90.2%) for T. aduncus to allow species specific monitoring using PAM based on echolocation clicks. The classification method developed would benefit from further refinement and may be improved by increasing the suite of acoustic parameters considered. It is anticipated that the methods eventually can be incorporated into PAM systems as species-specific classification algorithms, facilitating the use of PAM methodologies to monitor occurrence of specific delphinid species. Such information would provide researchers and managers with the foundational data needed to devise evidence-based species-specific conservation strategies, particularly in areas where species are threatened by anthropogenic activities. S1 Audio. Echolocation click trains of Indian Ocean humpback dolphin (Sousa plumbea). (ZIP) S1 Audio. Echolocation click trains of Indian Ocean humpback dolphin (Sousa plumbea). 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The Journal of the Acoustical Society of America. 2008; 124(1):609–24. https://doi.org/10.1121/1.2932059 PMID: 18647003 14. Author Contributions Conceptualization: Liangliang Yang, Per Berggren. Data curation: Liangliang Yang, Matt Sharpe, Andrew J. Temple, Per Berggren. Formal analysis: Liangliang Yang, Andrew J. Temple. Funding acquisition: Xiaomei Xu, Per Berggren. Investigation: Matt Sharpe, Andrew J. Temple, Narriman Jiddawi. Methodology: Liangliang Yang, Andrew J. Temple, Per Berggren. Resources: Narriman Jiddawi, Per Berggren. Software: Liangliang Yang. Data curation: Liangliang Yang, Matt Sharpe, Andrew J. Temple, Per Berggren. Formal analysis: Liangliang Yang, Andrew J. Temple. Data curation: Liangliang Yang, Matt Sharpe, Andrew J. Temple, Per Berggren. Software: Liangliang Yang. 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 PLOS ONE Description and classification of echolocation clicks of humpback and bottlenose dolphins Supervision: Xiaomei Xu, Per Berggren. Writing – original draft: Liangliang Yang, Per Berggren. Writing – review & editing: Liangliang Yang, Matt Sharpe, Andrew J. Temple, Xiaomei Xu, Per Berggren. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230319 March 13, 2020 References Kotsiantis SB, Zaharakis I, Pintelas P. Supervised machine learning: A review of classification tech- niques. Emerging artificial intelligence applications in computer engineering. 2007; 160:3–24. 26. Bittle M, Duncan A, editors. A review of current marine mammal detection and classification algorithms for use in automated passive acoustic monitoring. Annual Conference of the Australian Acoustical Soci- ety, Science, Technology and Amenity; 2013. 27. Valletta JJ, Torney C, Kings M, Thornton A, Madden J. 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https://openalex.org/W2753107405	https://europepmc.org/articles/pmc5574578?pdf=render	English	null	Mother-to-child transmission of HIV in Kenya: A cross-sectional analysis of the national database over nine years	PloS one	2,017	cc-by	9,185	RESEARCH ARTICLE OPEN ACCESS To describe factors associated with mother-to-child HIV transmission (MTCT) in Kenya and identify opportunities to increase testing/care coverage. Citation: Mwau M, Bwana P, Kithinji L, Ogollah F, Ochieng S, Akinyi C, et al. (2017) Mother-to-child transmission of HIV in Kenya: A cross-sectional analysis of the national database over nine years. PLoS ONE 12(8): e0183860. https://doi.org/ 10.1371/journal.pone.0183860 Matilu Mwau1,2, Priska Bwana2, Lucy Kithinji2, Francis Ogollah2, Samuel Ochieng2, Catherine Akinyi1, Maureen Adhiambo1, Fred Ogumbo1, Martin Sirengo3, Caroline Boeke4 Matilu Mwau1,2, Priska Bwana2, Lucy Kithinji2, Francis Ogollah2, Samuel Ochieng2, Catherine Akinyi1, Maureen Adhiambo1, Fred Ogumbo1, Martin Sirengo3, Caroline Boeke 1 Centre for Infectious and Parasitic Diseases Control Research, Kenya Medical Research Institute, Busia, Kenya, 2 Centre for Virus Research, Kenya Medical Research Institute, Nairobi, Kenya, 3 National AIDS and STIs Control Program, Ministry of Health, Nairobi, Kenya, 4 Clinton Health Access Initiative, Boston, Massachusetts, United States of America 1 Centre for Infectious and Parasitic Diseases Control Research, Kenya Medical Research Institute, Busia, Kenya, 2 Centre for Virus Research, Kenya Medical Research Institute, Nairobi, Kenya, 3 National AIDS and STIs Control Program, Ministry of Health, Nairobi, Kenya, 4 Clinton Health Access Initiative, Boston, Massachusetts, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * mmwau@kemri.org Methods Received: February 11, 2017 Accepted: August 11, 2017 Published: August 29, 2017 365,841 Kenyan infants were tested for HIV from January 2007-July 2015 and results, demographics, and treatment information were entered into a national database. HIV risk factors were assessed using multivariable logistic regression. Copyright: © 2017 Mwau 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: Sarah L. Pett, University of New South Wales, AUSTRALIA Editor: Sarah L. Pett, University of New South Wales, AUSTRALIA Design Cross-sectional analysis of national early infant diagnosis (EID) database. Mother to child transmission of HIV in Kenya Conclusion Competing interests: The authors declare no competing interests. The IT infrastructure was funded by Hewlett-Packard but the infrastructure funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The infrastructure funding does not alter our adherence to PLOS ONE policies on sharing data and materials. Despite impressive gains in Kenya’s PMTCT program, some HIV-infected infants present late and are not benefitting from PMTCT best practices. Efforts to identify these early and enforce evidence-based practice for PMTCT should be scaled up. Infant testing should be expanded in pediatric/outpatient departments, given high yields in these portals. Introduction Without antiretroviral therapy (ART), approximately 50% of HIV-infected infants die by the age of two[1]. However, the use of elective caesarian section, the provision of highly active anti- retroviral therapy (HAART) during pregnancy, and the avoidance of breastfeeding have reduced the HIV transmission rate to less than 2% and resulted in the near elimination of mother to child transmission (MTCT) [2, 3]. Unfortunately, barriers to healthcare and sub- optimal infant feeding and care practices still exist in resource-limited settings and continue to propagate transmission of HIV from mothers to infants. Identifying infected infants early and initiating them on ART as soon as possible after diagnosis is essential to slow the progression from HIV infection to AIDS and prolong the life of the patient[1]. Despite substantial progress in efforts to reduce MTCT of HIV in Kenya, HIV transmission through the end of breastfeeding remains at 17%, with approximately 13,000 new HIV infec- tions among children in 2014[4]. Option B+, an approach to prevent MTCT of HIV by offer- ing all HIV-positive pregnant women treatment for life and ART prophylaxis to all exposed infants, free of charge, has been scaled up through a phased rollout in Kenya starting in 2014; by October 2015 more than 90% of all sites with programs on prevention to mother-to-child transmission of HIV (PMTCT) or maternal, newborn, and child health had adopted it[5]. UNAIDS estimates suggest that greater than 90% of all pregnant women attending ANC in Kenya are tested for HIV[6]. However, gaps remain. Nearly half (42%) of women do not fully access antenatal care (at least 4 visits during pregnancy)[7], and 39% deliver outside of health- care facilities[8]. Access to care is poorest in rural areas. Due in large part to this gap, an esti- mated 33% of HIV-infected pregnant women are not on treatment and early infant diagnosis of HIV (EID) testing coverage by two months of age in exposed infants was 72% in 2014[4]. Better understanding of these gaps and the remaining risk factors for HIV infection among infants who get tested is essential to eliminate MTCT in Kenya and neighboring countries. Despite substantial progress in efforts to reduce MTCT of HIV in Kenya, HIV transmission through the end of breastfeeding remains at 17%, with approximately 13,000 new HIV infec- tions among children in 2014[4]. Results 11.1% of infants tested HIV positive in 2007–2010 and 6.9% in 2014–2015. Greater odds of infection were observed in females (OR: 1.08; 95% CI:1.05–1.11), older children (18–24 months vs. 6 weeks-2 months: 4.26; 95% CI:3.87–4.69), infants whose mothers received no PMTCT intervention (vs. HAART OR: 1.92; 95% CI:1.79–2.06), infants receiving no prophy- laxis (vs. nevirapine for 6 weeks OR: 2.76; 95% CI:2.51–3.05), and infants mixed breastfed (vs. exclusive breastfeeding OR: 1.39; 95% CI:1.30–1.49). In 2014–2015, 9.1% of infants had mothers who were not on treatment during pregnancy, 9.8% were not on prophylaxis, and 7.0% were mixed breastfed. Infants exposed to all three risky practices had a seven- fold higher odds of HIV infection compared to those exposed to recommended practices. The highest yield of HIV-positive infants were found through targeted testing of symptomatic infants in pediatric/outpatient departments (>15%); still, most infected infants were identified through PMTCT programs. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: The authors wish to thank the Centers for Disease Control and Prevention (CDC), USAID/ PEPFAR, and UNITAID for funding HIV diagnostics in Kenya. The IT infrastructure was funded by Hewlett-Packard. None of the funders had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Introduction Option B+, an approach to prevent MTCT of HIV by offer- ing all HIV-positive pregnant women treatment for life and ART prophylaxis to all exposed infants, free of charge, has been scaled up through a phased rollout in Kenya starting in 2014; by October 2015 more than 90% of all sites with programs on prevention to mother-to-child transmission of HIV (PMTCT) or maternal, newborn, and child health had adopted it[5]. transmission of HIV (PMTCT) or maternal, newborn, and child health had adopted it[5]. UNAIDS estimates suggest that greater than 90% of all pregnant women attending ANC in Kenya are tested for HIV[6]. However, gaps remain. Nearly half (42%) of women do not fully access antenatal care (at least 4 visits during pregnancy)[7], and 39% deliver outside of health- care facilities[8]. Access to care is poorest in rural areas. Due in large part to this gap, an esti- mated 33% of HIV-infected pregnant women are not on treatment and early infant diagnosis of HIV (EID) testing coverage by two months of age in exposed infants was 72% in 2014[4]. Better understanding of these gaps and the remaining risk factors for HIV infection among infants who get tested is essential to eliminate MTCT in Kenya and neighboring countries. This study used nearly a decade of nationwide data from a national HIV laboratory data- base of over 370,000 EID samples to assess the risk factors for HIV transmission in infants and to identify important predictors of HIV infection over time. We also assessed which facility entry points had the greatest yield for EID case detection of HIV-infected infants to inform resource allocation for HIV testing. While as a registry-based study, the data have some limita- tions, this is amongst the first studies of this scale examining risk factors for HIV infection in infants through the period of scale-up of Option B+ in sub-Saharan Africa. Study setting and design The EID program in Kenya is overseen by the Ministry of Health. As of 2007, there was tar- geted testing of HIV-exposed symptomatic infants; in 2008–2009, as more resources became available for testing, the guidelines changed to test all HIV-exposed infants. The infant HIV testing algorithm as of 2012 in Kenya was as follows: a maternal or infant HIV antibody test PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 2 / 15 Mother to child transmission of HIV in Kenya was conducted at first visit for all children of unknown HIV status aged <18 months to estab- lish HIV exposure status. If positive, an EID test was recommended. If the EID test was posi- tive, the infant was started on ART and if negative, the infant received an HIV antibody test at 9 months (or earlier if child developed symptoms suggestive of HIV). If the antibody test was positive at 9 months, the infant received a confirmatory EID test. If the EID test was negative, the HIV antibody test was repeated at 18 months or 6 weeks after cessation of breastfeeding in a child >18 months[9]. EID testing coverage by 2 months of age in HIV-exposed infants in Kenya was estimated to be below 50% in 2011 and increased to 72% in 2014[4]. In 2015, the algorithm was updated to recommend an EID test at 6 weeks or first contact after 6 weeks[10]. Seven laboratories form the testing network and laboratory request forms are compiled into a national database across the laboratories. Thus, the database covers nearly all infants receiving EID testing in the country. Data from the database can be viewed on a national dashboard (http://eid.nascop.org/). Mean turnaround time from sample collection to results dispatch from the laboratory has changed over time but was 17 days as of July 2015[11]. This cross-sec- tional analysis was based on a retrospective review of all data stored in the national HIV data- base between January 2007 and July 2015. Clinical and laboratory procedures Dried Blood Spots (DBS) were collected as part of routine care for infants with suspected or known HIV exposure as previously described[12]. Briefly, samples were collected under sterile conditions from infants using either a heel prick or finger prick depending on the age and weight of the infant. Sample collection occurred at several entry points including maternal and child health (MCH)/PMTCT clinics, the comprehensive clinic care (CCC)/patient support center (PSC), the outpatient department (OPD), and the pediatric ward. DBS filter papers were labeled and dried separately on a drying rack overnight. They were then packaged using glycine envelopes and sealed plastic bags under sterile conditions and sent to the testing labo- ratory by a courier system accompanied by a laboratory request form. CobasAmpliPrep/CobasTaqMan_HIV-1 Qual or Abbot real-time HIV automated PCR test procedures were conducted on each sample based on the manufacturer’s guidelines, as described previously[12–14]. The CobasAmpliPrep/CobasTaqMan_HIV-1 Qual uses one dried blood spot (70uL) while the Abbot real-time HIV automated PCR testing uses two spots (140uL) of blood for the test. All positive samples were retested to confirm their status while a request for recollection of a new sample was made in cases where the test failed or number of blood spots was not sufficient to allow repeat confirmatory testing for positive samples. Lab results were stored in an online accessible Laboratory Information System database provided by the Ministry of Health, along with the laboratory request form indicating the number of samples sent to the testing laboratory, the age and sex of the infant, mother’s HIV status, breastfeeding status, point of entry, medication given to the mother, and prophylaxis given to the baby. The database was retrospectively accessed to extract the relevant data and run these analyses. Statistical analysis Demographic data were summarized with descriptive statistics. The primary outcome was infant HIV status (infected vs. uninfected). We examined predictors of infant HIV status, including breastfeeding, age, prophylaxis, antiretroviral therapy and portal of entry. As a pre- liminary statistical analysis, continuous predictors were compared in relation to infant HIV result using Student’s T-tests if normally distributed or Mann-Whitney U tests if skewed. Cate- gorical data were compared using the Fisher’s exact test or the chi square test. Logistic regression was used to calculate univariate odds ratios of potential predictors in relation to infant HIV result and 95% confidence intervals. Variables that were significantly associated with infant HIV infection in univariate models were included in a multivariate logistic regression model. Finally, we combined all behavioral risk factors identified for infant HIV infection into one variable and assessed the increased odds of infection in an infant with multiple risk factors. The models accounted for clustering by health facilities using the vce (cluster) function for statistical analysis. Reference groups were selected based on the most common group and/or the standard of care in Kenya. The interaction between age and sex was examined and a Wald p-value for interaction was calculated. Separate logistic regression models were run by time period (2007–2010, prior to any Option B+ programs and when EID testing was limited and targeted; 2011–2013, when EID testing was scaled up and Option B+ was piloted at some sites; and 2014–2015, when Option B+ was rolled out nationally and EID coverage continued to expand) to assess differences over time as Kenya’s national testing and treatment program grew and developed. Significance levels were set at p<0.05. All analysis in this study was conducted using Stata Version 13 (StataCorp LP, College Station, Texas, USA). Study population A total of 370,196 samples were successfully collected from infants visiting health facilities across all regions in Kenya between January 2007 and July 2015 and tested in seven national laboratories. 362 samples had the same patient ID number, came from the same facility, were tested on the same date, and were from an individual of the same age and sex as another sam- ple; these were considered to be duplicate tests from the same infant and were excluded from the dataset. Due to improper DBS sample collection, packaging or labeling, 3228 samples were 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Mother to child transmission of HIV in Kenya rejected and were therefore excluded from the analysis. Patients with age listed as a negative value or greater than or equal to 2 years (n = 765) were excluded from the main analysis; a sec- ondary analysis was conducted setting age to missing in these individuals. Patients with miss- ing data on other predictor variables were included in a missing indicator category. 365,841 patients had a valid test result and were included in the tables and primary analyses. Quality assurance Kenya has a national quality assurance program for the seven molecular laboratories. The pro- gram supports external quality assurance, training/registration of users, and harmonisation of operating procedures. All seven labs are enrolled and participate in the CDC Atlanta profi- ciency testing program. They also participate in a quarterly inter-laboratory EQA programme. Ethical approval This study was approved by the Scientific Steering Committee and Ethical Review Committee of the Kenya Medical Research Institute (Protocol SSC No. 1066). The need for participant consent was waived. No identifiable patient information was included in the database, but facility-specific patient identification numbers were included. NASCOP considers the EID ser- vice as a national HIV program and a standard of care. Results Patient characteristics and univariate and multivariate logistic regression analyses are pre- sented in Table 1. Of the 365,841 infants in the dataset, 8.9% (32,441) were found to be HIV- infected. 50.4% of samples (of those with known sex) were from females. Data on sex was unavailable for 10.9% of samples. The median age at testing was 1.8 months (IQR: 1.5–5.0 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Mother to child transmission of HIV in Kenya Table 1. (Continued) Factor Univariate Multivariate N % HIV+ OR 95%CI OR 95%CI No breastfeeding 37,856 9.7% 1.63 1.49– 1.80 0.95 0.88– 1.02 Missing 104,528 10.6% 1.81 1.70– 1.92 0.98 0.92– 1.04 Entry point Comprehensive clinic care/patient support center 54,257 9.1% 1.27 1.17– 1.38 1.10 1.03– 1.17 Maternal and child health/PMTCT 208,834 7.3% Ref Ref Maternity 2,505 9.0% 1.26 1.05– 1.51 1.12 0.94– 1.33 Outpatient department 10,216 18.7% 2.93 2.68– 3.19 1.65 1.52– 1.79 Other 9,753 12.6% 1.83 1.64– 2.05 1.15 1.03– 1.28 Pediatric Ward 3,381 32.9% 6.23 5.26– 7.39 3.32 2.82– 3.92 Missing 76,895 10.2% 1.44 1.34– 1.55 0.92 0.87– 0.99 Test year 2007 1,548 31.5% 6.61 5.90– 7.41 3.72 2.85– 4.85 2008 18,257 10.1% 1.62 1.52– 1.72 1.00 0.89– 1.13 2009 19,116 11.7% 1.91 1.81– 2.03 1.37 1.21– 1.54 2010 57,584 10.7% 1.72 1.65– 1.81 1.12 1.03– 1.22 2011 58,213 10.0% 1.61 1.53– 1.69 1.08 0.99– 1.18 2012 55,063 8.2% 1.29 1.23– 1.36 0.91 0.86– 0.97 2013 55,070 8.0% 1.25 1.19– 1.31 0.99 0.94– 1.06 2014 58,495 7.1% 1.10 1.05– 1.16 0.96 0.91– 1.02 2015 42,495 6.5% Ref Ref Univariate and multivariable logistic regression accounting for clustering by health facility. The multivariable model was adjusted for all factors in this table. The missing indicator method was used to account for missing data. Abbreviations: AZT: zidovudine. 3TC: lamivudine. Sd: single dose. NVP: nevirapine. HAART hi hl ti ti t i l th PMTCT ti f th t hild t i i f HIV Univariate and multivariable logistic regression accounting for clustering by health facility. The multivariable model was adjusted for all factors in this table. The missing indicator method was used to account for missing data. Abbreviations: AZT: zidovudine. 3TC: lamivudine. Sd: single dose. NVP: nevirapine. HAART: highly active antiretroviral therapy. PMTCT: prevention of mother-to-child transmission of HIV. Univariate and multivariable logistic regression accounting for clustering by health facility. The multivariable model was adjusted for all factors in this table. The missing indicator method was used to account for missing data. Abbreviations: AZT: zidovudine. 3TC: lamivudine. Sd: single dose. NVP: nevirapine. HAART: highly active antiretroviral therapy. PMTCT: prevention of mother-to-child transmission of HIV. months). Age at testing did not differ substantially between the sexes. 51.5% mothers were confirmed to be on highly active antiretroviral therapy (HAART; out of those with data on treatment regimen) and an additional 4.7% on interrupted HAART. Mother to child transmission of HIV in Kenya Table 1. Predictive factors for HIV positivity in 365,841 infants testing for HIV in Kenya. Factor Univariate Multivariate N % HIV+ OR 95%CI OR 95%CI Gender Male 161,682 8.4% Ref Ref Female 164,259 8.9% 1.06 1.04– 1.09 1.08 1.05– 1.11 Missing 39,900 10.4% 1.26 1.19– 1.32 1.11 1.03– 1.20 Age group 0–6 weeks 47,968 10.1% 2.43 2.25– 2.63 1.72 1.59– 1.86 6 weeks-2 months 138,494 4.4% Ref Ref 2–6 months 86,877 9.4% 2.25 2.12– 2.39 1.76 1.68– 1.86 6–9 months 35,888 11.9% 2.93 2.73– 3.13 2.08 1.95– 2.22 9–18 months 46,834 16.3% 4.23 3.96– 4.51 3.05 2.89– 3.21 18–24 months 4,599 21.6% 5.98 5.36– 6.67 4.26 3.87– 4.69 Missing 5,181 8.3% 1.96 1.73– 2.22 1.47 1.26– 1.72 PMTCT Intervention AZT from 14 weeks of pregnancy or later; AZT+3TC+sdNVP during labor; AZT +3TC for 7 days postpartum 41,394 6.5% 1.13 1.06– 1.21 1.13 1.06– 1.20 HAART 125,166 5.8% Ref Ref Interrupted HAART (HAART until end of breastfeeding) 11,437 7.0% 1.22 1.09– 1.37 1.20 1.08– 1.33 None 43,837 15.2% 2.90 2.67– 3.16 1.92 1.79– 2.06 Other 12,711 8.6% 1.53 1.37– 1.72 1.23 1.10– 1.36 Single dose NVP Only 8,633 9.1% 1.62 1.45– 1.80 1.32 1.20– 1.45 Missing 122,663 10.7% 1.94 1.80– 2.10 1.53 1.43– 1.63 Infant Prophylaxis AZT+3TC for 7 days only 128 2.3% 0.57 0.17– 1.90 0.40 0.12– 1.35 NVP during breastfeeding 50,505 6.3% 1.58 1.45– 1.73 1.30 1.20– 1.41 NVP for 6 weeks (Mother on HAART or not breastfeeding) 67,429 4.0% Ref Ref None 19,889 21.7% 6.56 5.83– 7.39 2.76 2.51– 3.05 Other 5,689 8.4% 2.18 1.82– 2.60 1.46 1.25– 1.70 Single dose NVP+AZT+3TC 3,436 8.1% 2.09 1.74– 2.50 1.45 1.22– 1.73 Single dose NVP Only 21,541 5.9% 1.49 1.34– 1.65 1.19 1.09– 1.30 Missing 197,224 10.2% 2.71 2.48– 2.95 1.56 1.43– 1.70 Breastfeeding Exclusive breastfeeding 167,166 6.2% Ref Ref Mixed breastfeeding 23,682 15.2% 2.73 2.48– 3.00 1.39 1.30– 1.49 Breastfeeding, unspecified 32,609 11.3% 1.94 1.81– 2.08 1.13 1.05– 1.20 (Continued) (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 18.0% were on no treat- ment. 11.8% of infants were not on any HIV prophylaxis (among those with known prophylaxis status). Exclusive breastfeeding (EBF) was provided to 64.0% of infants (out of those with breastfeeding information), while 14.5% of infants were not breastfed. Mixed breastfeeding was practiced by 9.1% of infants, although only 60.4% of those infants were under 6 months of age and it is unknown whether the older children were previously exclusively breastfed and cur- rently complementary feeding or were previously mixed breastfed. In addition, mixed breast- feeding decreased over time. 12.5% of infants were breastfed but did not have information on whether it was exclusive or mixed (hereafter referred to as “unspecified breastfeeding”). PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 6 / 15 Mother to child transmission of HIV in Kenya Approximately 72.3% of samples were collected via PMTCT/maternal and child health (MCH) clinic samples, while 18.8% were collected from CCC/ PSC, 3.5% from OPD, 1.2% from the pediatric ward and 0.9% from the maternity ward. The vast majority of samples across entry points were from patients ages 1 to 2 months, although the entry points with the most extreme peak in that age range were from PMTCT/MCH and CCC/PSC. In multivariate analyses, the odds ratio for HIV infection in females was 1.08 (95% CI 1.05– 1.11) compared to males. When examining the sex differences in infection by age, there was a statistically significant difference between positivity in females and males through 6 months but no statistically significant difference in older children (p-value for interaction = 0.01). 10.1% of newborns less than 6 weeks of age were found to be infected with HIV. Compared to infants being tested at 6 weeks to 2 months (the majority of infants), the odds ratio of testing positive for HIV infection was 1.72 (95% CI: 1.59–1.86) in infants aged 0 to 6 weeks. However, most of those infants were listed as testing at 0 months, which may have represented a data entry error; sensitivity analyses in which these infants were considered to be missing age showed an attenuated association in this age group [OR changed to 1.28 (95% CI: 1.18–1.40)]. The percentage testing positive for infection was the lowest at 6 weeks to 2 months when the largest number of infants were tested, and then increased with age to 21.6% at 18 months to 2 years (76.3% of infants in this category were 18.0–18.5 months old). Compared to infants being tested at 6 weeks to 2 months, the odds ratio of testing positive for HIV infection was 1.76 (95% CI: 1.68–1.86) in infants 2 to 6 months, increasing to 4.26 (95% CI: 3.87–4.69) in infants 18–24 months. Infants whose mothers were not taking ART had nearly double the odds of the HIV infection when compared to those whose mothers were on treatment (multi- variable-adjusted OR 1.92; 95% CI 1.79–2.06). For infants not on any prophylaxis, the odds ratio for HIV infection was 2.76 (95% CI 2.51–3.05) when compared to those who were on nevirapine (NVP) for six weeks whose mothers were on ART or were not breastfeeding. The percentage of infants on nevirapine for 6 weeks that tested positive was 4.0% vs. 21.7% of infants on no prophylaxis. Among infants whose mothers were on HAART, the differences in HIV infection by infant prophylaxis regimen were smaller but still substantial; 3.3% for those on nevirapine for 6 weeks and mother breastfeeding while 11.8% for those on no prophylaxis. Infants who were categorized as mixed breastfed had 1.39 (95% CI: 1.30–1.49) times the odds of HIV infection when compared to exclusively breastfed infants. There was not a statistically significant difference in HIV infection between exclusively breastfeeding and not breastfeed- ing. The highest yield for detecting infected patients was observed in the pediatric ward and the outpatient department. For infants who sought health services at the pediatric ward, the odds ratio of HIV infection was 3.32 (95% CI 2.82–3.92), while for those seen at the outpa- tient departments the odds ratio was 1.65 (95% CI 1.52–1.79) when compared to the MCH- PMTCT. However, given the difference in the volume of patients tested in each entry point, only 3.4% (5.3% in 2014–2015) of the HIV-infected infants were identified via the pediatric ward and 5.9% (9.0% in 2014–2015) through the outpatient department, whereas 46.9% (55.8% in 2014–2015) of patients were identified through MCH/PMTCT. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Mother to child transmission of HIV in Kenya Table 2. Risk factors for HIV positivity in 2007–2010 (N = 96,505; 11.1% HIV-positive) vs. 2011–2013 (N = 168,346; 8.8% HIV-positive) vs. 2014–2015 (N = 100,990; 6.9% HIV-positive) among infants testing for HIV in Kenya. I d l bi i b ti l b h i f HIV t i i th dj t d dd ti In a model combining sub-optimal behaviors for HIV transmission, the adjusted odds ratio of HIV infection for infants who did not receive infant prophylaxis, were mixed fed, and whose mothers did not receive any form of antiretroviral therapy (N = 2,550) was 7.10 (95%CI 5.64–8.93) compared to infants who received nevirapine for 6 weeks, were not breastfed, and whose mothers were on HAART (N = 3,325), and the odds were similar when compared to infants who received nevirapine, were exclusively breastfed, and mothers were on HAART [OR: 6.91 (95% CI: 6.14–7.78); S1 Table]. Table 2 shows multivariable-adjusted model results stratified by early and recent time period. The percentage of infants receiving an EID test who tested positive for HIV infection PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 7 / 15 Mother to child transmission of HIV in Kenya Table 2. (Continued) Factor 2007–2010 2011–2013 2014–2015 N % HIV + OR 95%CI N % HIV + OR 95%CI N % HIV + OR 95%CI Maternal and child health/PMTCT 45,312 9.8% Ref 93,876 7.4% Ref 69,646 5.5% Ref Maternity 534 11.4% 1.19 0.82– 1.72 1,191 9.2% 1.07 0.86– 1.33 780 7.2% 0.96 0.71– 1.30 Outpatient department 1,210 15.4% 1.44 1.18– 1.75 6,537 16.9% 1.55 1.38– 1.73 2,469 25.2% 1.81 1.60– 2.04 Other 4,444 11.3% 1.09 0.96– 1.24 3,753 14.8% 1.40 1.19– 1.66 1,556 10.7% 0.79 0.58– 1.08 Pediatric Ward 1,128 26.1% 2.63 2.09– 3.32 1,337 33.7% 3.53 2.86– 4.35 916 40.1% 3.90 3.08– 4.93 Multivariable logistic regression accounting for clustering by health facility. A separate model was run for each time period. The multivariable model was adjusted for all factors in this table plus test year. The missing indicator method was used to account for missing data; to save space, missing categories are not shown. Abbreviations: AZT: zidovudine. 3TC: lamivudine. Sd: single dose. NVP: nevirapine. HAART: highly active antiretroviral therapy. PMTCT: prevention of mother-to-child transmission of HIV Multivariable logistic regression accounting for clustering by health facility. A separate model was run for each time period. The multivariable model was adjusted for all factors in this table plus test year. The missing indicator method was used to account for missing data; to save space, missing categories are not shown. Abbreviations: AZT: zidovudine. 3TC: lamivudine. Sd: single dose. NVP: nevirapine. HAART: highly active antiretroviral therapy. PMTCT: prevention of mother-to-child transmission of HIV https://doi.org/10.1371/journal.pone.0183860.t002 decreased over time; in 2007–2010, positivity was 11.1% while in 2014–2015, positivity was 6.9%. Most associations were consistent over time, although some associations varied in their strength. The proportion of mothers not on ART during pregnancy (among those with treatment data) decreased dramatically over time, from 30.1% in 2007–2010 to 9.1% in 2014–2015. The percentage of infants born to mothers on HAART who were HIV-infected decreased from 9.1% in 2007–2010 to 4.4% in 2014–2015. Infant prophylaxis was not widely available in 2007– 2010 so data was not routinely collected, but between 2011–2013 and 2014–2015 the propor- tion of infants receiving no prophylaxis decreased from 13.7% to 9.8%. Factor 2007–2010 2011–2013 2014–2015 N % HIV + OR 95%CI N % HIV + OR 95%CI N % HIV + OR 95%CI Gender Male 37,305 10.8% Ref 77,365 8.4% Ref 47,012 6.7% Ref Female 37,639 11.4% 1.07 1.03– 1.12 78,483 9.0% 1.09 1.05– 1.13 48,137 6.9% 1.08 1.02– 1.14 Age group 0–6 weeks 23,035 11.2% 1.50 1.31– 1.71 17,115 9.9% 1.76 1.54– 2.01 7,818 7.2% 1.79 1.59– 2.03 6 weeks-2 months 23,039 6.8% Ref 63,735 4.6% Ref 51,720 3.1% Ref 2–6 months 24,858 11.1% 1.57 1.42– 1.72 39,968 9.2% 1.71 1.60– 1.82 22,051 7.9% 2.05 1.88– 2.23 6–9 months 11,959 13.6% 2.02 1.82– 2.24 17,094 11.1% 1.89 1.75– 2.04 6,835 11.0% 2.21 1.98– 2.46 9–18 months 11,455 16.6% 2.55 2.25– 2.89 25,273 15.3% 2.76 2.58– 2.96 10,106 18.5% 4.05 3.64– 4.50 18–24 months 982 20.6% 3.37 2.70– 4.20 2,632 19.3% 3.60 3.17– 4.08 985 28.7% 7.02 5.81– 8.49 PMTCT Intervention AZT from 14 weeks of pregnancy or later; AZT+3TC+sdNVP during labor; AZT+3TC for 7 days postpartum 8,559 8.7% 1.06 0.95– 1.18 23,819 6.0% 1.15 1.05– 1.26 9,016 5.8% 1.22 1.07– 1.39 HAART 21,979 9.1% Ref 49,017 5.9% Ref 54,170 4.4% Ref Interrupted HAART (HAART until end of breastfeeding) 805 10.2% 0.90 0.71– 1.14 6,691 6.9% 1.24 1.09– 1.41 3,941 6.5% 1.25 1.06– 1.48 None 15,254 12.0% 1.44 1.29– 1.61 21,448 14.8% 2.06 1.86– 2.28 7,135 23.3% 2.40 2.16– 2.67 Other 1,042 13.0% 1.24 1.01– 1.52 8,428 8.2% 1.19 1.04– 1.37 3,241 8.4% 1.46 1.25– 1.71 Single dose NVP Only 3,100 10.1% 1.22 1.04– 1.43 4,307 9.0% 1.50 1.31– 1.71 1,226 6.9% 1.20 0.93– 1.56 Infant Prophylaxis AZT+3TC for 7 days only 0 - - 25 0.0% - - 103 2.9% 0.47 0.13– 1.68 NVP during breastfeeding 20 5.0% 0.59 0.03– 10.16 28,385 6.5% 1.26 1.14– 1.39 22,100 6.0% 1.26 1.13– 1.40 NVP for 6 weeks (Mother on HAART or not breastfeeding) 13 7.7% Ref 27,478 4.4% Ref 39,938 3.8% Ref None 35 11.4% 1.23 0.16– 9.34 11,731 20.8% 2.57 2.29– 2.89 8,123 23.0% 2.30 2.04– 2.58 Other 6 16.7% 1.52 0.08– 30.12 2,567 10.4% 1.51 1.22– 1.87 3,116 6.8% 1.28 1.04– 1.57 Single dose NVP+AZT+3TC 36 0.0% - - 2,349 6.8% 1.19 0.97– 1.45 1,051 11.2% 2.05 1.55– 2.71 Single dose NVP Only 19 0.0% - - 13,256 6.4% 1.23 1.10– 1.37 8,266 5.0% 1.05 0.92– 1.20 Breastfeeding Exclusive breastfeeding 13,199 12.4% Ref 83,080 6.5% Ref 70,887 4.7% Ref Mixed breastfeeding 5,112 9.7% 0.74 0.65– 0.83 12,330 14.6% 1.39 1.27– 1.52 6,240 21.1% 1.84 1.67– 2.03 Breastfeeding, unspecified 18,491 10.3% 0.79 0.71– 0.89 9,832 13.0% 1.25 1.13– 1.37 4,286 12.2% 1.17 1.03– 1.33 No breastfeeding 11,134 9.4% 0.66 0.59– 0.74 19,213 9.9% 1.02 0.93– 1.12 7,509 9.8% 1.03 0.88– 1.20 Entry point Comprehensive clinic care/patient support center 11,539 12.7% 1.22 1.12– 1.32 26,043 8.9% 1.03 0.94– 1.13 16,675 6.9% 1.01 0.91– 1.11 (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 8 / 15 The higher prevalence of HIV infection among older children compared to younger chil- dren was stronger after scale-up of Option B+ (adjusted OR comparing children aged 18–24 months to children 6 weeks to 2 months in 2014–2015: 7.02; 95% CI: 5.81–8.49). Mixed breastfeeding decreased from 10.7% of infants in 2007–2010 (plus 38.6% unspecified breastfeeding) to 7.0% (plus 4.8% unspecified breastfeeding) in 2014–2015. There may have been differences in the way that the breastfeeding categories were defined/reported over the years, as in 2007–2010”mixed breastfeeding” appeared to be protective compared to exclusive breastfeeding whereas in more recent years it was harmful. Not breastfeeding appeared protec- tive compared to exclusive breastfeeding in 2007–2010 (OR: 0.66; 95% CI: 0.59–0.74) while there was no association in 2014–2015. Finally, associations between entry point and HIV have changed over time, particularly in that the pediatric ward in more recent years had an extremely high yield for identifying infected patients. There were no substantial differences in the study findings when patients with age listed as a negative value or greater than or equal to 2 years were included in the analysis with age listed as missing. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Discussion For this study, we analysed a comprehensive national dataset from Kenya covering nearly a decade to describe the determinants of HIV status in infants below 2 years of age at testing. We observed greater odds of HIV infection in females, older children, infants whose mothers received no PMTCT intervention, infants receiving no HIV prophylaxis, and infants who were 9 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Mother to child transmission of HIV in Kenya mixed breastfed. Exposure to risky practices (mother not on HAART, no infant prophylaxis, mixed breastfeeding) was associated with a seven-fold higher odds of HIV-positivity compared to exposure to recommended practices (mother on HAART, infant on nevirapine for six weeks, no breastfeeding). With PMTCT/EID program growth and more patients in care, there has been a decrease in risky practices over time, but even the recent data suggests that some infants are not reached in time for prevention; these findings would likely be more striking in other countries in sub-Saharan Africa where PMTCT programs are not as strong. Finally, the pediatric ward and outpatient department had the highest testing yield across entry portals, but most HIV-infected infants were identified through PMTCT/MCH programs. Most importantly, many infants were not identified as infected until older ages and received sub-optimal prevention practices (lack of HAART for mother, infant prophylaxis, exclusive breastfeeding or exclusive replacement feeding). Option B+ has been scaled up in recent years and the number of new pediatric infections in Kenya has decreased by 29% from 2009 to 2014 [4]. Yet hundreds to thousands of infants are still becoming infected each year because their mothers are not enrolled in care. Barriers to testing and care include distance to health facility and transportation costs, facility inefficiencies, such as stock-outs and long wait times, and per- sistent shamefulness and stigma[15]. Expanded testing efforts in adolescent and early adult women and increased HIV self-testing availability may help to expand case-finding in women of childbearing age.[16] Many pregnant women do not come to health facilities for antenatal care until late in pregnancy[17] if at all; a high proportion of women deliver outside of facili- ties. HIV-infected women who deliver outside of facilities tend to have lower income and be less educated and less likely to be on treatment[18], meaning that their infants are at especially high risk. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Discussion Yet a recent study among women in Nairobi found that fear of stigma and discrimination around HIV was a persistent barrier to exclusive breastfeeding[33]. In this study, authors con- cluded that health education and counseling as well as male partner support helped to mitigate these issues. Other studies have shown that intensive counseling is a viable way to increase EBF in resource-limited settings[34], although in an intervention study in Nairobi, breastfeed- ing counseling alone did not increase EBF[35]. Regardless, there is more work to be done to reduce mixed breastfeeding in infants younger than 6 months. In this study we found that HIV positivity did not differ significantly between EBF and exclusive replacement feeding (ERF) after adjustment for other factors, even when adjusted for age. This was surprising, as exclusive replacement feeding should significantly reduce HIV transmission. It is possible that due to stigma, or the costs associated with ERF, some of those who said they provided replacement feeding for their infants also breastfed from time to time, leading to some measurement error in this category. Infants who entered care and treatment through outpatient departments or the pediatric wards were more likely to be HIV-infected than those entering through any other service point. This finding is not surprising, as those infants come to hospital due to illnesses which may include, or could be related to, HIV, and supports the latest Kenyan guidelines stating that all children presenting for care should be offered an HIV test[36]. In addition, testing in these infants was likely offered in a targeted fashion; for example, symptomatic infants were more likely to be tested. The high proportion of infected patients suggests that these entry points may be important for expanded infant testing to identify more HIV-exposed infants that are not reached by routine testing through PMTCT programs. In the future, EID detection efforts could be expanded further to better cover patients in alternative entry points such as OPD and pediatric wards to identify those who are missed by PMTCT testing programs, given the high yield in these areas. This is in line with current WHO recommendations, which state that in settings with generalized epidemics, HIV testing should be performed on infants with unknown HIV status who are admitted to inpatient and nutrition wards and offered to infants with unknown HIV status at outpatient and immunization clinics[37]. Increased resources for testing at these portals may be important. Discussion Pilot interventions that have been shown to improve PMTCT program coverage, retention, and quality include mHealth tools[19] such as SMS[20], rapid results initiatives[21], systems engineering approaches[22], and efforts to reduce health provider absenteeism[23]; these could be considered in areas struggling with program performance. In addition, there likely remain inconsistencies with health provider care among women and infants enrolled in PMTCT programs. A case-control study in Western Kenya observed that Infants were more likely to be infected with HIV if their provider did not follow maternal and infant ART guide- lines[24]. Ensuring reinforcement of guidelines through periodic re-trainings and supportive supervision, as well as strong supply chain management systems will be important to continue to strengthen the PMTCT program. We observed HIV infections in some infants despite their mothers being on HAART; the percentage of infected infants in this category declined over time as drug regimens changed to 4.4% in 2014–2015. Possible reasons for transmission in these infants include late treatment initiation in mothers, treatment nonadherence, lower efficacy on certain treatments, drug resistance, and stock-outs. This database does not contain information on these factors and therefore we are only able to speculate on the relative contribution of each one. Some mothers had interrupted HAART, indicating that non-adherence to treatment may be another barrier to care and testing. Strong adherence support programs are also important to minimize trans- mission during pregnancy. Innovative methods including community adherence groups[25] and transport reimbursement for low-income patients[26] may help to improve drug adher- ence and retention in PMTCT programs. New treatment regimens for mothers and prophy- laxis options for infants will likely influence transmission rates in the future. Thousands of infants are still exposed to the sub-optimal practice of mixed breastfeeding each year in Kenya, and these infants had worse outcomes compared to exclusively breastfed infants and infants not given any breastmilk. The dangers posed by mixed feeding have been well-described in the literature[27–31]. Besides pointing to a gap in program coverage, these findings illustrate a need for enhanced education of the public that this practice is detrimental. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 10 / 15 Mother to child transmission of HIV in Kenya The Kenyan National Demographic and Health Survey noted that by 2014 exclusive breast- feeding had increased to 61% in infants less than 6 months, a near doubling from 32% in 2009 [32]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Discussion However, it is important to note that the greatest numbers of cases are still detected through MCH/PMTCT programs, despite the lower yields in these portals, and these programs should continue this important work. Of note, a number of studies on improving Kenya’s EID program are recently completed or still ongoing[38–41]. Through continued national monitoring efforts in Kenya and scaling up such efforts in other countries, we can continue to work to minimize transmission of HIV to infants. Overall, we observed slightly higher odds of positivity in female infants compared to male infants from birth to six weeks. While this small difference could be due to chance, this phe- nomenon has been described previously and there are theories that female infants may be more susceptible to HIV infection in the in-utero and peripartum period [42–44]. While the exact mechanism is not known, hypotheses include chromosomal affinity and a higher rate of male intrauterine death among HIV-infected fetuses, leading to females being more likely to be HIV infected at birth. In this database, females did not appear to be testing later than males, so age at testing did not seem to explain the imbalance. Confirmation in other studies and fur- ther investigation is needed to better understand this phenomenon. This study has a number of limitations. Firstly, it is observational and cross-sectional; thus, causal relationships cannot be inferred. We did not have access to data on all predictors of infant HIV transmission and all potential confounders. In addition, the study data come from a national health registry system and were not collected for the purpose of a research study; PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 11 / 15 Mother to child transmission of HIV in Kenya thus, there were large amounts of missing data for some indicators and some inaccuracies are possible. Along those lines, given the vagueness of the patient identification numbers at some facilities, we were unable to assess tests from the same individuals or calculate a seroconversion rate among patients with multiple tests. Strengthening patient identification systems and data entry would further maximize the utility of this database. This analysis did not include out- come data on HIV-infected infants following diagnosis; linkage to care and retention in care for these infants is an important area for future research. Conclusions This study emphasizes the importance of national health registry systems to monitor the suc- cess of programs such as Option B+ and to inform testing for EID. Given the relative success of Kenya’s national program, the associations are likely to be even more striking in other coun- tries in sub-Saharan Africa with less successful PMTCT programs. We have confirmed risk factors previously seen on a smaller scale in this national dataset, and illustrated the impact of exposure to multiple risk factors overall and over time. The findings point to a need for resources to continue to reinforce best treatment and prophylaxis prescribing behavior, pro- mote exclusive breastfeeding, and test infants early in life and at testing portals with greater case-finding. Routine point of care testing will be important to dramatically reduce time to treatment initiation for HIV-infected infants. Most importantly, efforts must continue to reach patients that are not coming for PMTCT programs. Supporting information S1 Table. Sub-optimal behaviors in relation to HIV positivity in 365,841 infants testing for HIV in Kenya. Univariate and multivariate logistic regression accounting for clustering by health facility. The multivariable model was adjusted for sex, age, entry point, and year. The missing indicator method was used to account for missing data. Abbreviations: NVP: nevira- pine. HAART: highly active antiretroviral therapy. PMTCT: prevention of mother-to-child transmission of HIV. (DOCX) Discussion Finally, the data is based on infants who were tested for HIV, which, while covering >70% of exposed infants, does not include the infants outside of care who are at highest risk of HIV transmission. This also means that com- parisons between groups cannot be interpreted as nationally representative, do not necessarily represent transmission, and are in part a product of testing coverage. The strengths of this study include its large size, numerous risk factors examined, and comprehensiveness across Kenya over nearly a decade. Acknowledgments The authors wish to thank the Centers for Disease Control and Prevention (CDC), USAID/ PEPFAR, and UNITAID for funding HIV diagnostics in Kenya, as well as the Clinton Health Access Initiative for technical support. References 1. Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F, et al. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet. 2004; 364 (9441):1236–43. https://doi.org/10.1016/S0140-6736(04)17140-7 PMID: 15464184. 2. UNAIDS/WHO. UNAIDS Report on the Global AIDS Epidemic 2013. 3. Cooper ER, Charurat M, Mofenson L, Hanson IC, Pitt J, Diaz C, et al. Combination antiretroviral strate- gies for the treatment of pregnant HIV-1-infected women and prevention of perinatal HIV-1 transmis- sion. J Acquir Immune Defic Syndr. 2002; 29(5):484–94. 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Mechanism of cation binding to the glu- tamate transporter EAAC1 probed with mutation of the conserved amino acid residue Thr101. J Biol Chem. 2010; 285(23):17725–33. https://doi.org/10.1074/jbc.M110.121798 PMID: 20378543; PubMed Central PMCID: PMCPMC2878536. 12. Kageha S, Okoth V, Kadima S, Vihenda S, Okapesi E, Nyambura E, et al. Discrepant test findings in early infant diagnosis of HIV in a national reference laboratory in Kenya: challenges and opportunities for programs. J Trop Pediatr. 2012; 58(4):247–52. https://doi.org/10.1093/tropej/fmr076 PMID: 22052701. 13. Chang J, Omuomo K, Anyango E, Kingwara L, Basiye F, Morwabe A, et al. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in compari- son to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya. J Virol Methods. 2014; 204:25–30. https://doi.org/10.1016/j.jviromet.2014.03.010 PMID: 24726703; PubMed Central PMCID: PMCPMC4720144. 14. Lofgren SM, Morrissey AB, Chevallier CC, Malabeja AI, Edmonds S, Amos B, et al. Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote healthcare facilities. AIDS. 2009; 23(18):2459–66. https://doi.org/10.1097/QAD. 0b013e328331f702 PMID: 19741481; PubMed Central PMCID: PMCPMC2890230. 14. Project administration: Matilu Mwau, Lucy Kithinji. Resources: Matilu Mwau. Software: Matilu Mwau. Software: Matilu Mwau. Supervision: Matilu Mwau. Supervision: Matilu Mwau. Validation: Matilu Mwau. Validation: Matilu Mwau. Visualization: Matilu Mwau. Visualization: Matilu Mwau. Writing – original draft: Matilu Mwau, Priska Bwana, Caroline Boeke. Writing – review & editing: Matilu Mwau, Priska Bwana, Francis Ogollah, Samuel Ochieng, Catherine Akinyi, Maureen Adhiambo, Martin Sirengo, Caroline Boeke. Writing – review & editing: Matilu Mwau, Priska Bwana, Francis Ogollah, Samuel Ochieng, g g , , g , g, Catherine Akinyi, Maureen Adhiambo, Martin Sirengo, Caroline Boeke. Conceptualization: Matilu Mwau, Martin Sirengo. Conceptualization: Matilu Mwau, Martin Sirengo. Data curation: Matilu Mwau, Priska Bwana, Lucy Kithinji, Francis Ogollah, Samuel Ochieng, Catherine Akinyi, Maureen Adhiambo, Fred Ogumbo. Data curation: Matilu Mwau, Priska Bwana, Lucy Kithinji, Francis Ogollah, Samuel Ochieng, Catherine Akinyi, Maureen Adhiambo, Fred Ogumbo. Formal analysis: Matilu Mwau, Lucy Kithinji, Caroline Boeke. Formal analysis: Matilu Mwau, Lucy Kithinji, Caroline Boeke. 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183860 August 29, 2017 Mother to child transmission of HIV in Kenya Methodology: Matilu Mwau, Samuel Ochieng, Catherine Akinyi, Maureen Adhiambo, Fred Ogumbo. Methodology: Matilu Mwau, Samuel Ochieng, Catherine Akinyi, Maureen Adhiambo, Fred Ogumbo. 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https://openalex.org/W2515398420	https://lirias.kuleuven.be/bitstream/123456789/632286/2/Formanoy2016PAARI.pdf	English	null	Physical activity and relaxation in the work setting to reduce the need for recovery: what works for whom?	BMC public health	2,016	cc-by	12,219	Abstract Background: To recover from work stress, a worksite health program aimed at improving physical activity and relaxation may be valuable. However, not every program is effective for all participants, as would be expected within a “one size fits all” approach. The effectiveness of how the program is delivered may differ across individuals. The aim of this study was to identify subgroups for whom one intervention may be better suited than another by using a new method called QUalitative INteraction Trees (QUINT). Methods: Data were used from the “Be Active & Relax” study, in which 329 office workers participated. Two delivery modes of a worksite health program were given, a social environmental intervention (group motivational interviewing delivered by team leaders) and a physical environmental intervention (environmental modifications). The main outcome was change in Need for Recovery (NFR) from baseline to 12 month follow-up. The QUINT method was used to identify subgroups that benefitted more from either type of delivery mode, by incorporating moderator variables concerning sociodemographic, health, home, and work-related characteristics of the participants. Results: The mean improvement in NFR of younger office workers in the social environmental intervention group was significantly higher than younger office workers who did not receive the social environmental intervention (10.52; 95 % CI: 4.12, 16.92). Furthermore, the mean improvement in NFR of older office workers in the social environmental intervention group was significantly lower than older office workers who did not receive the social environmental intervention ( −10.65; 95 % CI: −19.35, −1.96). The results for the physical environmental intervention indicated that the mean improvement in NFR of office workers (regardless of age) who worked fewer hours overtime was significantly higher when they had received the physical environmental intervention than when they had not received this type of intervention (7.40; 95 % CI: 0.99, 13.81). Finally, for office workers who worked more hours overtime there was no effect of the physical environmental intervention. Conclusions: The results suggest that a social environmental intervention might be more beneficial for younger workers, and a physical environmental intervention might be more beneficial for employees with a few hours overtime to reduce the NFR. * Correspondence: erwintak2@gmail.com 1Netherlands Organisation for Applied Scientific Research TNO, Schipholweg 77, Leiden, The Netherlands Full list of author information is available at the end of the article Physical activity and relaxation in the work setting to reduce the need for recovery: what works for whom? Margriet A. G. Formanoy1, Elise Dusseldorp1,2,3, Jennifer K. Coffeng4, Iven Van Mechelen2, Cecile R. L. Boot4,5, Ingrid J. M. Hendriksen1,5 and Erwin C. P. M. Tak1* Margriet A. G. Formanoy1, Elise Dusseldorp1,2,3, Jennifer K. Coffeng4, Iven Van Mechelen2, Cecil Ingrid J. M. Hendriksen1,5 and Erwin C. P. M. Tak1* © 2016 The Author(s). 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. Formanoy et al. BMC Public Health (2016) 16:866 DOI 10.1186/s12889-016-3457-3 Formanoy et al. BMC Public Health (2016) 16:866 DOI 10.1186/s12889-016-3457-3 Background Not taking enough time to relax increased the NFR and was associated with exhaustion [14]. Furthermore, within a group of service employees, enjoyable and restful within workday breaks improved their need for recovery [15]. The results of the “Be Active & Relax” project showed that none of the delivery modes were effective in redu- cing the NFR [17]. Therefore, it was not recommended to implement the current interventions because the NFR did not significantly differ as a result of applying either delivery mode. And although some significant effects were found on work-related outcomes, these were only small (e.g., contextual performance, dedication, task per- formance, and absorption [21]). To identify subgroups for whom one intervention might be better suited than another, several statistical methods are available. Based on the characteristics of the partici- pants measured at baseline (so called moderator variables), the subgroups can be determined a priori (e.g., based on previous literature; also called confirmatory analysis) or a posteriori, that is, induced from the data (also called ex- ploratory analysis). The classical approaches to identify subgroups are analysis of variance with paired compari- sons, and multiple regression analysis with pre-defined interaction effects. A disadvantage of these methods is that they can handle only a small number of moderator vari- ables. For the situation of many potential moderator vari- ables that might interact with the treatment variable and no clear a priori hypotheses, several new methods were de- veloped in the last decade based on recursive partitioning (see [22] for an enumeration), among which QUalitative INteraction Trees (QUINT) [23, 24]. These new methods can handle a large number of potential moderators in the analysis. Most of these methods optimize treatment- subgroup interactions in general, while QUINT aims at only identifying subgroups that differ in direction of the treatment effect (so called qualitative treatment-subgroup interactions). Because our interest was in discovering such subgroups, QUINT was used in this study. However, some studies did not succeed in improving the need for recovery by a worksite health program [16] or the effect did not sustain in the long-term [17]. One possible cause of this phenomenon could be that the studies focused on the effect(s) of a worksite health pro- gram for all workers included in the study (i.e., the treat- ment main effect). However, the question is whether the assumption that a program is equally effective for all workers is realistic. Background Therefore, it might be very useful to define subgroups that may benefit more from a certain worksite health program. The present study focuses on the identification of such subgroups using data from the “Be Active & Relax” study. Work stress and work-related health problems are a major problem in modern organizations [1]. When chronically exposed to high levels of psychological job demands, job variety, little autonomy or support from others, it may lead to increased stress levels as well as health problems [1]. Work-related stress can cause substantial economic costs due to lost productivity and absenteeism [2]. A 2010 European survey (n = 21,703) showed that 28 % of the workers experienced work- related stress [3]. The worksite health program “Be Active & Relax” was developed to reduce the NFR in office workers [19] via the increase of daily physical activity and relaxation. The ef- fectiveness of the intervention was investigated in a trial using a 2 × 2 factorial design. The design factors were two different strategies of delivering the “Be Active & Relax” program, in other words, two delivery modes. One delivery mode was a “social environmental intervention”, which consisted of a counseling style that focused on behavioral change in groups and was derived from motivational interviewing at the individual level [20]. The other delivery mode was a “physical environmental inter- vention”, which consisted of encouraging physical activity and relaxing behavior by making changes in the physical environment. An early precursor for work-related health problems is a higher Need for Recovery (NFR; [4]). NFR is described as the need to recuperate and unwind from work- induced effort and represents the short-term workload effects of a day at work [5]. It can be seen as an inter- mediate variable between psychosocial work characteris- tics and work-related health problems [6, 7]. Several health behavior strategies were studied to re- duce work stress and work-related health problems, and/or NFR, among which physical activity and relax- ation. Evidence was found that physical activity is valu- able in unwinding from work [8, 9]. Sufficient physical activity, that is, three to five times a week for 15 to 45 min, resulted in lower work stress [10], reduced ab- senteeism [11], and improved job satisfaction [12]. In addition, participating in relaxation activities diverted the mind from work and improved self-esteem-feelings that are essential for recovery [13]. Trial registration: NTR2553 Keywords: Motivational interviewing, Social environmental intervention, Environmental modifications, Physical activity, Worksite health promotion, Relaxation program, Need for recovery, Work, QUINT * Correspondence: erwintak2@gmail.com 1Netherlands Organisation for Applied Scientific Research TNO, Schipholweg 77, Leiden, The Netherlands Full list of author information is available at the end of the article © 2016 The Author(s). 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. Formanoy et al. BMC Public Health (2016) 16:866 Formanoy et al. BMC Public Health (2016) 16:866 Page 2 of 15 Interventions The delivery modes of the physical activity and relax- ation intervention of the “Be Active & Relax” study were systematically developed [19] using a modified version of the intervention mapping protocol [25]. Two types of delivery modes were chosen: a social environmental intervention and a physical environmental intervention. g The research questions of the present study were: Background It may be that a “one size fits all” ap- proach is not suitable in this case. In other words, the effectiveness of an intervention may differ across indi- viduals. For example, Taris et al. [18] suggested that working overtime on a recurrent character may influ- ence the results of workplace health interventions in two different ways. First, as a result of working overtime, less time is available for recovery. Second, people who work overtime recurrently are prone to make “unhealthy choices” concerning lifestyle, such as smoking and con- suming high levels of alcohol. By incorporating (some of) these moderator variables into the design of the study, one can target the right population. Page 3 of 15 Page 3 of 15 Page 3 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Formanoy et al. BMC Public Health (2016) 16:866 The aim of this study was to identify which intervention in the “Be Active & Relax” study [19] - the social environ- mental or the physical environmental intervention-best suited which group of office workers. It was hypothesized that one or more moderator variables might influence the effect on the primary outcome measure, which was change in NFR from baseline to follow-up. Social environmental intervention (SEI) 1) Did the effect of the social environmental 1) Did the effect of the social environmental intervention differ for subgroups of workers and did the effect of the physical environmental intervention differ for subgroups of workers? Four group motivational interviewing sessions were con- ducted by the team leaders (i.e., after receiving a 2-day training) with the office workers of their own team. The main aim of the sessions was to stimulate physical activity and relaxation by group motivational interviewing ses- sions (e.g., during session number two, office workers were asked to fill in a worksheet stating their goals and subsequent rewards). The social environmental interven- tion was supported by a web-based social media platform. 2) More specifically, for both interventions, which subgroup of workers showed a positive effect (e.g., a better outcome in NFR) and which subgroup showed a negative effect (e.g., a worse outcome in NFR)? Study design and study population For this study, data were used from the “Be Active & Relax” study [19]. From September 2011 to September 2012, 329 office workers participated. They were all recruited at one business location of a Dutch financial service provider. All participants signed an informed consent. This study was a cluster-randomized controlled trial (with three data collection time points) to reduce the NFR in office workers using a physical activity and relaxation program. The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center (Amsterdam, The Netherlands). Add- itional details regarding the study design and methods of the “Be Active & Relax” study have been published else- where [19]. Methods Based on previous studies [26–32], the aim was to facili- tate daily physical activity and relaxation in the work en- vironment, by changing the coffee corners (by adding a bar with bar chairs and a giant wall poster visualizing a relaxing environment), the open office environment (exercise balls and curtains to reduce background noise), the meeting rooms (a standing table and a giant wall poster visualizing a relaxing environment), and the en- trance hall (a table tennis table, lounge chairs for infor- mal meetings, and footsteps on the floor to promote stair walking). The interventions PEI and the SEI are de- scribed in more detail elsewhere [19]. Study design and study population Outcome measure h The primary outcome measure of the study was NFR, which was assessed using the NFR after work scale [5]. The questionnaire for the participants was in Dutch. The scale is originally a Dutch scale and is validated for the Dutch population [5]. The scale has good reliability (Cronbach’s alpha = 0.88) and validity. The validity was demonstrated by estimating the associations of NFR with psychosocial risk factors (e.g., emotional load and physical exertion) [5]. Furthermore, it was shown that the NFR has satisfactory test-retest reliability (ICCs 0.68 to 0.80) and is sensitive to detect change in increase in work related fa- tigue due to the increase in working hours (effect size 0.40) [26]. The NFR after work scale is a sum score of 11 dichotomous items, expressed as a percentage, represent- ing short-term effects of a day at work. Example questions of this scale included: “I find it hard to relax at the end of a working day”, “when I get home from work, I need to be left in peace for a while”. A hundred percent NFR corre- sponds to a sum score of 11, and represents a very high need for recovery. Our outcome for the analyses is change A 2 by 2 full factorial design was applied, resulting in four experimental conditions (see Table 1). Condi- tions with the social environmental intervention are referred to as SEI+ and conditions without as SEI−. The same applies to the conditions with the physical environmental intervention (PEI+) or without (PEI−). Table 1 Two by two factorial design, the social environmental intervention (SEI) and the physical environmental intervention (PEI), including the number of participants per condition PEI Total PEI+ PEI− SEI SEI+ 63 94 157 SEI− 76 96 172 Total 139 190 329 Table 1 Two by two factorial design, the social environmental intervention (SEI) and the physical environmental intervention (PEI), including the number of participants per condition Formanoy et al. BMC Public Health (2016) 16:866 Page 4 of 15 Page 4 of 15 Page 4 of 15 in NFR from baseline to follow-up (at 12 months). The follow-up score at 12 months was taken to ascertain that the time period was long enough to be able to measure ef- fect of SEI and / or PEI. The follow-up score was sub- tracted from the baseline score. Moderator variables The “Be Active & Relax” study contained many potential moderator variables. From these variables, we selected the most relevant ones. These were chosen based on the expertise of the researchers, supplemented with modera- tors that were identified by a literature search. The search was performed in several databases (i.e., PubMed, PsycInfo and Picarta) using the following entries: work- site health promotion, intervention, exercise, workplace, relaxation, and need for recovery. This resulted in vari- ables concerning socio-demographic variables (e.g. age [33], sex, BMI), health and home related variables (e.g. physical activity in free time [34], mental health variables [35], detachment at home) and work related variables (e.g. vitality, team commitment, organizational commit- ment). For an overview, see Appendix 1. Their univariate effects on NFR were analyzed [16]. The algorithm of QUINT starts with all persons in one node (the root node). Then it searches for a moderator that optimizes the qualitative treatment-subgroup inter- action. The search is performed among all possible mod- erators, all possible split points on each moderator and all possible assignments of the leaves to the subgroups. The best combination of moderator, split point, and as- signment is used to split the total group into two nodes (the “current” leaves). For the next splits, the procedure is repeated in each of the current leaves, and now the combination of moderator, split point, current leaf node, and assignment of the new leaves to the partition classes is chosen that optimizes the qualitative treatment- subgroup interaction. Outcome measure h A positive score means an improvement and represents an absolute reduction in NFR from baseline to 12 months. The minimal relevant difference on the NFR after work scale was set at 12 [26]. subgroups of persons. This implies that the treatment ef- fect may be positive for one subgroup, and negative for another subgroup. As such, these types of interactions are essential for optimal treatment assignment. In a QUINT analysis, three types of subgroups are dis- tinguished that are involved in (a) qualitative treatment- subgroup interaction(s). We will elucidate these sub- groups for one of our treatment factors, that is, the social environmental intervention with categories SEI+ (n = 157) and SEI−(n = 172). A QUINT analysis parti- tions the total group of office workers in the following types of subgroups (i.e., partition classes): 1) a group of persons for whom SEI+ has on average a better outcome than SEI−; 2) a group for whom SEI−has a better out- come than SEI+, and 3) a group for whom it does not make any difference. The partitioning criterion of the QUINT algorithm takes into account the difference in treatment outcome and the number of participants in subgroups 1 and 2. The difference in treatment outcome can be specified as a treatment effect size (i.e., Cohen’s d) or as a crude difference in means; the corresponding partitioning criterion is called “effect size criterion” or “difference in means criterion”, respectively. Statistical analyses Introduction of QUINT The best pruned tree is the smallest tree that satisfies the one-standard-error rule, that is, it has a bias-corrected criterion value higher or equal to the maximum bias-corrected criterion value minus its stand- ard error. The pruning procedure is described in detail in Appendix 2 of an earlier paper [23]. In an extensive simulation study, it was shown that the inferential errors (Type I and Type II errors) of QUINT were small if the total sample size was higher or equal to 400, dmin equaled 0.30, and the true treatment effect size was large (i.e., \|d\| ≥1). If the sample size was lower or equal to 300, the recovery of tree complexity was acceptable only for smaller trees (i.e., trees with one or two splits) [23]. intervention. In both analyses, improvement in NFR was used as the outcome variable and all moderator variables were used as splitting candidates. The number of office workers used in this analysis was 304 (25 of the 329 of- fice workers were deleted due to missing values on one or more moderators). In the final QUINT analyses, the variable “general health” was deleted from the set of moderator variables, because eight office workers had a missing value only on this variable and this variable did not appear to be important (i.e., it was not selected as splitting variable). By deleting “general health”, the number of office workers in the final analysis increased to 312. As was mentioned before, two partitioning criteria can be used in a QUINT analysis, either the effect size criter- ion or the difference in means criterion. Our outcome measure can be treated as ordinal (i.e., ratings), as well as numeric (i.e., percentages); therefore, in a first series of QUINT analyses, the effect size criterion was used, and in a second series, the difference in means criterion was used. In this way, it could also be checked whether the solution was stable (i.e., the same) or not. In both series of analyses, a value of 25 was used as minimal sample size per intervention in a leaf, dmin was set at 0.299, and default values of the weights of the partition- ing criterion were used (see supplementary materials in Appendix 1). A large number of bootstrap samples (i.e., B = 1000) was used and the one-standard-error pruning rule. Statistical analyses Introduction of QUINT Using a new technique like QUINT has some advan- tages. Compared to the more classical approach of ana- lysis of variance (where the “one size fits all” paradigm is predominate), one advantage of the QUINT method is its ability to identify subgroups that differ in direction of the intervention effect. The results of QUINT give prac- tical indications on how to optimally assign workers to an intervention. Other advantages are that QUINT can handle a large number of moderator variables (in this study, 25 in total) and can easily identify higher order intervention-subgroup interactions, both of which are not possible when using the classical post hoc analysis. Furthermore, the bias-corrected pruning procedure of the method guarantees that no spurious interaction ef- fects will be found. The splitting process stops if the value of the partition- ing criterion cannot be increased anymore. Furthermore, several stopping criteria are taken into account during the splitting process, among which a qualitative inter- action condition (i.e., after the first split, the absolute value of the standardized mean difference in treatment outcome in each of the two leaves should exceed a crit- ical minimum value (dmin; the default value is 0.30; this can be seen as a check of whether a qualitative inter- action is present in the data), and a minimal sample size per treatment (i.e., in each leaf, a minimum number of participants is in each treatment group for reliable esti- mation of the treatment means; by default this number is 10 % of the treatment group sample size). After fitting the full tree (with the total number of leaves as L), the tree is pruned back using a bootstrap-based bias- correction procedure [22]. This procedure results in QUINT is based on a binary recursive partitioning algo- rithm, which is an algorithm that recursively splits the data in two groups and thus resulting in a binary tree. The partitioning criterion of QUINT maximizes qualitative treatment-subgroup interactions. In general, treatment- subgroup interactions imply that the effect of a treatment variable on an outcome variable depends on the levels of (an) other variable(s). These levels define subgroups of persons. In case of qualitative treatment-subgroup inter- actions, the treatment effect differs in direction for specific Formanoy et al. BMC Public Health (2016) 16:866 Page 5 of 15 Page 5 of 15 bias-corrected criterion values for trees with number of leaves is 1 to L. Application of QUINT to the data of the “Be Active & Relax” study The QUINT analyses were performed using the R-package “quint” [24, 36] in the R version 3.0.2 (R Core Team, 2013). Before starting these analyses, two pre-conditions were tested. The first condition of QUINT is that each person is randomly assigned to an intervention group; however, in the “Be Active & Relax” study, the social en- vironmental intervention was randomized at department (i.e., different service departments of a financial service provider) level, and the physical environmental interven- tion was stratified on department level, i.e., one stratum with environmental modifications and the other stratum without environmental modifications. To check if the nesting of office workers within depart- ments could be disregarded, the intra cluster correlation coefficient (ICC) was computed, using improvement in NFR as outcome variable, and the departments as clusters. The ICC was 0.02, indicating that the amount of variance attributed to the departments was very low, and could be neglected. The second condition of QUINT is that the data include only 2 intervention groups, whereas the “Be Active & Relax” study concerned a 2 by 2 design, implying four intervention groups. A re-analysis of the data of the “Be Active & Relax” study using a full factorial analysis of variance (i.e., including main effects of the social en- vironmental intervention and the physical environmental intervention, and the interaction effect between the inter- ventions) with improvement in NFR as outcome variable, revealed that the interaction effect was not significant (nor the two main effects). This result showed that the effect of the social environmental intervention on NFR did not de- pend on the physical environmental intervention. This allowed us to treat the two factors (i.e., the social environ- mental and the physical environmental intervention) sep- arately (e.g., analogous to the approach of Raveaud [37]). Statistical analyses Introduction of QUINT Furthermore, independent t-tests were performed in each leaf of the pruned tree to test the difference in means of the two groups. It should be noted that the sig- nificance level of these t-tests is somewhat inflated, due to the data-induced subgroups. Therefore, also the bias- corrected effect sizes in the leaves were estimated using the validation procedure for small data sets (recom- mended by Dusseldorp & Van Mechelen [23]). Main effects of the delivery modes The office workers in the social environmental interven- tion group showed a mean improvement in NFR of 3.82, and those who did not receive the social environmental intervention showed a mean improvement of 1.17 (Table 2); the difference in means was 2.65 (SD = 23.63; effect size d = 0.11), and the main effect of the social en- vironmental intervention was not significant (p >0.05; indicated by an independent t-test). The office workers in the physical environmental intervention group showed a mean improvement in NFR of 4.59, and those who did not receive the physical environmental inter- vention showed a mean increase of 0.85 (Table 2); the difference in means was 3.75 (SD = 23.60; effect size d = 0.16), and the main effect of the physical environmen- tal intervention was not significant (p > 0.05; indicated by To answer the two research questions, two QUINT analyses were performed; one for the social environmen- tal intervention and one for the physical environmental Formanoy et al. BMC Public Health (2016) 16:866 Page 6 of 15 Table 2 Descriptive statistics for all variables involved in re-analyses of data from the “Be Active & Relax” study. The potential moder- ators were all measured at baseline (i.e., before receiving a physical activity and relaxation program). The statistics are given for both delivery modes: the social intervention and the physical environmental intervention (N = 312) Table 2 Descriptive statistics for all variables involved in re-analyses of data from the “Be Active & Relax” study. The potential moder- ators were all measured at baseline (i.e., before receiving a physical activity and relaxation program). The statistics are given for both delivery modes: the social intervention and the physical environmental intervention (N = 312) delivery modes: the social intervention and the physical environmental intervention (N = 312) Delivery mode: social environmental intervention Delivery mode: physical environmental intervention Yes n = 149 No n = 163 Yes n = 132 No n = 180 Variable Range Mean (SD) Mean (SD) Mean (SD) Mean (SD) Outcome Improv. in need for recovery −100.0 81.82 3.82 (25.32) 1.17 (21.98) 4.59 (23.38) 0.85 (23.75) Potential moderators Need for recovery at baseline 0.00 100.00 30.42 (28.90) 30.36 (28.88) 32.18 (30.28) 29.08 (27.75) Age (in years) 19.00 63.00 42.46 (10.05) 41.77 (9.89) 41.63 (10.39) 42.45 (9.63) Sex (male vs. Main effects of the delivery modes female) 0 1 0.62 (0.48) 0.63 (0.48) 0.62 (0.49) 0.63 (0.48) Level of education 1.00 3.00 2.29 (0.86) 2.41 (0.77) 2.46 (0.76) 2.27 (0.84) Cohabiting (yes vs. no) 0 1 0.75 (0.43) 0.77 (0.42) 0.8 (0.40) 0.74 (0.44) Mother country (Neth. vs. other) 0 1 0.93 (0.26) 0.91 (0.29) 0.93 (0.25) 0.91 (0.29) Body Mass Index 17.10 39.19 25.18 (4.35) 24.87 (3.74) 24.61 (3.56) 25.31 (4.34) Mental Health 2.00 6.00 4.5 (0.72) 4.51 (0.73) 4.42 (0.69) 4.57 (0.74) Detachment at home 1.00 7.00 4.76 (1.33) 4.9 (1.35) 4.8 (1.39) 4.86 (1.31) Relaxation at home 2.00 7.00 5.16 (1.02) 5.25 (1.11) 5.05 (1.07) 5.33 (1.05) Physical activity (in MET-min.) 375 29610 7527 (4234) 7521 (3937) 7066 (4018) 7860 (4095) Vitality 2.00 7.00 5.00 (0.96) 5.06 (1.00) 4.92 (0.97) 5.11 (0.98) Team commitment 1.00 5.00 4.07 (0.65) 4.14 (0.68) 3.99 (0.64) 4.19 (0.67) Organizational commitment 2.57 5.00 4.00 (0.47) 4.08 (0.44) 3.97 (0.44) 4.09 (0.46) Supervisor support 1.00 4.00 2.87 (0.51 2.89 (0.48) 2.86 (0.53) 2.89 (0.47) Colleague support 2.00 4.00 3.09 (0.38) 3.09 (0.37) 3.05 (0.37) 3.12 (0.37) Job demands 1.50 4.00 2.82 (0.49) 2.71 (0.40) 2.78 (0.46) 2.75 (0.44) Decision authority 1.00 4.00 2.98 (0.53) 2.99 (0.54) 2.98 (0.56) 2.99 (0.52) Job insecurity 1.00 3.00 1.55 (0.39) 1.65 (0.48) 1.58 (0.42) 1.62 (0.45) Skill discretion 1.83 4.00 3.03 (0.37) 3.09 (0.37) 3.1 (0.39) 3.03 (0.35) Working overtime (in hrs. p. wk.) 0.00 40.00 2.85 (6.05) 3.19 (7.78) 2.74 (6.70) 3.25 (7.22) Detachment at work 1.00 7.00 3.48 (1.39) 3.54 (1.34) 3.46 (1.30) 3.54 (1.41) Relaxation at work 1.00 7.00 3.53 (1.25) 3.69 (1.31) 3.45 (1.19) 3.74 (1.33) Walking during lunch 1 5 2.78 (1.45) 2.94 (1.47) 2.86 (1.39) 2.87 (1.52) Active during lunch 1 4 1.92 (1.04) 1.91 (1.04) 1.83 (0.98) 1.97 (1.08) around 300), the smaller pruned trees were preferred to the larger ones to take a conservative approach. There- fore, the results of the first series of analysis are de- scribed below and presented in Figs. 1 and 2, those from the second series are presented in Appendix 2. an independent t-test). The mean values (or percentages) on all included moderator variables for the groups with and without each delivery mode are shown in Table 2. Results of the QUINT analyses The first series of QUINT analyses resulted in the same full trees as the second series of analyses. However, the pruning results were different: the pruned trees from the effect size criterion were smaller than those from the difference in mean criterion. Because the sample size of this study was relatively small for a QUINT analysis (i.e., The result for the social environmental intervention was a pruned tree with two leaves. The split of the tree (Fig. 1) involved the variable “Age” and a split point of 46.5 years. It should be noted that in the leaves of Fig. 1, the effect sizes d are expressed as the standardized mean difference between the group with the social Formanoy et al. BMC Public Health (2016) 16:866 Page 7 of 15 Fig. 1 Pruned tree for social environmental intervention with moderator variable “Age” with two leaves (File ‘Fig. 1 pruned.png’). Legend: Pruned tree involving the variable Age and a split point of 46.5 years; the effect sizes d are expressed as the standardized mean difference between the group with the social environmental intervention (SEI+) and the group without (SEI−); For the leaf assigned to P1 (i.e., the left green leaf) the effect size d is positive, while for the leaf assigned to P2, the effect size d is negative Fig. 1 Pruned tree for social environmental intervention with moderator variable “Age” with two leaves (File ‘Fig. 1 pruned.png’). Legend: Pruned tree involving the variable Age and a split point of 46.5 years; the effect sizes d are expressed as the standardized mean difference between the group with the social environmental intervention (SEI+) and the group without (SEI−); For the leaf assigned to P1 (i.e., the left green leaf) the effect size d is positive, while for the leaf assigned to P2, the effect size d is negative means = 10.52; 95 % CI: 4.12, 16.92); bias-corrected effect size d = 0.32, Table 3). Furthermore, the results showed that older office workers (> 46.5 years) who received the social environ- mental intervention had a worse outcome than older of- fice workers who did not receive the social environmental intervention (Leaf 2 in Fig. 1; difference in means = -10.65; 95 % CI: −19.35, −1.96); bias-corrected effect size d = -0.22, Table 3). environmental intervention (SEI+) and the group without (SEI−). Results of the QUINT analyses As a consequence, for the leaf assigned to P1 (i.e., the left green leaf), the effect size d is positive, while for the leaf assigned to P2, the ef- fect size d is negative. The results indicated that youn- ger office workers (≤46.5 years) who received the social environmental intervention had a better outcome in NFR than younger office workers who did not receive the social environmental intervention (Leaf 1 in Fig. 1; difference in means = 10.52; 95 % CI: 4.12, 16.92); bias-corrected effect size d = 0.32, Table 3). Furthermore, the results showed that older office workers (> 46.5 years) who received the social environ- mental intervention had a worse outcome than older of- fice workers who did not receive the social environmental intervention (Leaf 2 in Fig. 1; difference in means = -10.65; 95 % CI: −19.35, −1.96); bias-corrected effect size d = -0.22, Table 3). Fig. 2 Pruned tree for physical environmental intervention with moderator variable “Working overtime” with two leaves (File ‘Fig. 2 pruned.png’). Legend: Pruned tree involving the variable Working overtime and a split point at 2.25 h indicating that office workers who worked fewer hours overtime (≤2.25) had a better outcome with the physical environmental intervention than without the physical environmental intervention (Leaf 1) and office workers who worked more hours overtime (> 2.25) had a worse outcome with the physical environmental intervention than without (Leaf 2) Fig. 2 Pruned tree for physical environmental intervention with moderator variable “Working overtime” with two leaves (File ‘Fig. 2 pruned.png’). Legend: Pruned tree involving the variable Working overtime and a split point at 2.25 h indicating that office workers who worked fewer hours overtime (≤2.25) had a better outcome with the physical environmental intervention than without the physical environmental intervention (Leaf 1) and office workers who worked more hours overtime (> 2.25) had a worse outcome with the physical environmental intervention than without (Leaf 2) Formanoy et al. BMC Public Health (2016) 16:866 Page 8 of 15 Table 3 Descriptive statistics in the leaves of the quint results for the social environmental intervention (SEI; Fig. 1) and the physical environmental intervention (PEI; Fig. 2). Discussion This study examined which subgroups benefit more from which delivery mode of a physical activity and re- laxation program during work on NFR. The results from the QUINT analysis suggest that a social environmental intervention might be more beneficial for younger workers, and a physical environmental intervention might be more beneficial for employees with a only few hours overtime. Furthermore, Hughes et al. [39] found an individual- ized program to be more effective than a program where older university workers could choose health modules on their own, and received generic health e-mail tips. Participants from the individualized intervention re- ported a borderline significant reduction in percentage energy from fat at 6 months (p = .063) and a significant reduction at 12 months (p < .05) and the group reported significantly more minutes of moderate physical activity than did controls at 6 and 12 months (p < .05). At last, a significant decline in waist circumference at 6 months was achieved by the intervention group that was main- tained at 12 months (p < .05). Stress and smoking did not change significantly. These results underline the claims for individualizing the approach for older office workers. Age Several studies found age to be an important factor in the effectiveness of worksite health programs [38, 39]. The review by Rongen et al. [38] demonstrated that the effectiveness of workplace health promotion programs was larger in younger populations (mean age of <40, with a difference in effect size of -0.17; 95 % CI -0.23, −0.17). Specifically, 18 studies were compared that had studied an intervention aimed at a healthy lifestyle (such as physical activity, healthy nu- trition, weight loss, or smoking cessation). The deliv- ery mode varied from a tailored fitness program to counseling sessions focusing on physical activity and nutrition. The authors of this review advised to target specific age populations in worksite interventions. The study also found workplace health promotion programs to be more effective in white collar workers. Results of the QUINT analyses The mean values and standard deviations on improvement in Need for Recovery (NFR) are displayed (i.e., a higher score reflects a larger reduction in NFR from baseline to 12 month follow-up), and the treatment outcome diff differences n Mean SD n Mean SD Difference in means (95 % CI) Bias-corrected effect size d Fig. 1 SEI+ SEI− Leaf 1 90 8.29 22.27 107 −2.23 23.20 10.52 (4.12, 16.92)* 0.32 Leaf 2 59 −3.00 28.22 56 7.66 17.89 −10.65 (−19.35, -1.96)* −0.22 Fig. 2 PEI+ PEI− Leaf 1 103 6.15 23.90 128 −1.25 25.39 7.40 (0.99, 13.81)* 0.13 Leaf 2 29 −0.94 20.90 52 6.01 18.35 −6.95 (−16.26, 2.36) −0.08 Note. CI confidence interval; *p < .05, estimated by independent t-test latter difference concerned an increase of NFR in a non-experimental design and was determined over a longer period (2 years). The result for the physical environmental interven- tion was also a pruned tree with two leaves. The split of the tree involved the variable “Working overtime” and a split point at 2.25 h (Fig. 2). The results indi- cated that office workers who worked fewer hours overtime (≤2.25) had a better outcome with the physical environmental intervention than without the physical environmental intervention (Leaf 1 in Fig. 2; difference in means = 7.40; 95 % CI: 0.99, 13.81); bias- corrected effect size d = 0.13). Furthermore, the re- sults showed that office workers who worked more hours overtime (> 2.25) had a worse outcome with the physical environmental intervention than without, but this effect was not significant (Leaf 2 in Fig. 2; difference in means = −6.95; 95 % CI: −16.26, 2.36); bias-corrected effect size d = −0.08, Table 3). Comparison with other studies Observed change in NFR Although the “Be Active & Relax” study demonstrated no significant effect on the main outcome NFR [16], differential treatment efficacy was found in our study. The changes in NFR found from baseline to 12- month follow-up in the intervention subgroups (i.e., 8.29 for the social environmental and 6.15 for the physical environmental intervention) were somewhat lower than the change found by De Croon et al. [26], that is, a difference in NFR of 12.6. However, the Page 9 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Page 9 of 15 The social environmental intervention had a negative effect on older workers. It could be that younger em- ployees had a more positive attitude toward the social environmental intervention than older employees. fruits and vegetables in full-time workers. Working overtime simply reduces the amount of time and en- ergy available to be physically active [18]. It could be that the participants in this study who worked more hours overtime did not make as much use of the environmental modifications as the partici- pants who did not work overtime because of time constraints. As a result, working overtime may have interfered with fully benefitting from the environmen- tal modifications. Office workers who work overtime on a regular basis probably need a different interven- tion to change their lifestyle and make them more physically active. It may be advisable for organizations to pay attention to the work process when working overtime is structural. Office workers who work over- time structurally may be a vulnerable group. Not only do they have stress as a result of working extra hours, but they may also be less susceptible for interventions aimed at physical activity and relaxation. Furthermore, there is some evidence that being a good role model as a team leader in terms of healthy behaviors may be important for those offering the social environmental intervention to support physical activity and relaxation [16]. In addition, other charac- teristics of the team leaders (i.e., age, gender, ethni- city, or level of education) might have influenced the effectiveness of the social environmental intervention. Finally, the older participants in our study (> 46.5 years old) may have been healthier than the office workers who have left the job (healthy worker effect). The baseline NFR score of the older participants was relatively low (i.e., 28.0) and did not differ signifi- cantly from the younger ones. Limitations of this study y There are some limitations of this study. First, the compliance of the sample. Not all participants in the “Be Active & Relax” study fully participated in the so- cial and in the physical environmental interventions [42]. The reach (% of participants that attended at least one sessions or used at least one element) for the social and physical environmental intervention ranged from 45 to 76 % [42]. A barrier for not at- tending the social environmental intervention sessions were, for example, having a holiday. An important barrier for the participants for not using the physic- ally activating and relaxing elements in the physical environmental intervention was office workers indicat- ing that they did not have enough time. Although the level of reach is comparable to other worksite health programs (mostly below 50 % [43]); this could have underestimated the results in the QUINT analysis. Comparison with other studies Observed change in NFR This finding is in con- trast with the studies of Kiss et al. [40] and Mohren et al. [41], who found that older workers had a higher NFR than younger workers. Kiss et al. [40] found that the mean recovery score (40.9) was sig- nificantly higher in the group of the older public sector workers as compared to the mean score (33.6) of younger workers (p < 0.05). Mohren et al. [41] studied day workers and discovered that the highest levels of NFR were observed in the age group of 46– 55 years. The relative risk for developing elevated NFR was highest in the age groups 36–45 years (RR 1.30; 1.07–1.58) and 46–55 years (RR 1.25; 1.03– 1.52) in men and 46–55 years (RR 1.36; 1.04–1.77) in women when compared to the reference group. Looking at the evaluation of the “Be Active & Relax” study [42], it is remarkable that the element in the environmental modifications that was most phys- ically activating (e.g. table tennis table) was not used much. It was shown that the percentage that used the table tennis table at least once on a scale from 0 (never) to 5 (always) was on average 17 % of the 35 participants that were evaluated. Implications for research and practice The implications of the results of our study are three- fold. Firstly, the results suggest that a “one size fits all” approach does not hold for the worksite health program “Be Active and Relax”. For other worksite health programs, this commonly used approach may also not work. Instead, we recommend the developers of such programs to carefully consider possibilities to tailor the program to specific subgroups. Secondly, the study shows that an advanced exploratory ana- lyses method, like QUINT, is able to indicate for which subgroups of workers a worksite health pro- gram is beneficial and for which subgroups it is not. And thirdly, the results suggest that group motiv- ational interviewing by team leaders is not an appro- priate strategy for older workers to reduce their need for recovery. In addition, changing the work environ- ment to facilitate daily physical activity and relaxation at the worksite is not appropriate for workers who work more hours overtime. More research is needed to investigate which type of health program would work for these specific subgroups of office workers. Based on the results, we advise to take age and work- ing overtime into account when developing a health program for office workers, and also when designing health intervention evaluation studies. A second limitation of the study was that the exist- ence of possible subgroups that benefit more from the combination of the social and physical environ- mental intervention was not explored due to sample size restrictions. Although the statistical analyses did not show the need for doing this (due to a non- significant interaction effect between the social envir- onmental intervention and the physical environmental intervention), it cannot be ruled out that subgroups exist that might benefit especially from the combined intervention. The generalizability of the interventions to other work environments is questionable, because the inter- ventions were specifically tailored to the target popu- lation by using intervention mapping. Furthermore, our study population consisted of 60 % males and 57 % was highly educated, which does not represent the general Dutch working population. This was due to the fact that the financial service provider’s work- force involves mainly highly educated, male and white collar employees. Finally, the “Be Active & Relax” study was not a full RCT. The social environmental intervention group was randomized, but the physical environmental inter- vention was not (for details, see [19]). Working overtime k Working overtime means that a person is putting in more hours than agreed upon in his or her contract. Although the threshold for working overtime to bene- fit from environmental modifications in this study is rather low (on average 2.25 h per week), underlying mechanisms may explain why office workers who work overtime do not benefit fully from the delivery mode environmental modifications. Research indicates two possible mechanisms for overtime work causing an increase in stress levels [18]. The first is that high levels of overtime may lead to lack of recovery because of the shorter periods of rest between working days. This can affect recovery time and increase the exposure time to work stress, in turn resulting in adverse health and well-being. The second mechanism affects health indirectly and concerns behavioral decisions and habits (i.e., lifestyle behaviors) of workers. Taris et al. found overtime to lead to lower levels of physical activity and intake of Although no significant effect was found on the main outcome measure of NFR in the study by Cof- feng et al. [16], secondary outcome measures did dif- fer significantly. In the combined environmental intervention group (n = 92), exhaustion and vigorous physical activities decreased significantly, and small breaks at work and active commuting increased sig- nificantly compared to the control group. The social Page 10 of 15 Page 10 of 15 Formanoy et al. BMC Public Health (2016) 16:866 environmental intervention group (n = 118) showed a significant reduction in exhaustion, and sedentary be- havior at work, and a significant increase in small breaks at work and leisure activities compared to the control group. In the physical environmental interven- tion group (n = 96), stair climbing at work and active commuting significantly increased, and sedentary be- havior at work decreased significantly compared to the control group. research, larger sample sizes are recommended to confirm our hypotheses. The software of QUINT al- lows the user to choose between several parameters, for example, the minimum sample size per interven- tion condition in a leaf and the total number of bootstrap samples used for the pruning procedure. In the present study, we found that results were more stable using a minimum intervention sample size of 25 (instead of the default value of 10 %), and a total number of bootstrap samples of 1000 (instead of the default value of 25). Implications for research and practice Therefore, cau- tiousness is needed in the interpretation of the results of the physical environmental intervention; the benefi- cial effect for office workers with a few hours over- time might not have been caused by the intervention, due to the non-random assignment. Working overtime k However, generalizability of recommending the social environmental intervention for younger office workers and using the physical environmental intervention for office workers who do not work overtime should be interpreted cautiously. Conclusions This study was conducted to explore whether subgroups may benefit more from a social or a physical environ- mental intervention to reduce the NFR by using the QUINT method. Specifically, the results suggest possible roles for age and working overtime: younger workers benefitted (in terms of a reduced need for recovery) from a so- cial environmental intervention and employees who worked fewer hours overtime benefitted from a phys- ical environmental intervention. It is recommended to incorporate age and working overtime as stratification variables in future research into the need for recovery among office workers to confirm these results, and to tailor the interventions to specific groups. Methodological issues In the present study, the QUINT analyses were per- formed using the effect size criterion and the differ- ence in means criterion. Both analyses led to the same full trees, but different pruned trees. Due to the relatively low sample size (N = 312) for a QUINT analysis, we took a conservative approach and pre- ferred the smaller trees (for detecting complex inter- actions, N ≥400 is recommended [23]). In future Page 11 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Table 4 The potential moderators were all measured at baseline (i.e., before receiving a physical activity and relaxation program) and are divided into background variables, health and home related variables and work related variables # it α or r Example of an item with answer categories and score range Questionnaire Studies indicating a possible modifying effect Background Age 1 N/A “What is your date of birth?” N/A Steenstra (2009) [33]; Demou (2012) [48], Bauman (2012) [49] Sex 1 N/A “What is your gender?”; 2 categories “man” or “woman” N/A Demou (2012) [48], Bauman (2012) [49] Level of education 1 N/A “What is the highest level of education you have completed?; 8-point scale from 1”no education” to 8 “university degree” N/A Cohabiting 1 N/A “What is your current marital status?”; 6 categories (such as married, living with another, etc) N/A Mother country 1 N/A “In what country were you born?”; 2 categories: the Netherlands or other N/A Health/home BMI 2 N/A “What is your weight?” en “How tall are you?” N/A Colkesen (2011) [34] General health 1 N/A “In general, how would you say your health is?”; 5-point scale from 1 “excellent” to 5 “poor” Rand-36; Van der Zee (1993) [44] Demou (2012) [48], Colkesen (2011) [34], Bauman (2012) [49] Mental health 9 α = 0.90 “Did you feel full of pep?”; 6- point scale from 1 “none of the time” to 6 “all of the time” Rand-36; Van der Zee (1993) [44] Demou (2012) [48]; Dishman (2010) [35] Detachment at home 4 α = 0.94 “After work, I don’t think about work at all”; 7-point scale from 1 “never” to 7 “almost always” The recovery experience questionnaire; Sonnentag (2007) [14] and De Bloom (2011) [45] Physical activity 7 r = 0,97 “Do you walk/cycle to work? On how many days per week? What is the average time it takes? Dutch version of the Job Content Questionnaire Karasek (1998) [47] Methodological issues Rate the intensity: light, moderate, vigorous” Short Questionnaire to Assess Health Enhancing Physical Activity (SQUASH); Wendel-Vos (2003) [46] Colkesen (2011) [34] Relaxation at home 4 α = 0.92 “After work, I do relaxing things”; 7-point scale from 1 “never” to 7 “almost always” The recovery experience questionnaire; Sonnentag (2007) [14] and De Bloom (2011) [45] Work related Vitality 5 α = 0.83 “At my work I feel bursting with energy”; 6-point scale from 1 “never” to 6 “always” Part of Utrecht Work engagement Scale (UWES); Demerouti (2001) Strijk (2012) [17] Team commitment 3 α = 0.82 “What I feel for this team – I am proud”; 5-point scale from 1 “to- tally agree” to 5 “totally disagree” Organizational commitment 8 α = 0.79 “I find it important that my job go’s well”; 5-point scale from 1 “strongly agree” to 5 “strongly disagree” Meyer (2002) [50]; Lohela (2009) [51] Supervisor support 4 α = 0.78 “My supervisor is concerned about the welfare of those under him”; 4-point scale from 1 “strongly agree” to 4 “strongly disagree” Dutch version of the Job Content Questionnaire Karasek (1998) [47] Choi (2011) [52] Appendix 1 Relevant moderator variables measured in the “Be Active & Relax” Project Appendix 1 Relevant moderator variables measured in the “Be Active & Relax” Project The recovery experience questionnaire; Sonnentag (2007) [14] and De Bloom (2011) [45] Part of Utrecht Work engagement Scale (UWES); Demerouti (2001) Strijk (2012) [17] Meyer (2002) [50]; Lohela (2009) [51] Choi (2011) [52] Dutch version of the Job Content Questionnaire Karasek (1998) [47] Formanoy et al. Methodological issues BMC Public Health (2016) 16:866 Page 12 of 15 Table 4 The potential moderators were all measured at baseline (i.e., before receiving a physical activity and relaxation program) and are divided into background variables, health and home related variables and work related variables (Continued) Colleague support 4 α = 0.76 “People I work with are competent in doing their jobs”; 4-point scale from 1 “strongly agree” to 4 “strongly disagree” Dutch version of the Job Content Questionnaire Karasek (1998) [47] Choi (2011) [52] Job demands 5 α =0.70 “My job requires working very fast”; 4-point scale from 1 “strongly agree” to 4 “strongly disagree ” Dutch version of the Job Content Questionnaire Karasek (1998) [47] Lohela (2009) [51], Van Laethem (2013) [53] Decision authority 3 α =0.68 “My job allows me to make a lot of decisions on my own”; 4-point scale from 1 “ strongly agree” to 4 “strongly disagree ” Dutch version of the Job Content Questionnaire Karasek (1998) [47] Mullan (1985) [54] Skill discretion 6 α =0.67 “My job requires that I learn new things”; 4-point scale from 1 “strongly agree” to 4 “strongly disagree ” Dutch version of the Job Content Questionnaire Karasek (1998) [47] (WEBA) Job insecurity 3 α =0.68 “During the past year were you in a situation where you faced job loss or layoff?”; 3-point scale from 1 “no” to “actually layed off” Dutch version of the Job Content Questionnaire Karasek (1998) [47] Karlsson (2010) [55], Lohela (2009) [51] Working overtime 1 N/A “On average, how many hours of overtime do you put in per week?” Taris (2011) [18] Detachment at work 4 α = 0.93 “During a break at work I forget about my work”; 7-point scale from 1 “never” to 7 “almost always” Adapted question (at work was added) of “The recovery experience questionnaire”; Sonnentag (2007) [14] and De Bloom (2011) [45] Relaxation at work 4 α = 0.88 “During a break at work I use the time to relax”; 7-point scale from 1 “never” to 7 “almost always” Adapted question (at work was added) of “The recovery experience questionnaire”; Sonnentag (2007) [14] and De Bloom (2011) [45] Notes: # it number of items, α Cronbach’s α, r Pearson’s correlation coefficient, N/A not applicable Dutch version of the Job Content Questionnaire Karasek (1998) [47] Karlsson (2010) [55], Lohela (2009) [51] Taris (2011) [18] Acknowledgements W ld lik t th g We would like to thank all teamleaders and employees who participated in the “Be Active & Relax study. The results showed that the social intervention was especially effective for the younger employees with a high Organization commitment (Leaf 2 in Fig. 3; bias-corrected effects were d = 0.38 and difference in means = 10.40). Furthermore the social environmental intervention was Appendix 2 counter-effective for older persons working less than one hour of overtime (Leaf 3 in Fig. 3; d = -0.97; bias-corrected effects were d = −0.67; difference in means = −16.29). Results of the second series of analyses Results of the second series of analyses The full tree that resulted from the QUINT analysis for the social environmental intervention and for the physical environmental intervention using the Differ- ence in means criterion was equal to the full trees using the Effect size criterion (the results from the latter were described in the Results section). However, the pruning procedure resulted in larger trees for both the social environmental intervention and the physical environmental intervention, that is, trees with four leaves (see Figs. 3 and 4). For the social en- vironmental intervention, additional splitting variables were Organization commitment and Working over- time (Fig. 3). For the physical environmental intervention, additional splitting variables were Team commitment and Physical activity (Fig. 4). The results showed that the physical en- vironmental intervention was especially effective for the employees working less hours overtime with a high Team commitment and a lower level of Physical activity (Leaf 2; bias-corrected effects were d = 0.42 and differ- ence in means = 11.40). Funding Thi j Funding This project was funded by Fonds Nuts Ohra. The contribution of Iven van Mechelen was funded by the Research Fund KU Leuven (Award Number: GOA/15/003) and the Belgian Interuniversity Attraction Poles programme (Award Number: IAP/P7/06). g This project was funded by Fonds Nuts Ohra. The contribution of Iven van Mechelen was funded by the Research Fund KU Leuven (Award Number: GOA/15/003) and the Belgian Interuniversity Attraction Poles programme (Award Number: IAP/P7/06). Page 13 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Fig. 4 QUINT solution for the delivery mode “physical environmental intervention”, using the Difference in means criterion ig. 4 QUINT solution for the delivery mode “physical environmental intervention”, using the Difference in means criterion Fig. 3 QUINT solution for the delivery mode “social environmental intervention”, using the Difference in means criterion ig. 3 QUINT solution for the delivery mode “social environmental intervention”, using the Difference in means criterion Page 14 of 15 Page 14 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Authors’ contributions MAGF and ED wrote the initial manuscript. ED and IVM performed the data analysis, with input from MAGF and ECPMT. ECPMT had a role in supervision. JKC, CRLB en IJMH were responsible for the management of the trial and data collection. All authors contributed to the further writing and approved the final version of the manuscript. 13. Hahn VC, Binnewies C, Sonnentag S, Mozja EJ. Learning how to recover from job stress: effects of a recovery training program on recovery, recovery-related self-efficacy, and well-being. J Occup Health Psych. 2011;16(2):202–16. 14. Sonnentag S, Fritz C. The recovery experience questionnaire: development and validation of a measure for assessing recuperation and unwinding from work. J Occup Health Psych. 2007;12:204–21. 14. Sonnentag S, Fritz C. The recovery experience questionnaire: development and validation of a measure for assessing recuperation and unwinding from work. J Occup Health Psych. 2007;12:204–21. Ethics approval and consent to participate For this study, data were used from the “Be Active & Relax” study [19]. From September 2011 to September 2012, 329 office workers participated. They were all recruited at one business location of a Dutch financial service provider. All participants signed an informed consent. This study was a cluster-randomized controlled trial (with three data collection time points) to reduce the NFR in office workers using a physical activity and relaxation program. The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center (Amsterdam, The Netherlands). Additional details regarding the study design and methods of the “Be Active & Relax” study have been published elsewhere [19]. 17. Strijk JE, Proper KI, Van der Beek AJ, Van Mechelen W. A worksite vitality intervention to improve older workers’ lifestyle and vitality-related outcomes: results of a randomised controlled trial. J Epidemiol Community Health. 2012;66(11):1071–8. doi:10.1136/jech-2011-200626. 18. Taris TW, Ybema JF, Beckers DG, Verheijden MW, Geurts SA, Kompier MA. Investigating the associations among overtime work, health behaviors, and health: a longitudinal study among full-time employees. Int J Behav Med. 2011;18(4):352–60. 19. Coffeng JK, Hendriksen IJM, Duijts SF, Proper KI, van Mechelen W, Boot CRL. The development of the be active & relax ‘Vitality in Practice’ (VIP) project and design of an RCT to reduce the need for recovery in office employees. BMC Public Health. 2012;12(1):592–617. 19. Coffeng JK, Hendriksen IJM, Duijts SF, Proper KI, van Mechelen W, Boot CRL. The development of the be active & relax ‘Vitality in Practice’ (VIP) project and design of an RCT to reduce the need for recovery in office employees. BMC Public Health. 2012;12(1):592–617. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. 16. Coffeng JK, Boot CR, Duits SF, Twisk JW, Mechelen W, Hendriksen IJ. Effectiveness of a worksite social & physical environment intervention on need for recovery, physical activity and relaxation; results of a randomized controlled trial. PLoS One. 2014;9(12):e114860. Availability of data and materials 9. Rook JW, Zijlstra FRH. The contribution of various types of activities to recovery. Eur J Work Organ Psychol. 2006;15:218–40. The Data Protection officer of VU University Medical Center has ethical restrictions to making our data publicly accessible, because the data can lead to personally identifiable health information. The data includes the name of the company, age, education, being overweight, and other personal characteristics. The readers may contact Cécile Boot (crl.boot@vumc.nl) to request the data. The data will be available upon request to all interested researchers. 10. Norris R, Carroll D, Cochrane R. The effects of aerobic and anaerobic training on fitness, blood pressure, and psychological stress and well-being. J Psychosom Res. 1990;34:367–75. 11. Lechner L, de Vries H. Effects of an employee fitness program on reduced absenteeism. J Occup Environ Med. 1997;39(9):827–31. 12. Rosenfeld O, Tenenbaum G, Ruskin H. 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https://openalex.org/W3181087707	https://www.researchsquare.com/article/rs-701769/latest.pdf	English	null	Responsiveness to change in health status of the EQ-5D in patients treated for depression and anxiety	Research Square (Research Square)	2,021	cc-by	8,315	Conclusion The EQ-5D showed good agreement with clinical measures evaluating symptom change, and correctly identifying recovered patients. These findings indicate that the EQ-5D may be appropriately responsive to change in patients with depression and anxiety disorders, although replication in other clinical samples is needed. Results Effect sizes were large for the BAI, the BDI-II, the EQ-5D value and the EQ VAS, ranging from d = 1.07 to d = 1.84. SRM ranged from .93 – 1.67. Spearman’s correlation showed moderate agreement between the EQ-5D value and the BDI-II (rs = - .48), and the BAI (rs = - .41). The EQ-5D consistently identified “Recovered” patients versus “Improved” or “Unchanged” in the ROC analyses with AUROC ranging from .72 – .84. Purpose The EQ-5D is a commonly used generic measure of health but evidence on its responsiveness to change in mental health is limited. This study aimed to explore EQ-5D responsiveness in patients receiving treatment for depression and anxiety. Research article Posted Date: July 13th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Page 1/21 Page 1/21 Page 1/21 Methods Patient data (N = 416) were collected at baseline and at end of treatment in an observational study in a Norwegian outpatient clinic. Responsiveness in the EQ-5D was compared to change in the Beck Depression Inventory-II (BDI-II) and the Beck Anxiety Inventory (BAI). Effect sizes (Cohen’s d), Standardised response mean (SRM), and Spearman’s correlation were calculated. Patients were classified as “Recovered”, “Improved”, or “Unchanged” during treatment using the BDI-II and the BAI. ROC analyses determined whether the EQ-5D could correctly classify patient outcomes. Plain English Summary The EQ-5D is a questionnaire that people fill in to report their subjective health. It is often used in clinics or hospitals to better understand how patients are affected by their illnesses, and if their health improves after treatment. For this information to be trustworthy, we need to verify how accurately the EQ-5D measures health for the particular patients we want to use it with. This is often done by comparing EQ-5D scores with scores from other questionnaires. For example, if we want to use the EQ-5D with a group of patients with depression, we compare the scores of the EQ-5D with scores from questionnaires that are commonly used to measure depression symptoms. Introduction One out of every two people will experience a mental health problem during their lifetime and mental ill health is a leading cause of global disease burden [1]. Between 2010 and 2030, mental illness is projected to cost $ 16.1 trillion worldwide, putting it on par with cardiovascular disease [2]. Depression and anxiety disorders account for 40.5 % and 14.6 % of the disability-adjusted life-years that are due to mental illness, making them the most costly mental health problems [3]. This substantial burden may still be underestimated [4], in part because of difficulty in measuring the wide ranging effects these disorders have on health and functioning [5]. At the recommendation of decision-making bodies such as the National institute of Health and Care excellence (NICE), generic measures are increasingly used to capture health status [6, 7]. Mental disorders like depression and anxiety have broad, negative impact on quality of life and wellbeing that may not be adequately reflected by condition-specific measures [5, 8]. Generic measures may thus be a valuable supplement to measures of primary symptoms as they capture broader concepts of health. These instruments can also help compare burden of disease and impact of interventions for different patient groups, such as in cost-benefit analyses, making them useful tools for decision-makers, researchers, and clinicians [6]. To adequately fill this role, it must be demonstrated that the generic measure in question can accurately record health status in the relevant patient population. One of the most commonly used generic measures of self-reported health is the EQ-5D [6]. The EQ-5D records health status across five dimensions: Mobility, Self-care, Usual activities, Pain / discomfort, and Anxiety / depression [6]. The previous version of the EQ-5D used three levels of severity and showed good psychometric properties in depression, but mixed results in anxiety disorders [9]. A recent review evaluated the properties of the newer five-level version of the EQ-5D across multiple patient groups [10]. Three studies of patients with mental health problems were included, showing moderate to good correlation between condition-specific measures and the five-level EQ-5D in cross-sectional designs [11– 13]. These studies did not include data on responsiveness [10]. Responsiveness is often defined as an instruments ability to detect clinically significant change over time [9, 14]. Page 2/21 In this study, we compared the scores of the EQ-5D with scores from questionnaires measuring symptoms of depression and anxiety. Their performances were similar, and the EQ-5D scores could also correctly identify which patients had recovered during treatment. This implies that the EQ-5D can be a useful tool for understanding the impact of depression and anxiety and can help in decision-making regarding these patients. Introduction Two criteria have been suggested for defining what constitutes “clinically significant change”: That the magnitude of change be statistically reliable, and that patients end up in a clinical range that renders them indistinguishable from the normal population, i.e. they have recovered [15]. Responsiveness according to these criteria is not a fixed parameter, but will likely vary according to populations and context [16]. This makes it necessary to Page 3/21 Page 3/21 Page 3/21 investigate responsiveness across multiple patient groups. One study did find reasonable validity and moderate responsiveness in anxiety on the three-level EQ-5D [17]. But only a few studies have examined this aspect of the five-level EQ-5D in depression and anxiety [10]. One such study found that using only the Anxiety/depression dimension of the EQ-5D did not adequately capture responsiveness in anxiety and depression for patients treated in a general internal medicine ward [18]. Another study found that the EQ-5D could adequately screen for depression and anxiety by distinguishing between severity levels in patients with type 2 diabetes. This was true of both the Anxiety/depression dimension and the overall EQ-5D value [19]. However, this was a cross-sectional design, and the ability of the EQ-5D to detect change over time in patients with depression and anxiety is not established, and was specifically targeted by a review of the literature as a future research priority [10]. Investigating this aspect of the EQ-5D is imperative in establishing its usefulness in this patient group, particularly in clinical settings. To our knowledge, ours is the first study to examine the responsiveness of the five-level EQ-5D in patients treated for depression and anxiety as their primary diagnoses. In line with recommendations and methodology used in previous studies, we explored responsiveness of the EQ-5D by comparing change from start to end of intervention with change in condition-specific measures [16, 18, 19]. The aim of the study was thus to test the following hypotheses: 1) that the EQ-5D shows similar range in effect size and an at least moderate correlation with change scores in condition-specific measures, and 2) that the EQ-5D can identify patients classified as “Recovered” by condition-specific measures at end of treatment. Study context Data were collected at in a naturalistic observational study that ran from May 2017 – March 2020 at the Department of Mental Health and Substance Abuse, Diakonhjemmet Hospital in Oslo, Norway. The clinic is part of the national health service, and the study is part of the project “The Norwegian studies of psychological treatments and work (NOR-WORK)”. Patients are referred by their general practitioners and screened by a clinical psychologist using anamnestic information, the Beck Depression Inventory-II (BDI- II), the Beck Anxiety Inventory (BAI), and the MINI-International Neuropsychiatric Interview [20–22]. Diagnoses are set according to the International Classification of Diseases 10 (ICD-10) [23]. Inclusion criteria for the present study were that the patient was an adult of working age (18–70 years) with clinically significant levels of depression and anxiety operationalised as follows: Patients with a primary depression diagnosis had to have a minimum score of 14 on the BDI-II, and patients with a primary anxiety diagnosis had to have a minimum score of 16 on the Beck Anxiety Inventory BAI. In addition to primary depression or anxiety diagnoses, patients with adjustment disorder and mixed anxiety and depression were included in the study. Adjustment disorder is sometimes referred to as “situational depression”, underlining its close relationship with depressive disorders [23]. Similarly, patients with a Page 4/21 Page 4/21 mixed anxiety and depressive disorder were included as the diagnosis is comprised of symptoms of anxiety and depression. Exclusion criteria were severe mental illness such as bipolar disorder, high risk of suicide, engaging in active substance abuse, or suffering from cluster A or B personality disorder. All patients who signed a written consent form and completed treatment, including filling in questionnaires at baseline and at end of treatment, were included (N = 416). The current study thus focused on patients who completed treatment. Patients received either Metacognitive therapy (MCT) or Cognitive behavioural therapy (CBT) according to diagnose specific manuals [24, 25], and average duration of treatment was 10.11 sessions (SD 3.93). Previous research has shown that half the patients are on sick leave when referred, and treatment thus also includes interventions aimed at helping patients return to work [26]. Instruments Clinical and sociodemographic data were collected at baseline and end of treatment from patient journals and from self-report questionnaires. The EQ-5D: The EQ-5D questionnaire firstly asks respondents to rate their current health on five dimensions: Mobility, Self-Care, Usual activities, Pain / discomfort, and Anxiety / depression on a severity scale from 1 (“No problems”) to 5 (“Severe problems”). The combined severity ratings give an EQ-5D profile, e.g. “11111” in the case of “No problem” on all five dimensions. This health profile can be converted to the EQ-5D value using preference-based weights. A value of 0.00 indicates death and 1.00 indicates perfect health. The EQ-5D value can be used to calculate quality-adjusted life-years (QALYs), i.e. a score of 1.00 for one year equals one QALY. The preference-based weights used to convert responses to EQ-5D values are often referred to as “value sets”. A study is underway to acquire a Norwegian set, but is not yet completed [27]. This study used the crosswalk system recommended by NICE for converting EQ- 5D profiles to EQ-5D values [28, 29]. For the EQ-5D value, healthy people generally report scores close to 1.00. In a recent survey of the Norwegian general population, the mean EQ-5D value in a postal survey was .848 [30]. The second part of the EQ-5D asks patients to rate their health on a 20 cm visual analogue scale (VAS) where the bottom (“0”) indicates worst possible health, and the top (“100”) indicates best possible health. Although it is related to the EQ-5D profile and the value scores, it does not measure the same construct. For instance, the EQ VAS score has been shown to decline with age even for people whose EQ-5D profile show no problems (“11111”) [6]. The Beck Depression Inventory-II (BDI-II) is a 21 item questionnaire measuring severity of symptoms over the last two weeks on a scale from 0–3, giving a total sum score of 0–63. Examples include feeling sad and change in appetite or sleep. Suggested scoring indicates that 0–13 reflects minimal symptoms, 14– 19 mild, 20–28 moderate, and 29–63 severe symptoms [21]. The BDI-II has been found to be psychometrically sound in depression[31], Chronbach’s α in the current study was .86. Page 5/21 The Beck Anxiety Inventory (BAI) is a self-report measure of anxiety severity over the last week. As with the BDI-II, anxiety symptoms (e.g. Statistical analyses Descriptive statistics on age, gender, education level and diagnosis were compiled at baseline. Distribution of scores on the EQ-5D dimensions were calculated in percentages at baseline and at end of treatment and analysed using a non-parametric test of trends developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Mean scores and standard deviations at baseline and end of treatment were calculated. Change (∆) during treatment was calculated for the BAI, the BDI-II, the EQ-5D values, and the EQ VAS by subtracting score at end of treatment from baseline score. Effect sizes (ES) from baseline to end of treatment were calculated using Cohen’s d. Values < .50 are considered small, ≥ .50 < .80 moderate, and ≥ .80 large [34]. We also calculated the standardised response mean (SRM), defined as the mean change in score from baseline to end of treatment divided by the standard deviation of change in scores [35]. It is suggested that interpretation of the SRM should take correlation between scores at baseline and end of treatment into account. For example, SRM > 0.8 can be interpreted as large if this correlation < .50, moderate if correlation > .50 [36]. Agreement between the change scores on the four measures were also analysed with Spearman’s correlation. We interpreted Spearman’s correlation as weak if rs 0 – .39, moderate if rs .40 − .69, and strong if rs < .70 [37]. Using the BAI and the BDI-II, the patients were classified according to treatment response. With a minimum score of 14 on the BDI-II for depression patients and 16 on the BAI for anxiety patients at baseline, the following classification was used: Patients were classified as “Deteriorated” if their scores increased by 9 points or more from baseline to end of treatment. If the change was less than 9 points in either direction, they were classified as “Unchanged”. If the scores decreased by 9 points or more patients were classified as “Improved”. Finally, patients were classified as “Recovered” if their score decreased by 9 points or more and their final score was below clinical threshold (i.e. 14 for the BDI-II and 16 for the BAI) [38, 39]. We ran ROC curve analyses to determine how well the EQ-5D value scores could classify patients according to the clinical criteria of the BDI-II and the BAI: Recovered versus Improved, Recovered versus Unchanged, and Improved versus Unchanged. Instruments “Heart pounding or racing” or feeling “nervous”) are scored on a severity range from 0–3, giving a total sum score of 0–63. Suggested scoring indicates that 0–15 reflects mild symptoms, 16–25 moderate, and 26–63 severe symptoms. The BAI has demonstrated good psychometric properties [32], Chronbach’s α in the current study was .90. Statistical analyses This was done with both the BDI-II and the BAI for the total sample, and separately with the BDI-II for the patients with depression disorders, and with the BAI for patients with anxiety disorders. Using the EQ-5D value at end of treatment as classifier, we computed area under the curve (AUROC). AUROC was interpreted as < .50 useless test, .51–.69 poor test, .70 – .79 fair test, .80 – .89 good test, .90 – .99 excellent test, 1.00 perfect test [40]. We also computed cut-off values for recovery using Youden’s index (J), which chooses the values with highest combined sensitivity and specificity [41]. Page 6/21 Missing data in the study was typically low, > 5% on individual items for all measures. Generally accepted methods for handling missing data are applicable to the EQ-5D [6]. Missing data on individual items in the current study were replaced by weighted means, a method developed for treating missing data in depression cohorts [42]. All analyses were carried out using STATA 16.1[43] Ethical considerations All patients included in the study gave written, informed consent to participate. The study is classified as health service research under Norwegian regulation. The Norwegian Data Protection Agency has in such cases designated that treatment providers (i.e. hospitals) are responsible for proper data management. Data collection and security in the present study was managed by Diakonhjemmet Hospital, and approval of data handling was granted by Oslo University Hospital, approval number 2015/15606. The study was carried out in accordance with the principles of the Helsinki declaration. Results Characteristics of included patients (N = 416) at baseline are shown in Table 1. The study only recorded primary diagnoses, comorbidity was not recorded. The majority of patients had either a primary depression or anxiety diagnosis, the remaining patients were diagnosed with either mixed anxiety / depression, or adjustment disorder. The most prevalent single diagnoses were F32 Major depressive disorder, single episode (n = 114, 26.8 %), F 33 Major depressive disorder, recurrent (n = 97, 22.8 %), and F 41.1 Generalised anxiety disorder (n = 86, 20.2 %). Page 7/21 Table 1 Demographic characteristics and diagnoses of patients at baseline (N = 416) Mean SD n % Age, years 37.66 10.65 Gender Female Male 299 71.88 117 28.13 Education Primary / Secondary Higher education ≤ 4 yrs Higher education > 4 yrs 70 17.16 151 37.01 187 45.83 Primary diagnosis Depression disorder Anxiety disorder Mixed anxiety / depression Adjustment disorder 216 51.92 161 38.70 24 6.77 15 3.61 Change in depression, anxiety and the EQ-5D during treatment > .80 on all instruments. Correlation between baseline scores and scores at end of treatment were < .50 on the BDI-II (rs = .39), EQ-5D utility (rs = .34), and the EQ-VAS (rs = .31), but > .50 on the BAI (rs = .51). This indicates that the SRM was large for the BDI-II, EQ-5D utility, and the EQ VAS, whilst moderate for the BAI. Change in depression, anxiety and the EQ-5D during treatment Of the 216 patients with depression diagnoses, 146 (67.59 %) were “Recovered” at end of treatment, 31 (14.35 %) were “Improved”, and 39 were (18.05 %) were “Unchanged”. Of the 161 patients with anxiety disorder diagnoses, 109 (67.70 %) were “Recovered” at end of treatment, 14 (8.69 %) were “Improved”, and 38 were (23.60 %) were “Unchanged”. Overall, two patients in the sample were “Deteriorated” on the BAI at end of treatment, both were diagnosed with adjustment disorder. Four patients were “Deteriorated” on the BDI-II, three of which were diagnosed with adjustment disorder, and one with anxiety disorder. No patients with anxiety diagnoses were “Deteriorated” on the BAI at end of treatment, and no patients with depression diagnoses were “Deteriorated” on the BDI-II at end of treatment. Table 2 shows the distribution of scores on the EQ-5D dimensions at baseline, and after end of treatment. All dimensions had at least some patients reporting problems at baseline. Cuzick’s non-parametric test of trends showed that all dimensions saw significant improvement from baseline to end of treatment [33]. The symptom scores reported on the BDI-II and the BAI at baseline in Table 3 indicate moderate levels of depression and anxiety. Patients saw a marked improvement in symptoms over the observation period. Cohen’s d was > .80 on all measures from baseline to end of treatment. Similarly, all SRM showed values Page 8/21 Page 8/21 > .80 on all instruments. Correlation between baseline scores and scores at end of treatment were < .50 on the BDI-II (rs = .39), EQ-5D utility (rs = .34), and the EQ-VAS (rs = .31), but > .50 on the BAI (rs = .51). This indicates that the SRM was large for the BDI-II, EQ-5D utility, and the EQ VAS, whilst moderate for the BAI. > .80 on all instruments. Correlation between baseline scores and scores at end of treatment were < .50 on the BDI-II (rs = .39), EQ-5D utility (rs = .34), and the EQ-VAS (rs = .31), but > .50 on the BAI (rs = .51). This indicates that the SRM was large for the BDI-II, EQ-5D utility, and the EQ VAS, whilst moderate for the BAI. Change in depression, anxiety and the EQ-5D during treatment The P-values denote significance of a non-parametric test of trends across ordered groups, developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Table 3 Instrument scores at baseline and end of treatment with ES and SRM (N = 416) Baseline End of treatment d SRM Mean SD Mean SD Anxiety (BAI) 18.48 10.27 6.49 6.47 1.39 1.35 Depression (BDI-II) 26.42 8.77 10.13 8.84 1.84 1.67 EQ-5D value .630 .189 .816 .153 1.07 .93 EQ VAS 54.58 17.13 74.71 14.80 1.25 1.06 Note. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II; ES, Effect Size (reported in Cohen’s d); SRM, Standardised response mean. Correlation of change scores Baseline End of treatment p value 4 0.97 3 0.73 Anxiety / depression No problems Slight problems Moderate problems Severe problems Extremely anxious or depressed 13 3.17 160 38.83 < .001 106 25.85 187 44.39 179 43.66 53 12.86 105 25.61 11 2.67 7 1.71 1 0.24 Note. Proportion of patients reporting the various levels of severity on the EQ-5D dimensions at baseline, and at end of treatment. The P-values denote significance of a non-parametric test of trends across ordered groups, developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Note. Proportion of patients reporting the various levels of severity on the EQ-5D dimensions at baseline, and at end of treatment. The P-values denote significance of a non-parametric test of trends across ordered groups, developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Table 3 Instrument scores at baseline and end of treatment with ES and SRM (N = 416) Baseline End of treatment d SRM Mean SD Mean SD Anxiety (BAI) 18.48 10.27 6.49 6.47 1.39 1.35 Depression (BDI-II) 26.42 8.77 10.13 8.84 1.84 1.67 EQ-5D value .630 .189 .816 .153 1.07 .93 EQ VAS 54.58 17.13 74.71 14.80 1.25 1.06 Note. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II; ES, Effect Size (reported in Cohen’s d); SRM, Standardised response mean. Table 3 Note. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II; ES, Effect Size (reported in Cohen’s d); SRM, Standardised response mean. Change in depression, anxiety and the EQ-5D during treatment Page 9/21 Table 2 Table 2 P 10/21 Distribution of EQ-5D dimensions as reported by patients (N = 416) Baseline End of treatment p value n % n % Mobility No problems Slight problems Moderate problems Severe problems Unable to walk about 323 78.59 364 88.35 < .001 62 15.09 39 9.47 20 4.87 9 2.18 6 1.46 . . . . . . Self-care No problems Slight problems Moderate problems Severe problems Unable to wash or dress 350 85.37 398 96.60 < .001 52 12.68 11 2.67 6 1.46 2 0.49 2 0.49 1 0.49 . . . . Usual activities No problems Slight problems Moderate problems Severe problems Unable to do usual activities 83 20.19 258 62.93 < .001 161 39.17 111 27.07 113 27.49 35 8.54 50 12.17 6 1.46 4 0.97 . . Pain / discomfort No problems Slight problems Moderate problems Severe problems Extreme pain or discomfort 93 22.63 206 50.00 < .001 177 43.07 151 36.65 113 27.49 45 10.92 24 5.84 7 1.70 Note. Proportion of patients reporting the various levels of severity on the EQ-5D dimensions at baseline, and at end of treatment. The P-values denote significance of a non-parametric test of trends across ordered groups, developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Distribution of EQ-5D dimensions as reported by patients (N = 416) Note. Proportion of patients reporting the various levels of severity on the EQ-5D dimensions at baseline, and at end of treatment. The P-values denote significance of a non-parametric test of trends across ordered groups, developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Note. Proportion of patients reporting the various levels of severity on the EQ-5D dimensions at baseline, and at end of treatment. The P-values denote significance of a non-parametric test of trends across ordered groups, developed by Cuzick, similar to the Wilcoxon rank-sum test [33]. Page 10/21 Page 10/21 Baseline End of treatment p value 4 0.97 3 0.73 Anxiety / depression No problems Slight problems Moderate problems Severe problems Extremely anxious or depressed 13 3.17 160 38.83 < .001 106 25.85 187 44.39 179 43.66 53 12.86 105 25.61 11 2.67 7 1.71 1 0.24 Note. Proportion of patients reporting the various levels of severity on the EQ-5D dimensions at baseline, and at end of treatment. Correlation of change scores Spearman’s rank order correlations are shown in Table 4. Note that the BAI and the BDI-II indicate worse health status with higher scores, whereas the reverse is true for the EQ-5D value and the EQ VAS. The EQ- 5D value change showed moderate correlations with both the BDI-II, the BAI, and the EQ VAS. A weak correlation was seen between the EQ VAS and the BAI. Page 11/21 Table 4 Spearman’s correlation of change scores (N = 416) BAI ∆ BDI-II ∆ EQ-5D value ∆ BAI ∆ BDI-II ∆ .48 EQ-5D value ∆ − .41 − .48 EQ VAS ∆ − .32 − .56 .51 Note. Change scores calculated by subtracting the score at end of treatment from the score at baseline. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II. Spearman’s correlation 0 – .39 are considered weak, .40 − .69 moderate [37]. Spearman’s correlation of change scores (N = 416) Note. Change scores calculated by subtracting the score at end of treatment from the score at baseline. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II. Spearman’s correlation 0 – .39 are considered weak, .40 − .69 moderate [37]. Note. Change scores calculated by subtracting the score at end of treatment from the score at baseline. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II. Spearman’s correlation 0 – .39 are considered weak, .40 − .69 moderate [37]. ROC curve analysis For the total sample, the ROC curve analysis showed that the EQ-5D value consistently distinguished between “Recovered” and “Improved” or “Unchanged” patients according the BDI-II or BAI, AUROC ranging from .72 – .84 (Table 5). The AUC did not adequately distinguish between “Improved” and “Unchanged” on either measure, AUROC ranged from .49 – .61. Page 12/21 Table 5 Area under the receiver operating characteristic curve (AUROC) using non-parametric ROC analysed (N = 416) BDI-II BAI n AUROC SE 95 % CI n AUROC SE 95 % CI Overall Recovered vs. Unchanged Recovered vs. Improved Improved vs. Unchanged 329 0.81 .032 (0.75– 0.88) 215 0.82 .039 (0.75– 0.90) 314 0.78 .035 (0.71– 0.85) 210 0.72 .061 (0.60– 0.84) 121 0.52 .052 (0.48– 0.68) 49 0.64 .079 (0.49– 0.80) Depression diagnoses Recovered vs. Unchanged Recovered vs. Improved Improved vs. Unchanged 184 0.80 .045 (0.71– 0.88) 176 0.75 .047 (0.65– 0.84) 68 0.61 .070 (0.47– 0.75) Anxiety diagnoses Recovered vs. Unchanged Recovered vs. Improved Improved vs. Unchanged 147 0.83 (0.71– 0.95) 123 0.84 (0.70– 0.97) 52 0.53 (0.29– 0.77) Note. Area under the curve (AUROC) reported for receiver operating analyses. The BAI and the BDI-II were used as classifiers of whether patients were “Recovered”, “Improved”, or “Unchanged” at end of treatment. Analyses were run using the BAI and the BDI-II for the whole sample, using the BDI-II for only the patients with depression diagnoses, and using the BAI for only the patients with anxiety Note. Area under the curve (AUROC) reported for receiver operating analyses. The BAI and the BDI-II were used as classifiers of whether patients were “Recovered”, “Improved”, or “Unchanged” at end of treatment. Analyses were run using the BAI and the BDI-II for the whole sample, using the BDI-II for only the patients with depression diagnoses, and using the BAI for only the patients with anxiety diagnoses. Abbreviations: BAI, the Beck Anxiety Inventory; BDI-II, the Beck Depression Inventory-II. Page 13/21 The same pattern repeated when patients scores were analysed according to diagnoses. For patients with depression, the AUC was excellent when distinguishing between “Recovered” and “Unchanged” (.80) and acceptable distinguishing “Recovered” from “Improved” (.75), but ineffective separating “Improved” and “Unchanged” (.61). For patients with anxiety, the AUC showed excellent classification for both “Recovered” versus “Unchanged” (.83) and Recovered” versus “Improved” (.84). The test did not distinguish between “Improved” versus “Unchanged” (.53). ROC curve analysis Youden’s index indicated that an EQ-5D value of .768 had the highest combined sensitivity and specificity when identifying recovered patients. The value was the same for both depression and anxiety (Table 6). Table 6 The central range of operating characteristics of the EQ-5D value post-treatment for identifying recovered versus non-recovered patients (N = 416) EQ-5D cut-off Sensitivity Specificity J Correctly classified Depression .740 84.72 % 62.69 % 0.47 77.73 .750 81.25 % 62.69 % 0.44 75.36 .768 80.56 % 64.18 % 0.45 75.36 .819 74.31 % 67.16 % 0.41 72.04 .827 74.31 % 68.66 % 0.43 72.51 Anxiety .740 84.75 % 80.00 % 0.65 83.76 .750 83.70 % 80.00 % 0.64 82.91 .768 83.70 % 84.00 % 0.68 83.76 .819 63.04 % 84.00 % 0.47 67.52 .827 60.87 % 84.00 % 0.45 65.81 Note. J = Youden’s index. Depression and Anxiety recovery measured and classified using the BDI-II and the BAI, respectively. Note. J = Youden’s index. Depression and Anxiety recovery measured and classified using the BDI-II and the BAI, respectively. Discussion Our aim was to explore the responsiveness of the EQ-5D in patients receiving treatment for depression and anxiety. This was done by comparing change in the EQ-5D to change in the disorder-specific measures BDI-II and BAI. We hypothesised that the EQ-5D should show magnitude of change as the BDI-II and BAI during treatment. The ES was large (d > .80) for all measures, ranging from Cohen’s d 1.07–1.84. For the SRM, which accounts for variability in treatment response by dividing change scores by the Page 14/21 Page 14/21 standard deviation of change scores, the BDI-II, the EQ-5D value and the EQ VAS all showed large magnitude of change. The BAI showed moderate magnitude of change on the SRM when accounting for its higher correlation between baseline and end of treatment scores. Furthermore, the EQ-5D value change scores showed moderate correlation with the BDI-II and the BAI change scores. The hypothesis that the EQ-5D should show similar magnitude of change as the condition-specific measures during treatment thus seems confirmed. We then examined if the EQ-5D utility could correctly classify patients deemed as “Recovered” according to the condition-specific measures. Results from the ROC analyses indicate that this was the case: AUROC were from fair to good on all analyses when distinguishing “Recovered” patients from “Improved” or “Unchanged”. This was true for the total sample (AUROC .72 – .82), for patients with depression (AUROC .75 and .80), and for patients with anxiety (AUROC .83 and .84). In a similarly consistent pattern, the EQ-5D showed poor ability to distinguish between “Improved” and “Unchanged” patients for the total sample, for depression, and for anxiety, (AUROC .52 – .61). The ability of the EQ-5D to consistently identify recovered patients indicates that our second hypothesis was confirmed. We also calculated Youden’s index, as this may be informative for clinicians and serve as a reference for future research. For recovery from both depression and anxiety, cut-off point as defined by highest combined sensitivity and specificity was an EQ-5D utility ≥ .768 at end of treatment. Data on the responsiveness of the five-level version of the EQ-5D in mental health is limited, though cross- sectional measures have indicated moderate to good correlation with condition-specific measures [10]. Comparing to the three-level version, one study found moderate responsiveness to anxiety disorders. Similar to the present study, patients were classified as having either “more”, “constant”, or “less anxiety” according to the BAI. Discussion This suggests that the EQ-5D can be a valid and useful tool for evaluating impact of disease and benefit of treatment for these patients, for instance through estimating QALYs. It also suggests that the EQ-5D can be useful in clinical setting, such as evaluating treatment outcomes. Discussion T-tests showed significant differences in change scores for the EQ-5D value and the EQ VAS. However, that study found that the SRM were moderate to small, and ES were large for the EQ-5D value only when patients were deteriorated [17]. Reviews of the literature on the three-level version have indicated reasonable responsiveness in depression and anxiety [9], suggesting that the five-level may have similar properties. One recent study compared the responsiveness of the three-level and five-level versions of Anxiety/depression dimension for mental health patients. Although the five-level version was found to be more responsive, both showed limited ability to capture changes in mental health [18]. The Anxiety/depression dimension did show significant change from baseline to end of treatment in the present study. Future research may determine if it is useful as a standalone tool. A previous cross-sectional study did find that the EQ-5D value could screen for depression and anxiety in patients with type 2 diabetes [19]. In the present study, the EQ-5D value showed similar performance in a longitudinal design in patients with depression and anxiety as primary diagnoses. That the EQ-5D value may perform better than the Anxiety / depression dimension alone is perhaps reasonable, as it may better capture the wide-ranging impact of depression and anxiety on health and quality of life [4, 5]. Page 15/21 Page 15/21 The EQ-5D is increasingly used when evaluating health status in surveys and clinical trials [6], and decision-making bodies recommend its use in evaluating health technologies [7, 44]. Demonstrating its validity in diverse patient groups is therefore essential for assisting sound decision-making when allocating health care resources. In this study, the EQ-5D showed good responsiveness to change for patients with depression and anxiety. This suggests that the EQ-5D can be a valid and useful tool for evaluating impact of disease and benefit of treatment for these patients, for instance through estimating QALYs. It also suggests that the EQ-5D can be useful in clinical setting, such as evaluating treatment outcomes. The EQ-5D is increasingly used when evaluating health status in surveys and clinical trials [6], and decision-making bodies recommend its use in evaluating health technologies [7, 44]. Demonstrating its validity in diverse patient groups is therefore essential for assisting sound decision-making when allocating health care resources. In this study, the EQ-5D showed good responsiveness to change for patients with depression and anxiety. Conflict of interest None declared. Code availability Any inquiry can be directed to kenneth.sandin@diakonsyk.no Availability of data material The patient data used for this study is not readily available as the patients have not given consent for use or distribution beyond the research at Diakonhjemmet Hospital. Inquiries can be directed to kenneth.sandin@diakonsyk.no. Strengths and limitations The main strength of the study is adding to a limited evidence-base concerning the responsiveness of the five-level version of the EQ-5D in patients with depression and anxiety. The study included a fairly large clinical sample who were assessed and diagnosed by clinical psychologists before entering treatment. We can thus be reasonably certain of the clinical characteristics of the sample. The study took part in a national health service clinic, suggesting that these patients are somewhat representative of clinical populations with depression and anxiety in Norway. The patients saw substantial treatment gains as reflected by the large ES and SRM, which gave an opportunity for evaluating the ability of the EQ-5D to identify recovered patients. Several limitations to the study have to be considered. The study only included patients who completed treatment, and treatment gains were large. The study could therefore not evaluate the ability of the EQ-5D to detect smaller changes, that still may be of importance to patients. A related limitation is that the large rate of recovered patients in the study meant that “Unchanged” patients formed a small subgroup. The findings concerning the unchanged patients should be treated with caution. We also lack adequate data to determine if the EQ-5D would be equally responsive to deterioration as improvement during treatment. The study also lacked data on comorbidity. The current study uses the UK value set for converting to EQ-5D value scores, as there is currently no Norwegian value set available. Choice of value sets has shown to influence the estimation of QALYs, which suggests that it would be useful to replicate the present findings when a Norwegian value set is available [13]. As new measures of health status become available, such as the Recovering Quality of Life (ReQoL) or capability-oriented instruments, it will be important to compare and contrast these against the EQ-5D to judge which instrument is best suited for patients with depression and anxiety [45, 46]. There is evidence that a wide range of outcomes that are important to patients with mental health problems are not adequately captured by commonly used instruments [5, 8]. Further research is needed to assess whether the EQ-5D could reflect key changes in a wider range of outcomes [5], or if other instruments or bolt-on dimensions may be better for capturing psycho-social factors of importance to patients [47]. Page 16/21 Page 16/21 Page 16/21 Authors’ contribution All authors contributed to the conceptualisation and design of the study. KS led the writing of the manuscript and is the principal author of the funding application. GS contributed to conceptualisation, analyses, and writing. RGHG, KO, MTB, and SER contributed to design of study and intervention, data management, and writing. OH is the project manager and contributed to design, analyses and writing. All authors read and approved the final manuscript. Funding The study was primarily sponsored by Diakonhjemmet Hospital, with additional funding from the Southern and Eastern Norway Regional Health Authority, and Stiftelsen Dam (2018/FO201448), a not for profit trust that funds health research and innovation projects. Conclusion The findings in this study suggest that the EQ-5D may be responsive to change in health status for patients receiving treatment for depression and anxiety. The EQ-5D showed similar magnitude of change as the condition-specific measures and was also able to consistently identify patients who had recovered from depression and anxiety. Responsiveness of the EQ-5D is likely sensitive to context, and these findings should be replicated in other samples. Still, these findings suggest that the EQ-5D may be a useful tool for evaluating outcomes of treatment for patients with depression and anxiety. Consent for publication All participants gave written, informed consent for use of data for research and publication. Ethics approval This study was approved as a health service study by the Norwegian Data Protection Authority. The data used in the present study is part of ongoing routine data collection, and no further approval is needed beyond consent from the individual patient. Data collection and security in the present study was Page 17/21 managed by Diakonhjemmet Hospital, and approval of data handling was granted by Oslo University Hospital, approval number 2015/15606. managed by Diakonhjemmet Hospital, and approval of data handling was granted by Oslo University Hospital, approval number 2015/15606. Acknowledgement The authors would like to thank the patients, next of kin, and user representatives for their participation in the study, and for their valuable feedback. We would also like to thank the staff at the Division of Mental Health and Substance Abuse at Diakonhjemmet Hospital. References 1. OECD. Health at a Glance 2019: OECD Indicators. Paris: OECD Publishing; 2019. 2. Bloom DE, Cafiero ET, Jané-Llopis E, Abrahams-Gessel S, Bloom LR, Fathima S, Feigl AB, Gaziano T, Mowafi M, Pandya A, Prettner K, Rosenberg L, Seligman B, Stein AZ, Weinstein C. The Global Economic Burden of Noncommunicable Diseases. Geneva; 2011. 2. 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https://openalex.org/W2989451497	https://europepmc.org/articles/pmc6935675?pdf=render	English	null	How much liver does a person need?	Clinical case reports	2,019	cc-by	825	K E Y W O R D S abdominal compression, liver cyst C L I N I C A L I M A G E C L I N I C A L I M A G E Igor Alexander Harsch Igor Alexander Harsch Department of Internal Medicine II, Division of Endocrinology and Metabolism, Thuringia Clinic “Georgius Agricola”, Saalfeld, Germany Correspondence Correspondence Igor Alexander Harsch, Department of Internal Medicine II, Division of Endocrinology and Metabolism, Thuringia Clinic “Georgius Agricola”, Rainweg 68, D-07318 Saalfeld/Saale, Germany. Email: iharsch@thueringen-kliniken.de K E Y W O R D S abdominal compression, liver cyst K E Y W O R D S abdominal compression, liver cyst Abstract In patients who had no medical care for years, it is wise to expect the unexpected. Here, a gigantic liver cyst compressed abdominal organs, vessels, and the gut. The patient is thus far doing well in a nursing home, exemplifying the low amount of residual liver tissue necessary for survival. Received: 12 September 2019 \| Revised: 10 October 2019 \| Accepted: 21 October 2019 Received: 12 September 2019 \| Revised: 10 October 2019 \| Accepted: 21 October 2019 Received: 12 September 2019 \| Revised: 10 October 2019 \| Accepted: 21 October 2019 DOI: 10.1002/ccr3.2552 2598 \| wileyonlinelibrary.com/journal/ccr3 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. \| This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. Igor Alexander Harsch https://orcid. org/0000-0003-2716-0066 Igor Alexander Harsch https://orcid. org/0000-0003-2716-0066 Igor Alexander Harsch https://orcid. org/0000-0003-2716-0066 REFERENCES The author is grateful to Roman Harsch (BA Translation) for language editing. 1. Larssen TB, Rørvik J, Hoff SR, Horn A, Rodendahl K. The occur- rence of asymptomatic and symptomatic simple hepatic cysts. A prospective, hospital-based study. Clin Radiol. 2005;60:1026-1029. 1. Larssen TB, Rørvik J, Hoff SR, Horn A, Rodendahl K. The occur- rence of asymptomatic and symptomatic simple hepatic cysts. A prospective, hospital-based study. Clin Radiol. 2005;60:1026-1029. CONFLICT OF INTEREST 2. Guglielmi A, Ruzzenente A, Conci S, Valdegamberi A, Iacono C. How much remnant is enough in liver resection? Dig Surg. 2012;29:6-17. There are no conflicts of interest. AUTHOR CONTRIBUTIONS IAH wrote the article and has accountability for all aspects of the work. How to cite this article: Harsch IA. How much liver does a person need? Clin Case Rep. 2019;7:2598– 2599. https://doi.org/10.1002/ccr3.2552 How to cite this article: Harsch IA. How much liver does a person need? Clin Case Rep. 2019;7:2598– 2599. https://doi.org/10.1002/ccr3.2552 1 \| CASE REPORT 20%-30% is considered sufficient for functional maintenance in healthy people.2 Unfortunately, self-care by the patient was no longer possible; he was transferred to a nursing home. He Liver cysts are not an uncommon finding. In elderly patients without medical care for long time, they may reach critical di- ameters combined with a compression of abdominal organs, vessels, and the gut. Other potential problems are rupture or bleeding of the cyst(s) or an infection of the cyst(s). FIGURE 1 Computed tomography of the abdomen, coronal section: Huge liver cyst with displacement of the upper abdominal organs, gastrointestinal tract, and intra-abdominal vessels An 82-year-old patient suffered a syncope and was found bedraggled at home in February 2019. The patient had hardly any social contacts in recent years and had not been to a doctor for a long time. He had anemia, but normal serum transaminase and blood coagulation levels. Only cholines- terase was reduced with 77 μkat/L (normal range: 89-215). An ultrasonography revealed a huge liver cyst, the computed tomography scan of the abdomen revealed the cystic lesion sized 25 × 19 cm, filling almost the entire right lobe of the liver (Figure 1). It caused a diaphragmatic elevation on the right and a compression of abdominal organs. There was no hint of malignancy in other organs. The patient declined a puncture to achieve at least a transient volume relief. Surgery was not possible in his general condition. Liver cysts are frequent: In an ultrasound screening by Larssen et al1 of 1541 patients, cysts are described in 11.3%. In more than 90% of cases, they were 3 cm in diameter or smaller, the incidence increasing with age. Usually, no ther- apy is required. In liver surgery, a residual tissue mass of FIGURE 1 Computed tomography of the abdomen, coronal section: Huge liver cyst with displacement of the upper abdominal organs, gastrointestinal tract, and intra-abdominal vessels Clin Case Rep. 2019;7:2598–2599. 2599 HARSCH is doing well there, and the case described here exemplifies how little liver remnant parenchyma is needed to survive. ETHICAL APPROVAL The patient gave written consent to report his case and the imaging.
https://openalex.org/W2922806121	https://cnrs.hal.science/hal-03100146/document	English	null	Pixel: a content management platform for quantitative omics data	PeerJ	2,019	cc-by	9,754	To cite this version: Thomas Denecker, William Durand, Julien Maupetit, Charles Hébert, Jean-Michel Camadro, et al.. Pixel: a content management platform for quantitative omics data. PeerJ, 2019, 7, pp.e6623. 10.7717/peerj.6623. hal-03100146 Pixel: a content management platform for quantitative omics data as Denecker, William Durand, Julien Maupetit, Charles Hébert, Jean-Michel Camadro, Pierre Poulain, Gaëlle Lelandais Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03100146 https://cnrs.hal.science/hal-03100146v1 Submitted on 6 Jan 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. Distributed under a Creative Commons Attribution 4.0 International License ABSTRACT Background. In biology, high-throughput experimental technologies, also referred as ‘‘omics’’ technologies, are increasingly used in research laboratories. Several thousands of gene expression measurements can be obtained in a single experiment. Researchers are routinely facing the challenge to annotate, store, explore and mine all the biological information they have at their disposal. We present here the Pixel web application (Pixel Web App), an original content management platform to help people involved in a multi-omics biological project. Methods. The Pixel Web App is built with open source technologies and hosted on the collaborative development platform GitHub (https://github.com/Candihub/pixel). It is pp p g collaborative development platform GitHub (https://github.com/Candihub/pixel). It is written in Python using the Django framework and stores all the data in a PostgreSQL database. It is developed in the open and licensed under the BSD 3-clause license. The Pixel Web App is also heavily tested with both unit and functional tests, a strong code coverage and continuous integration provided by CircleCI. To ease the development and the deployment of the Pixel Web App, Docker and Docker Compose are used to bundle the application as well as its dependencies. Submitted 5 October 2018 Accepted 14 February 2019 Published 27 March 2019 Corresponding authors Pierre Poulain, pierre.poulain@univ-paris-diderot.fr Gaëlle Lelandais, gaelle.lelandais@u-psud.fr Academic editor Gerard Lazo Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.6623 Copyright Submitted 5 October 2018 Accepted 14 February 2019 Published 27 March 2019 Corresponding authors Pierre Poulain, pierre.poulain@univ-paris-diderot.fr Gaëlle Lelandais, gaelle.lelandais@u-psud.fr Academic editor Gerard Lazo Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.6623 Results. The Pixel Web App offers researchers an intuitive way to annotate, store, explore and mine their multi-omics results. It can be installed on a personal computer or on a server to fit the needs of many users. In addition, anyone can enhance the application to better suit their needs, either by contributing directly on GitHub (encouraged) or by extending Pixel on their own. The Pixel Web App does not provide any computational programs to analyze the data. Still, it helps to rapidly explore and mine existing results and holds a strategic position in the management of research data. Subjects Computational Biology, Genomics, Data Science Keywords Data cycle analyses, Omics, Open source, Pixel Web App INTRODUCTION How to cite this article Denecker T, Durand W, Maupetit J, Hébert C, Camadro J-M, Poulain P, Lelandais G. 2019. Pixel: a content man- agement platform for quantitative omics data. PeerJ 7:e6623 http://doi.org/10.7717/peerj.6623 Pixel: a content management platform for quantitative omics data Thomas Denecker1,, William Durand2,, Julien Maupetit2,, Charles Hébert3, Jean-Michel Camadro4, Pierre Poulain4, and Gaëlle Lelandais1,* Thomas Denecker1,, William Durand2,, Julien Maupetit2,, Charles Hébert3, Jean-Michel Camadro4, Pierre Poulain4, and Gaëlle Lelandais1,* 1 CEA, CNRS, Univ. Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France 2 TailorDev SAS, Clermont-Ferrand, France 3 BIOROSETICS, Houilles, France 4 CNRS, Univ. Paris Diderot, Institut Jacques Monod (IJM), Paris, France * These authors contributed equally to this work. 4 CNRS, Univ. Paris Diderot, Institut Jacques Monod (IJM), Paris, France * These authors contributed equally to this work. ABSTRACT Subjects Computational Biology, Genomics, Data Science Keywords Data cycle analyses, Omics, Open source, Pixel Web App INTRODUCTION ‘‘Secondary data’’ are produced upon analysis of primary data. It can be the counts of reads per genes for HTS results or the abundance values per proteins for MS results. In multi-omics datasets analysis, combining secondary data is essential to answer specific biological questions. It can be typically, the identification of differentially expressed genes (or proteins) between several cell growth conditions from transcriptomics (or proteomics) datasets, or the identification of cellular functions that are over-represented in a list of genes (or proteins). In that respect, secondary data can be analysed and re-analysed within a multitude of analytical strategies, introducing the idea of data analysis cycle. The researcher is thus constantly facing the challenge to properly annotate, store, explore and mine all the biological data he/she has at his/her disposal in a multi-omics project. This challenge is directly related to the ability to extract as much information as possible from the produced data, but also to the crucial question of doing reproducible research. A Nature’s survey presented in 2016 indicates that more than 70% of the questioned researchers already experienced an impossibility to reproduce published results, and more than half of them were not able to reproduce their own experiments (Baker, 2016) This MinION, Blow, 2013). The number of HT applications in biology has grown so rapidly in the past decade that it is hard to not feel overwhelmed (Hadfield & Retief, 2018) (‘‘The data deluge, 2012’’). It seems possible to address in any organism, any biological question through an ‘‘omics’’ perspective, providing the right HT material and method are found. If HTS is often put at the forefront of ‘‘omics’’ technologies (essentially genomics and transcriptomics, Reuter, Spacek & Snyder, 2015), other technologies must be considered. Mass spectrometry (MS) for instance, enables HT identification and quantification of proteins (proteomics). Metabolomics and lipidomics are other derived applications of MS to characterize quantitative changes in small-molecular weight cellular components (Smith et al., 2014). Together, they all account for complementary ‘‘omics area’’ with the advantage to quantify distinct levels of cellular components (transcripts, proteins, metabolites, etc.). q y p ( p , p , , ) Integration of datasets issued from different HT technologies (termed as multi-omics datasets) represents a challenging task from a statistical and methodological point of view (Huang, Chaudhary & Garmire, 2017). It implies the manipulation of two different types of data. INTRODUCTION The first type is the ‘‘primary data’’, which correspond to raw experimental results. It can be FASTQ files for sequencing technology (Cock et al., 2010) or mzML files for MS (Martens et al., 2011). These files can be stored in public repositories such as SRA (Leinonen, Sugawara & Shumway, 2011), GEO (Clough & Barrett, 2016), PRIDE (Martens et al., 2005) or PeptideAtlas (Desiere et al., 2006). Analyses of primary data rely on standard bioinformatics protocols that for instance, perform quality controls, correct experimental bias or convert files from a specific format to another. A popular tool to analyse primary data is Galaxy (Afgan et al., 2016), which is an open web-based platform. ‘‘Secondary data’’ are produced upon analysis of primary data. It can be the counts of reads per genes for HTS results or the abundance values per proteins for MS results. In multi-omics datasets analysis, combining secondary data is essential to answer specific biological questions. It can be typically, the identification of differentially expressed genes (or proteins) between several cell growth conditions from transcriptomics (or proteomics) datasets, or the identification of cellular functions that are over-represented in a list of genes (or proteins). In that respect, secondary data can be analysed and re-analysed within a multitude of analytical strategies, introducing the idea of data analysis cycle. The researcher is thus constantly facing the challenge to properly annotate, store, explore and mine all the biological data he/she has at his/her disposal in a multi-omics project. This challenge is directly related to the ability to extract as much information as possible from the produced data, but also to the crucial question of doing reproducible research. A Nature’s survey presented in 2016 indicates that more than 70% of the questioned researchers already experienced an impossibility to reproduce published results, and more than half of them were not able to reproduce their own experiments (Baker, 2016). This last point is intriguing. If experimental biology can be subjected to random fluctuations hardly difficult to control, computational biology should not. Running the same software on the same input data is expected to give the same results. In practice, replication in computational science is harder than people generally think (see Mesnard & Barba, 2017 as an illustration). It requires to adopt good practices for reproducible-research on a daily basis, and not only when the final results are about to be published. Initiatives Denecker et al. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 INTRODUCTION In biology, high throughput (HT) experimental technologies—also referred as ‘‘omics’’— are routinely used in an increasing number of research teams. Financial costs associated to HT experiments have been considerably reduced in the last decade (Hayden, 2014) and the trend in HT sequencing (HTS) is now to acquire benchtop machines designed for individual research laboratories (for instance Illumina NextSeq500 or Oxford Nanopore Technologies Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS How to cite this article Denecker T, Durand W, Maupetit J, Hébert C, Camadro J-M, Poulain P, Lelandais G. 2019. Pixel: a content man- agement platform for quantitative omics data. PeerJ 7:e6623 http://doi.org/10.7717/peerj.6623 MinION, Blow, 2013). The number of HT applications in biology has grown so rapidly in the past decade that it is hard to not feel overwhelmed (Hadfield & Retief, 2018) (‘‘The data deluge, 2012’’). It seems possible to address in any organism, any biological question through an ‘‘omics’’ perspective, providing the right HT material and method are found. If HTS is often put at the forefront of ‘‘omics’’ technologies (essentially genomics and transcriptomics, Reuter, Spacek & Snyder, 2015), other technologies must be considered. Mass spectrometry (MS) for instance, enables HT identification and quantification of proteins (proteomics). Metabolomics and lipidomics are other derived applications of MS to characterize quantitative changes in small-molecular weight cellular components (Smith et al., 2014). Together, they all account for complementary ‘‘omics area’’ with the advantage to quantify distinct levels of cellular components (transcripts, proteins, metabolites, etc.). Integration of datasets issued from different HT technologies (termed as multi-omics datasets) represents a challenging task from a statistical and methodological point of view (Huang, Chaudhary & Garmire, 2017). It implies the manipulation of two different types of data. The first type is the ‘‘primary data’’, which correspond to raw experimental results. It can be FASTQ files for sequencing technology (Cock et al., 2010) or mzML files for MS (Martens et al., 2011). These files can be stored in public repositories such as SRA (Leinonen, Sugawara & Shumway, 2011), GEO (Clough & Barrett, 2016), PRIDE (Martens et al., 2005) or PeptideAtlas (Desiere et al., 2006). Analyses of primary data rely on standard bioinformatics protocols that for instance, perform quality controls, correct experimental bias or convert files from a specific format to another. A popular tool to analyse primary data is Galaxy (Afgan et al., 2016), which is an open web-based platform. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 INTRODUCTION (2019), PeerJ, DOI 10.7717/peerj.6623 2/18 to improve computational reproducibility exists (Peng, 2011; Stodden, Guo & Ma, 2013; to improve computational reproducibility exists (Peng, 2011; Stodden, Guo & Ma, 2013; to improve computational reproducibility exists (Peng, 2011; Stodden, Guo & Ma, 2013; Vasilevsky et al., 2017; Rougier et al., 2017; Stodden, Seiler & Ma, 2018), and today it is clear that the data alone are not enough to sustain scientific claims. Comments, explanations, software source codes and tests are prerequisites to ensure that an original research can be replicated by anyone, anytime, anywhere. Vasilevsky et al., 2017; Rougier et al., 2017; Stodden, Seiler & Ma, 2018), and today it is clear that the data alone are not enough to sustain scientific claims. Comments, explanations, software source codes and tests are prerequisites to ensure that an original research can be replicated by anyone, anytime, anywhere. We developed the Pixel web application (Pixel Web App) with these ideas in mind. It is a content management platform to help the researchers involved in a multi-omics biological project, to collaboratively work with their HT data. The Pixel Web App does not store the primary data. It is rather focused on annotation, storage and exploration of secondary data (see Fig. 1). These explorations represent critical steps to answer biological questions and need to be carefully annotated and recorded to be further exploited in the context of new biological questions. The Pixel Web App helps the researcher to specify necessary information required to replicate multi-omics results. We added an original hierarchical system of tags, which allows to easily explore and select multi-omics results stored in the system and to use them for new interpretations. The Pixel Web App can be installed on any individual computer (for a single researcher for instance), or on a web server for collaborative work between several researchers or research teams. The entire software has been developed with high quality programming standards and complies to major rules of open-source development (Taschuk & Wilson, 2017). The Pixel project is available on GitHub at https://github.com/Candihub/pixel, where full source code and detailed documentation are provided. We present in this article the Pixel Web App design and implementation. We provide a simple case study, emblematic of our daily use of the Pixel Web App, with the exploration of results issued from transcriptomics and proteomics experiments performed in the pathogenic yeast Candida glabrata. MATERIAL AND METHODS Stack overview The Pixel Web App provides researchers an intuitive way to annotate, store, explore and mine their secondary data analyses, in multi-omics biological projects. It is built upon mainstream open source technologies (see Fig. 2). Source code is hosted on the collaborative development platform GitHub (https://github.com/) and continuous integration is provided by CircleCI (https://circleci.com/). More precisely, the Pixel Web App uses the Python Django framework. This framework is based on a model- template-view architecture pattern, and data are stored in a PostgreSQL (https: //www.postgresql.org/) database. We have built a docker image for the Pixel Web App. Other containers, Nginx (to serve the Django application) and PostgreSQL rely on official docker images. Each installation/deployment will result in the creation/execution of three docker instances: one for the Pixel Web App, one for the PostgreSQL database and one for the Nginx web server. In case of multiple installations, each trio of docker instances is fully isolated, meaning that data are not shared across multiple Pixel Web App installations. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 3/18 Figure 1 Dataset flow through the Pixel Web App. (A) Different types of datasets, which are managed in a multi-omics biological project. Primary and secondary datasets are two types of information arising from HT experimental technologies (see ‘Introduction’). Only secondary data and their associated Pixel Sets are stored in the Pixel Web App. Note that several Pixel Sets can emerge from multiple secondary data analyses. They comprise quantitative values (Value) together with quality scores (QS) for several hundred of different ‘‘Omics Units’’ elements (for instance mRNA or proteins, see the main text). Omics Units are identified with a unique identifier (ID). (B) Screenshot of the home page of the Pixel web interface. (C) Schematic representation of the data analysis cycles that surrounds the integration of Pixel Sets in the Pixel Web App (see the main text). Full-size DOI: 10.7717/peerj.6623/fig-1 Figure 1 Dataset flow through the Pixel Web App. (A) Different types of datasets, which are managed in a multi-omics biological project. Primary and secondary datasets are two types of information arising from HT experimental technologies (see ‘Introduction’). Only secondary data and their associated Pixel Sets are stored in the Pixel Web App. Note that several Pixel Sets can emerge from multiple secondary data analyses. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Stack overview They comprise quantitative values (Value) together with quality scores (QS) for several hundred of different ‘‘Omics Units’’ elements (for instance mRNA or proteins, see the main text). Omics Units are identified with a unique identifier (ID). (B) Screenshot of the home page of the Pixel web interface. (C) Schematic representation of the data analysis cycles that surrounds the integration of Pixel Sets in the Pixel Web App (see the main text). Full-size DOI: 10.7717/peerj.6623/fig-1 4/18 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 4/18 Figure 2 Stack overview of the Pixel Web App. Open source solutions used to develop Pixel are shown here. They are respectively used for the software development and test (blue section), the data storage (green section) and the web application for both staging and production (orange section). Full-size DOI: 10.7717/peerj.6623/fig-2 Figure 2 Stack overview of the Pixel Web App. Open source solutions used to develop Pixel are shown here. They are respectively used for the software development and test (blue section), the data storage (green section) and the web application for both staging and production (orange section). Full-size DOI: 10.7717/peerj.6623/fig-2 Technical considerations Docker images The Pixel Web App is built on containerization paradigm (see Fig. 2). It relies on Docker (https://www.docker.com/), i.e., a tool which packages an application and its dependencies in an image that will be run as a container. Docker helps developers to build self-contained images to run a software. These images are downloaded on the host system and used to build the Pixel Web App. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Functionalities to annotate, store, explore and mine Pixel Sets Pixel Sets are obtained from secondary data analyses (see Fig. 1A). Their manipulation with the Pixel Web App consists in (i) their annotation, (ii) their storage in a database, (iii) their exploration and (iv) their mining (see Fig. 1C). This represents a cycle of multiple data analyses, which is essential in any multi-omics biological project. These different steps are detailed in the following. Minimal conﬁguration and dependencies The Pixel Web App can be deployed on Linux and MacOS operating systems (OS). Deployment on Windows is possible, but this situation will not be described here. Minimal requirements are: (i) 64 bits Unix-based OS (Linux/MacOS), (ii) Docker community edition >v18, (iii) Internet access (required in order to download the Docker images) and (iv) [optional] a web server (Apache or Nginx) configured as a reverse proxy. 5/18 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 5/18 A step-by-step tutorial to deploy the Pixel Web App can be found in the project repository (https://github.com/Candihub/pixel/blob/master/docs-install/how-to-install.md) t th ith d l i t T i thi i t th f ll i t together with a deploy script. To summarize, this script runs the following steps: • Pull a tagged image of Pixel (web, see docker-composer file), • Start all instances (web, db and proxy) recreating the proxy and web instances. Collect all static files from the Django app. These files will be served by the proxy instance. • Migrate the database schema if needed (to preserve existing data). Note that further technical considerations and full documentation can be found on GitHub repository associated to the Pixel project (https://github.com/Candihub/pixel/ tree/master/docs). RESULTS Functionalities to annotate, store, explore and mine Pixel Sets Installation A step-by-step tutorial to deploy the Pixel Web App can be found in the project repository (https://github.com/Candihub/pixel/blob/master/docs-install/how-to-install.md) together with a deploy script. To summarize, this script runs the following steps: RESULTS Deﬁnition of terms: Omics Unit, Pixel and Pixel Set , In the Pixel Web App, the term ‘‘Omics Unit’’ refers to any cellular component, from any organism, which is of interest for the user. The type of Omics Unit depends on the HT experimental technology (transcriptomic, proteomic, metabolomic, etc.) from which primary and secondary datasets were collected and derived (Fig. 1A). In this context, classical Omics Units can be transcripts or proteins, but any other cellular component can be defined as, for instance, genomic regions with ‘‘peaks’’ in case of ChIPseq data analyses (Merhej et al., 2014). A ‘‘Pixel’’ refers to a quantitative measurement of a cellular activity associated to a single Omics Unit, together with a quality score (see Fig. 1A). Quantitative measurement and quality score are results of statistical analyses performed on secondary datasets, e.g., search for differentially expressed genes (Seyednasrollah, Laiho & Elo, 2015). A set of Pixels obtained from a single secondary data analysis of HT experimental results is referred as a ‘‘Pixel Set’’ (see Fig. 1A). Pixel Sets represent the central information in the Pixel Web App and functionalities to annotate, store, explore and mine multi-omics biological data were designed according to this concept (see below). In the Pixel Web App, the term ‘‘Omics Unit’’ refers to any cellular component, from any organism, which is of interest for the user. The type of Omics Unit depends on the HT experimental technology (transcriptomic, proteomic, metabolomic, etc.) from which primary and secondary datasets were collected and derived (Fig. 1A). In this context, classical Omics Units can be transcripts or proteins, but any other cellular component can be defined as, for instance, genomic regions with ‘‘peaks’’ in case of ChIPseq data analyses (Merhej et al., 2014). A ‘‘Pixel’’ refers to a quantitative measurement of a cellular activity associated to a single Omics Unit, together with a quality score (see Fig. 1A). Quantitative measurement and quality score are results of statistical analyses performed on secondary datasets, e.g., search for differentially expressed genes (Seyednasrollah, Laiho & Elo, 2015). A set of Pixels obtained from a single secondary data analysis of HT experimental results is referred as a ‘‘Pixel Set’’ (see Fig. 1A). Pixel Sets represent the central information in the Pixel Web App and functionalities to annotate, store, explore and mine multi-omics biological data were designed according to this concept (see below). Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Annotation of Pixel Sets (A) New data-sets are submitted following a dedicated workflow that comprised 6 successive actions named ‘‘Download’’, ‘‘Upload’’, ‘‘Meta’’, ‘‘Validation’’, ‘‘Tags’’ and ‘‘Import archive’’ (see 1). Several files are required (see 2): the secondary data from which the Pixel Sets were calculated, the notebook in which the procedure to compute Pixel Sets from secondary data is described and the Pixel Set files (2 files in this example). A progression bar allows the user to follow the sequence of the submission process. (B) Excel spreadsheet in which annotations of Pixel Sets are written. Information related to the Experiment (see 1), the Analysis (see 2) and the Pixel datasets (see 3) is required. Note that this file must be downloaded at the first step of the submission process (‘‘Download’’, see A), allowing several cells to be pre-filled with annotations stored in the database (see 4 as an illustration, with Omics area information). (C) All information filled in the Excel file (see B) is extracted and can be modified anytime through a dedicated web page as shown here. User can edit the Pixel Set (see 1), edit the analysis(see 2), edit the experiment (see 3) and add ‘‘Tags’’ (see Figure 4 Procedure to import new Pixel Sets in the Pixel Web App. (A) New data-sets are submitted following a dedicated workflow that comprised 6 successive actions named ‘‘Download’’, ‘‘Upload’’, ‘‘Meta’’, ‘‘Validation’’, ‘‘Tags’’ and ‘‘Import archive’’ (see 1). Several files are required (see 2): the secondary data from which the Pixel Sets were calculated, the notebook in which the procedure to compute Pixel Sets from secondary data is described and the Pixel Set files (2 files in this example). A progression bar allows the user to follow the sequence of the submission process. (B) Excel spreadsheet in which annotations of Pixel Sets are written. Information related to the Experiment (see 1), the Analysis (see 2) and the Pixel datasets (see 3) is required. Note that this file must be downloaded at the first step of the submission process (‘‘Download’’, see A), allowing several cells to be pre-filled with annotations stored in the database (see 4 as an illustration, with Omics area information). (C) All information filled in the Excel file (see B) is extracted and can be modified anytime through a dedicated web page as shown here. Annotation of Pixel Sets Annotation of Pixel Sets consists in tracking important details of Pixel Set production. For that, Pixel Sets are associated with metadata, i.e., Supplemental Information linked to the Pixel Sets. We defined minimal information necessary for relevant annotations of Pixel Sets (see Fig. 3). ‘‘Species’’, ‘‘Strain’’, ‘‘Omics Unit Type’’ and ‘‘Omics Area’’ are mandatory information that must be specified before a new Pixel Set submission (highlighted in blue, Fig. 3). They refer to general information related to the multi-omics biological project on Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 6/18 Figure 3 Data modelling in the Pixel Web App. The Pixel Set is the central information (see Fig. 1A), the corresponding table in the model is highlighted in red. Information that is required before Pixel Set im- port in the Pixel Web App is surrounded in blue, whereas information required during Pixel Set import is highlighted in orange. Other tables are automatically updated during the Pixel Web App data analysis life cycle (see Fig. 1C). An enlarged version of this picture together with full documentation is available online https://github.com/Candihub/pixel/blob/master/docs/pixel-db.pdf. Full-size DOI: 10.7717/peerj.6623/fig-3 Figure 3 Data modelling in the Pixel Web App. The Pixel Set is the central information (see Fig. 1A), the corresponding table in the model is highlighted in red. Information that is required before Pixel Set im- port in the Pixel Web App is surrounded in blue, whereas information required during Pixel Set import is highlighted in orange. Other tables are automatically updated during the Pixel Web App data analysis life cycle (see Fig. 1C). An enlarged version of this picture together with full documentation is available online https://github.com/Candihub/pixel/blob/master/docs/pixel-db.pdf. Full-size DOI: 10.7717/peerj.6623/fig-3 which the researcher is working on: (i) the studied organism and its genetic background (Species and Strain, e.g., Candida glabrata and ATCC2001), (ii) the type of monitored cellular components (Omics Unit Type, e.g., mRNA, protein) and (iii) the nature of the experimental HT technology (Omics Area, e.g., RNA sequencing, mass spectrometry). All Omics Units must be declared in the Pixel Web App before new Pixel Set submission. They must be defined with a short description and a link to a reference database. ‘‘Experiment’’ and ‘‘Analysis’’ are Pixel Set mandatory information, input during the submission of new Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 7/18 Figure 4 Procedure to import new Pixel Sets in the Pixel Web App. Annotation of Pixel Sets User can edit the Pixel Set (see 1), edit the analysis(see 2), edit the experiment (see 3) and add ‘‘Tags’’ (see 4). The Tags are of interest to further explore Pixel Sets in the Pixel Web App. F ll i DOI / j 66 /fi Pixel Sets in the Pixel Web App (highlighted in orange, Fig. 3). They include, respectively, the detailed description of the experimental strategy that was applied to generate primary and secondary data sets (Experiment) and the detailed description of the computational procedures that were applied to obtain Pixel Sets from secondary data set (Analysis). Information regarding the researcher who performed the analyses is referred as ‘‘Pixeler’’. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Storage of Pixel Sets in the database Import of new Pixel Sets in the Pixel Web App requires the user to follow a workflow for data submission. It corresponds to six successive steps that are explained below (Fig. 4A). 1. The ‘‘Download’’ step consists in downloading a template Excel file from the Pixel Web App (see Fig. 4B). In this file, multiple-choice selections are proposed for ‘‘Species’’, ‘‘Strain’’, ‘‘Omics Unit Type’’ and ‘‘Omics Area’’ fields. These choices Import of new Pixel Sets in the Pixel Web App requires the user to follow a workflow for data submission. It corresponds to six successive steps that are explained below (Fig. 4A). 1. The ‘‘Download’’ step consists in downloading a template Excel file from the Pixel Web App (see Fig. 4B). In this file, multiple-choice selections are proposed for ‘‘Species’’, ‘‘Strain’’, ‘‘Omics Unit Type’’ and ‘‘Omics Area’’ fields. These choices Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 8/18 reflect what is currently available in the database and can be easily expanded. User must fill other annotation fields related to the ‘‘Experiment’’, ‘‘Analysis’’ and ‘‘Pixeler’’ information. The Excel file is next bundled into a ZIP archive with the secondary data file (in tab-separated values format), the user notebook (R markdown (https://rmarkdown.rstudio.com/) or Jupyter notebook (http://jupyter.org/) for instance) that contains the code used to produce the Pixel Sets from the secondary data file. y (https://rmarkdown.rstudio.com/) or Jupyter notebook (http://jupyter.org/) for instance) that contains the code used to produce the Pixel Sets from the secondary data file. 2. The ‘‘Upload’’ step consists in uploading the ZIP file in the Pixel Web App. 3. The step ‘‘Meta’’ consists in running an automatic check of the imported file integrity (md5sum checks are performed, Excel file version is verified, etc.). Note that no information is imported in the database at this stage, but a careful inspection of all Omics Units listed in the submitted Pixel Sets is done. This is why Omics Units need to be pre-registered in the Pixel Web App (see previous section). 4. In ‘‘Annotation’’ step, the annotations of Pixel Sets found in the Excel file (see Fig. 4C) are controlled and validated by the user. 5. Next, the ‘‘Tags’’ step is optional. It gives the opportunity to the user to add tags to the new Pixel Sets (see Fig. 4C), that could be helpful for further Pixel Set explorations (see next section). 5. Next, the ‘‘Tags’’ step is optional. Storage of Pixel Sets in the database It gives the opportunity to the user to add tags to the new Pixel Sets (see Fig. 4C), that could be helpful for further Pixel Set explorations (see next section). 6. The final step ‘‘Import archive’’ consists in importing all Pixel Sets in the database, together with annotations and tags. Note that the procedure of importing metadata as an Excel file has been inspired from the import procedure widely used in GEO (Clough & Barrett, 2016). Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Exploration of Pixel Sets p The Pixel Web App aims to help researchers to mine and integrate multiple Pixel Sets stored in the system. We developed a dedicated web interface to explore all the Pixel Sets stored in a particular Pixel instance (see Fig. 5). The upper part named ‘‘Selection’’ lists a group of Pixel Sets selected by the user for further explorations (Fig. 5A). The middle part named ‘‘Filters’’ lists the Pixel database contents regarding the Species, Omics Unit Types, Omics Areas and Tags annotation fields. The user can select information (Candida glabrata and modified pH here), search and filter the Pixel Sets stored in the database (Fig. 5B). The lower part is a more flexible search field in which keywords can be type. These keywords are searched in the Analysis and Experiment detailed description fields as illustrated here with LIMMA. The web interface also comprised detailed information for the selected subset of Pixel Sets with for instance, distributions of values and quality scores and a list of individual Omics Unit shown at the bottom of the page (Fig. 5C). Note that tags have been implemented to offer to the user a versatile yet robust annotation of Pixel Sets. They are defined during the import process, but they can be modified at any time through the Pixel web interface. Once searched, matching Pixel Sets are gathered in a table that can be exported. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 A case study in the pathogenic yeast Candida glabrata A case study in the pathogenic yeast Candida glabrata The yeast Candida glabrata (C. glabrata) is a fungal pathogen of human (Bolotin-Fukuhara & Fairhead, 2014). It has been reported as the second most frequent cause of invasive The yeast Candida glabrata (C. glabrata) is a fungal pathogen of human (Bolotin-Fukuhara & Fairhead, 2014). It has been reported as the second most frequent cause of invasive 9/18 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Figure 5 Functionalities to explore the Pixel Sets stored in the Pixel Web App. (A) Screenshot of the exploration menu available via the web interface. (B) Screenshot of the table that comprises all Pixel Sets, which match the filter criteria (see A). Particular Pixel Sets can be selected here (for instance ‘‘Pixel_C10.txt’’ and ‘‘Pixel_C60.txt’’). They will therefore appear in the ‘‘Selection’’ list (see A). (C) Screenshot of the web interface that gives detailed information for the selected subset of Pixel Sets (see A). Distribution of values and quality scores are shown and individual Omics Unit are listed at the bottom of the page. Full-size DOI: 10.7717/peerj.6623/fig-5 Figure 5 Functionalities to explore the Pixel Sets stored in the Pixel Web App. (A) Screenshot of the exploration menu available via the web interface. (B) Screenshot of the table that comprises all Pixel Sets, which match the filter criteria (see A). Particular Pixel Sets can be selected here (for instance ‘‘Pixel_C10.txt’’ and ‘‘Pixel_C60.txt’’). They will therefore appear in the ‘‘Selection’’ list (see A). (C) Screenshot of the web interface that gives detailed information for the selected subset of Pixel Sets (see A). Distribution of values and quality scores are shown and individual Omics Unit are listed at the bottom of the page. Full-size DOI: 10.7717/peerj.6623/fig-5 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 infections due to Candida species, i.e., candidemia, arising especially in patients with compromised immunity (HIV virus infection, cancer treatment, organ transplantation, etc.). Candidemia remains a major cause of morbidity and mortality in the healthcare structures (Horn et al., 2009; Pfaller et al., 2012). The genome of Candida glabrata has been published in 2004 (Dujon et al., 2004). Its size is 12.3 Mb with 13 chromosomes and is composed of ∼5,200 coding regions. Our research team is familiar with functional genomic studies in C. glabrata. A case study in the pathogenic yeast Candida glabrata In collaboration with experimental biologists, we published in the past ten years half dozen of articles, in which HT technologies were used (Lelandais et al., 2008; Goudot et al., 2011; Merhej et al., 2015; Merhej et al., 2016; Thiébaut et al., 2017). In our lab, the Pixel Web App is installed locally and store all the necessary genomics annotations to manage any multi-omics datasets in this species. As a case study, we decided to present how the Pixel Web App can be helpful to answer a specific biological question with only a few mouse clicks. As a biological question, we wanted to identify the genes in the entire C. glabrata genome: (i) which are annotated as involved in the yeast pathogenicity and (ii) for which the expression is significantly modified in response to an environmental stress induced by alkaline pH. Indeed, during a human host infection, C. glabrata has to face important pH fluctuations (see Ullah et al., 2013; Brunke & Hube, 2013; Linde et al., 2015 for more detailed information). Understanding the molecular processes that allow the pathogenic yeast C. glabrata to adapt extreme pH situations is therefore of medical interest to better understand host-pathogen interaction (Linde et al., 2015). In a paper published in 2015, Linde et al. (2015) provided a detailed RNAseq based analysis of the transcriptional landscape of C. glabrata in several growth conditions, including pH shift experiments. The primary dataset (RNAseq fastq files) is available in the Gene Expression Omnibus (Clough & Barrett, 2016) under accession number GSE61606. The secondary dataset (log2 Fold Change values) is available in Table S1 on the journal website (https://academic.oup.com/nar/article/43/3/1392/2411170). A first Pixel Set (labelled A) was created from this secondary dataset, annotated and imported into our Pixel Web App instance, following the procedure previously described. The associated ZIP archive is provided as Supplemental Information, along with all the details related to the experiment set up and the analysis. The Pixel Set A thus illustrates how publicly available data can be managed with the Pixel Web App. In our laboratory, we performed mass spectrometry experiments that also include pH shift (ZIP archive of the data is provided as Supplemental Information). Secondary dataset issued from these experiments leads to the Pixel Set B. Pixel Sets A and B comprise 5,253 Pixels and 1,879 Pixels (Fig. 6). Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 A case study in the pathogenic yeast Candida glabrata Transcriptomics (Pixel Set A) and proteomics (Pixel Set B) are interesting complementary multi-omics information that can be easily associated and compared with the Pixel Web App. In that respect, tags allowed to rapidly retrieve them using the web interface, applying the keywords ‘‘Candida glabrata’’ and ‘‘alkaline pH’’ (Fig. 6, Step 1). As we wanted to limit the analysis to the C. glabrata genes potentially involved in the yeast pathogenesis, a filter could be used to only retain the Omics Units for which the keyword ‘‘pathogenicity’’ is written in their description field (see Fig. 6, Step 2). As a result, a few numbers of Pixels were thus selected, respectively 17 in Pixel Set A and 6 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 11/18 Figure 6 Case study in the pathogenic yeast Candida glabrata. Our Pixel Web App was explored with the keywords ‘‘Candida glabrata’’ and ‘‘alkaline pH’’. Two Pixel Sets were thus identified because of their tags. Two other tags were identical between the two Pixel Sets (‘‘WT’’ and ‘‘logFC’’), indicating that (i) C. glabrata strains are the same, i.e., Wild Type, and (ii) Pixel values are of the same type, i.e., log Fold Change. Notably Pixel Set A is based on transcriptomics experiments (RNAseq, see the main text), whereas Pixel Set B is based on proteomics experiments (mass spectrometry, see the main text). Omics Unit were next explored using the keyword ‘‘pathogenesis’’ resulting in the identification of 17 Pixels (respectively 6 Pixels) in transcriptomics (respectively proteomics) results. They were combined and exported from the Pixel Web App, hence starting a new data analysis cycle. Full-size DOI: 10.7717/peerj.6623/fig-6 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 12/18 Figure 6 Case study in the pathogenic yeast Candida glabrata. Our Pixel Web App was explored with the keywords ‘‘Candida glabrata’’ and ‘‘alkaline pH’’. Two Pixel Sets were thus identified because of their tags. Two other tags were identical between the two Pixel Sets (‘‘WT’’ and ‘‘logFC’’), indicating that (i) C. glabrata strains are the same, i.e., Wild Type, and (ii) Pixel values are of the same type, i.e., log Fold Change. Notably Pixel Set A is based on transcriptomics experiments (RNAseq, see the main text), whereas Pixel Set B is based on proteomics experiments (mass spectrometry, see the main text). A case study in the pathogenic yeast Candida glabrata LogFC values measured in transcriptomic (Pixel Set A) and proteomic (Pixel Set B) experiments are shown in the third and fourth columns. Quality scores (QS) are following logFC values. They are p-values coming from the differential analysis of logFC replicates. The entire table of multi- pixel sets is available in Supplementary Data. Table 1 Detailed information regarding the Omics Unit identified in the C. glabrata case study. The two first columns give Omics Unit infor- mation as described in the Candida Genome Database (Skrzypek et al., 2017). All the description fields comprise the keyword ‘‘pathogenesis’’ (in bold). LogFC values measured in transcriptomic (Pixel Set A) and proteomic (Pixel Set B) experiments are shown in the third and fourth columns. Quality scores (QS) are following logFC values. They are p-values coming from the differential analysis of logFC replicates. The entire table of multi- pixel sets is available in Supplementary Data. mation as described in the Candida Genome Database (Skrzypek et al., 2017). All the description fields comprise the keyword ‘‘pathogenesis’’ (in bold). LogFC values measured in transcriptomic (Pixel Set A) and proteomic (Pixel Set B) experiments are shown in the third and fourth columns. Quality scores (QS) are following logFC values. They are p-values coming from the differential analysis of logFC replicates. The entire table of multi- pixel sets is available in Supplementary Data. Omics unit Description A B A (QS) B (QS) 1. CAGL0F04807g Ortholog(s) have role in pathogenesis and cell surface, hyphal cell wall, integral component of mitochondrial outer membrane, plasma membrane localization 1,09 1,81 2,23E−19 7,31E−05 2. CAGL0F06457g Ortholog(s) have role in fungal-type cell wall organization or biogenesis, mitochondrial outer membrane translocase complex assembly, pathogenesis, phospholipid transport, protein import into mitochondrial outer membrane 0,30 0,19 4,14E−02 2,65E−01 3. CAGL0I02970g Ortholog(s) have delta14-sterol reductase activity and role in cellular response to drug, ergosterol biosynthetic process, filamentous growth of a population of unicellular organisms in response to biotic stimulus, pathogenesis 0,90 −2,64 4,65E−16 2,19E−05 4. CAGL0I10516g Ortholog(s) have role in fungal-type cell wall organization, pathogenesis and cytoplasm, eisosome, integral component of plasma membrane, membrane raft localization 1,50 0,57 8,29E−60 1,16E−02 5. CAGL0L08448g Ortholog(s) have role in actin cytoskeleton organization, eisosome assembly, negative regulation of protein phosphorylation, negative regulation of sphingolipid biosynthetic process and pathogenesis 1,67 −0,57 1,77E−75 7,04E−03 in Pixel Set B. The last step consists in combining the mRNA and protein information (see Fig. 6, Step 3). Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 A case study in the pathogenic yeast Candida glabrata Omics Unit were next explored using the keyword ‘‘pathogenesis’’ resulting in the identification of 17 Pixels (respectively 6 Pixels) in transcriptomics (respectively proteomics) results. They were combined and exported from the Pixel Web App, hence starting a new data analysis cycle. Figure 6 Case study in the pathogenic yeast Candida glabrata. Our Pixel Web App was explored with the keywords ‘‘Candida glabrata’’ and ‘‘alkaline pH’’. Two Pixel Sets were thus identified because of their tags. Two other tags were identical between the two Pixel Sets (‘‘WT’’ and ‘‘logFC’’), indicating that (i) C. glabrata strains are the same, i.e., Wild Type, and (ii) Pixel values are of the same type, i.e., log Fold Change. Notably Pixel Set A is based on transcriptomics experiments (RNAseq, see the main text), whereas Pixel Set B is based on proteomics experiments (mass spectrometry, see the main text). Omics Unit were next explored using the keyword ‘‘pathogenesis’’ resulting in the identification of 17 Pixels (respectively 6 Pixels) in transcriptomics (respectively proteomics) results. They were combined and exported from the Pixel Web App, hence starting a new data analysis cycle. F ll i DOI 10 7717/ j 6623/fi 6 Figure 6 Case study in the pathogenic yeast Candida glabrata. Our Pixel Web App was explored with the keywords ‘‘Candida glabrata’’ and ‘‘alkaline pH’’. Two Pixel Sets were thus identified because of their tags. Two other tags were identical between the two Pixel Sets (‘‘WT’’ and ‘‘logFC’’), indicating that (i) C. glabrata strains are the same, i.e., Wild Type, and (ii) Pixel values are of the same type, i.e., log Fold Change. Notably Pixel Set A is based on transcriptomics experiments (RNAseq, see the main text), whereas Pixel Set B is based on proteomics experiments (mass spectrometry, see the main text). Omics Unit were next explored using the keyword ‘‘pathogenesis’’ resulting in the identification of 17 Pixels (respectively 6 Pixels) in transcriptomics (respectively proteomics) results. They were combined and exported from the Pixel Web App, hence starting a new data analysis cycle. F ll i DOI 10 7717/ j 6623/fi 6 Full-size DOI: 10.7717/peerj.6623/fig-6 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Table 1 Detailed information regarding the Omics Unit identified in the C. glabrata case study. The two first columns give Omics Unit infor- mation as described in the Candida Genome Database (Skrzypek et al., 2017). All the description fields comprise the keyword ‘‘pathogenesis’’ (in bold). DISCUSSION In this article, we introduced the principle and the main functionalities of the Pixel Web App. With this application, our aim was to develop a tool to support on a daily basis, the biological data mining in our multi-omics research projects. It is our experience that research studies in which HT experimental strategies are applied, require much more time to analyse and interpret the data, than to experimentally generate the data. Testing multiple bioinformatics tools and statistical approaches is a critical step to fully understand the meaning of a biological dataset and in this context, the annotation, the storage and the ability to easily explore all results obtained in a laboratory can be the decisive steps to the success of the entire multi-omics project. The data modelling around which the Pixel Web App was developed has been conceived to find a compromise between a too detailed and precise description of the data (which could discourage the researchers from systematically using the application after each of their analyses) and a too short and approximate description of the data (which could prevent the perfect reproduction of the results by anyone). Also, attention has been paid to allow heterogeneous data, i.e., different Omics Unit Type quantified in different Omics Area, to be stored in a coherent and flexible way. The Pixel Web App does not provide any computational programs to analyse the data. Still, it allows to explore existing results in a laboratory and to rapidly combine them for further investigations (using for instance the Galaxy platform or any other data analysis tool). Therefore, the Pixel Web App holds a strategic position in the data management in a research laboratory, i.e., as the starting point but also at the final point of all new data explorations. It also helps data analysis reproducibility and gives a constant feedback regarding the frequency of the data analysis cycles; the nature of the import and export data sets as well as full associated annotations. It is thus expected that the content of different Pixel Web App instance will evolve with time, according to the type of information stored in the system and the scientific interests of a research team. Software availability Pixel is released under the open-source 3-Clause BSD license (https://opensource. org/licenses/BSD-3-Clause). Its source code can be freely downloaded from the GitHub repository of the project: https://github.com/Candihub/pixel. In addition, the present version of Pixel (4.0.4) is also archived in the digital repository Zenodo (https://doi.org/10.5281/zenodo.1434316). A case study in the pathogenic yeast Candida glabrata For that a table comprising the multi-pixel sets can be automatically generated and easily exported. We present Table 1: five genes for which logFC values were obtained both at the mRNA and the protein levels, and for which statistical p-values were significant (<0.05). Notably two genes (CAGL0I02970g and CAGL0L08448g, lines 3 and 5 in Table 1) exhibited opposite logFC values, i.e., induction was observed at the mRNA level whereas repression was observed at the protein levels. Such observations can arise from post-translational regulation processes or from possible experimental noise, which could explain approximative mRNA or protein quantifications. In both cases, further experimental investigations are required. The three other genes (CAGL0F04807g, CAGL0F06457g and CAGL0I10516g, underlined in grey in Table 1) exhibited multi-omics coherent results and significant inductions were observed at the mRNA and protein levels. Again, further experimental investigations are required to fully validated these observations. Still, it is worth noting that the gene CAGL0F04807g, is described as ‘‘uncharacterized’’ in the Candida Genome Database (http://www.candidagenome.org/cgi- bin/locus.pl?locus=CAGL0F04807g&organism=C_glabrata_CBS138). Considering that logFC values for this gene are particularly high (>1), such an observation represents a good starting point to refine the functional annotation of this gene, clearly supporting the hypothesis that it has a role in the ability of C. glabrata to deal with varying pH situations. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 13/18 Competing Interests William Durand and Julien Maupetit are employed by TailorDev SAS. Charles Hébert is employed by Biorosetics. CONCLUSION The Pixel Web App is freely available to any interested parties. The initial installation on a personal workstation required IT support from a bioinformatician, but once this is done, all administration tasks can be performed through the Web Interface. This is of interest for user with a few technical skills. We chose to work exclusively with open source technologies and our GitHub repository is publicly accessible (https://github.com/Candihub/pixel). We 14/18 Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 thus hope that the overall quality of the Pixel Web App source code and documentation will be guaranteed over time, through the shared contributions of other developers. thus hope that the overall quality of the Pixel Web App source code and documentation will be guaranteed over time, through the shared contributions of other developers. thus hope that the overall quality of the Pixel Web App source code and documentation will be guaranteed over time, through the shared contributions of other developers. Author Contributions • Thomas Denecker performed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • William Durand conceived and designed the experiments, performed the experiments, approved the final draft. • Julien Maupetit and Charles Hébert conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, approved the final draft. • Julien Maupetit and Charles Hébert conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, approved the final draft. • Jean-Michel Camadro analyzed the data, approved the final draft. • Jean-Michel Camadro analyzed the data, approved the final draft. • Pierre Poulain conceived and designed the experiments, contributed reagents/material- s/analysis tools, authored or reviewed drafts of the paper, approved the final draft. • Gaëlle Lelandais conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. Funding This work was funded by the Agence Nationale pour la Recherche (CANDIHUB project, grant number ANR-14-CE14-0018-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability The following information was supplied regarding data availability: Source code can be freely downloaded from the GitHub repository of the project: https://github.com/Candihub/pixel. Source code can be freely downloaded from the GitHub repository of the project: https://github.com/Candihub/pixel. The present version of Pixel (4.0.4) is also archived at Zenodo: The present version of Pixel (4.0.4) is also archived at Zenodo: Durand, William, Maupetit, Julien, Denecker, Thomas, Hébert, Charles, Poulain, Pierre, & Lelandais, Gaëlle. (2018, September 24). Pixel (v4.0.4): Integration of smart ‘omics’ data (Version 4.0.4). Zenodo. http://doi.org/10.5281/zenodo.1434316. Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Agence Nationale pour la Recherche (CANDIHUB project: ANR-14-CE14-0018-02. Denecker et al. (2019), PeerJ, DOI 10.7717/peerj.6623 Supplemental Information pp Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.6623#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.6623#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.6623#supplemental-information. peerj.6623#supplemental-information. Denecker et al. 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https://openalex.org/W4361941413	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Data_from_Perioperative_ctDNA-Based_Molecular_Residual_Disease_Detection_for_Non_Small_Cell_Lung_Cancer_A_Prospective_Multicenter_Cohort_Study_LUNGCA-1_/22487895/1/files/39939495.pdf	English	null	Supplementary Data from Perioperative ctDNA-Based Molecular Residual Disease Detection for Non–Small Cell Lung Cancer: A Prospective Multicenter Cohort Study (LUNGCA-1)	null	2,023	cc-by	56	Figure S1. Somatic mutation types detected in tumor tissues. SNV, single nucleotide variant; CNV, copy number variation. Figure S1. Somatic mutation types detected in tumor tissues. SNV, single nucleotide variant; CNV, copy number variation. Figure S1. Somatic mutation types detected in tumor tissues. SNV, single nucleotide variant; CNV, copy number variation. variant; CNV, copy number variation.
https://openalex.org/W373840403	http://sedici.unlp.edu.ar/bitstream/handle/10915/140834/020-155-951-377-594.pdf-PDFA.pdf.pdf?sequence=1	English	null	null	Transition metal chemistry	1,997	cc-by	3,052	Equilibrium studies The equilibrium constant for complexation between the reactants was determined from absorbance change measurements at constant pH and folic acid concen- tration 1 10ÿ4 M. The NiII ion concentration ranged between 5 10ÿ4 and 0:3 M. Measurements were made at 390 nm. Typical absorbance changes at 390 nm for dif- ferent NiII ion concentrations at pH 6.0 are shown in Figure 2. Kinetics The kinetic study was carried out on a Durrum D-110 stopped-¯ow apparatus at 25 C. Reagent grade NiSO4 6H2O (Merck), folic acid (Merck), KNO3 (Merck) and bromothymol blue (Merck) were used as supplied. In all experiments the NiII concentration greatly exceeded that of folic acid in order to preserve pseudo-®rst order conditions. The NiII ion concentra- tion was between 1 10ÿ3 and 0:3 M. The folic acid concentration was 1 10ÿ4 M. Summary The kinetics of the complexation of NiII by pteroylglu- tamic acid have been studied in the 5±45 C range, the ionic strength 0:6 M being regulated with KNO3, in the 5.5±7.0 pH range, using the stopped-¯ow method. Un- der the experimental conditions two processes were observed. The faster process was detected in the milli- second range and is associated with the reaction between NiII and the ligand. The slower is observed within a few seconds. Complementary equilibrium studies were made at 25 C. The results are consistent with the formation of a 1:1 complex between the reactants, and a mechanism is proposed to account for the observed behaviour. Equi- librium constants for the NiII plus pteroylglutamic acid system, as well as activation parameters, are reported. à Member of CICPBA, Argentina. § Author to whom all correspondence should be directed. g à Member of CICPBA, Argentina. * Member of CONICET, Argentina. à 0340±4285 Ó 1997 Chapman & Hall Introduction Pteroylglutamic acid (folic acid) and related substances exhibit numerous dissociation equilibria in the 1±9 pH range1;2. Folic acid behaves as an acid owing to the presence of a phenolic group and two carboxylic groups (Figure 1). Several methods (u.v.±vis., 1H- and 13C- n.m.r. studies2;3) have been developed to measure pK values for folic acid. Equilibrium constants for the re- action between folic acid and some divalent metal ions have been reported in the literature1;4;5. Owing to a combination of factors, such as the sol- ubility of folic acid and its complexes in acid solution and the hydrolysis of the metal ion, kinetic studies were performed in the 5.6±7.3 pH range. The pH of each solution was adjusted before mixing and this is the pH reported. Absorbance changes were recorded at 390 nm. Some experiments were performed in the presence of 2 10ÿ5 M of bromothymol blue at 460 and 615 nm. No in¯uence of folic acid concentration was detected in the present study. In most of the experi- ments the ionic strength was 0:6 M (regulated by adding KNO3). Under all conditions, two processes were ob- served. No absorbance changes were observed in blank experiments. Thermodynamic and spectroscopic data are available in the literature for this vitamin, but kinetic studies of its interaction with divalent transition metal ions are scarce. To the best of our knowledge, the interaction with cobalt(II) is the only kinetic study reported6. Moreover, the equilibrium data available in the litera- ture either exhibit scattering or do not ®t the experi- mental conditions under which the present studies were performed. The principal aim of this work was to elucidate the kinetics and the mechanism of complexation between nickel(II) and pteroylglutamic acid. 0340±4285 Ó 1997 Chapman & Hall Figure 1. Folic acid molecule. Figure 2. Typical absorbance changes versus [Ni2+] at pH 7. * Member of CONICET, Argentina. à Member of CICPBA, Argentina. § Author to whom all correspondence should be directed. Figure 2. Typical absorbance changes versus [Ni2+] at pH 7. Figure 1. Folic acid molecule. * Member of CONICET, Argentina. à Member of CICPBA, Argentina. Figure 1. Folic acid molecule. Figure 1. Folic acid molecule. * Member of CONICET, Argentina. à * Member of CONICET, Argentina. * Member of CONICET, Argentina. g à Member of CICPBA, Argentina. Figure 2. Typical absorbance changes versus [Ni2+] at pH 7. Kinetic study of nickel(II) complexation by pteroylglutamic acid AndreÂ s Thomas, Ezequiel Wolcan, Mario R. FeÂ lizà and Alberto L. Capparelli*,§ Instituto de Investigaciones FisicoquõÂmicas TeoÂricas y Aplicadas (INIFTA), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, (1900) La Plata, RepuÂblica Argentina Transition Met. Chem., 22, 541±544 (1997) NiII complexation by pteroylglutamic acid 541 NiII complexation by pteroylglutamic acid Transition Met. Chem., 22, 541±544 (1997) 541 Introduction g § Author to whom all correspondence should be directed. 0340±4285 Ó 1997 Chapman & Hall Transition Met. Chem., 22, 541±544 (1997) 542 Capparelli et al. Figure 3. Behaviour of k1app versus [NiII]. Kinetic results Two well time-resolved processes are detected in the present system. Both follow a ®rst order law and are pH independent, as seen in Table 1. They are discussed separately. The temperature dependence of the fast process was measured in the linear region shown in Figure 3 and at high concentrations of NiII ion. These results are pre- sented in Figure 5. The activation energies E1app and E0 1app are 45.3 and 49:4 kJ molÿ1, respectively. Equilibrium study Equilibrium study The experimental procedure (spectrophotometric titra- tion) for measuring equilibrium constants has been reported elsewhere(6). The results obtained in the nickel- (II)±folic acid system can be interpreted according to the following complexation scheme: Table 2. Experimental rate constants at dierent NiII concentrations; folic acid concentration 1 10ÿ4 M Table 2. Experimental rate constants at dierent NiII concentrations; folic acid concentration 1 10ÿ4 M with b=a 8:16 10ÿ3 s Mÿ1 and 1=a 9:97 10ÿ5 Mÿ1 [NiII] (M) Slow process Fast process k2app sÿ1 k1app sÿ1 0.005 0.155 36.5 0.010 0.161 52.0 0.020 0.197 69.9 0.030 0.175 90.1 0.039 88.0 0.050 0.192 0.100 0.143 106.0 0.145 122.2 0.200 0.195 117.7 A nonlinear regression analysis leads to the following values for the equilibrium constants involved in this complexation: K1 20:8 M ÿ1, K2 2:0 10ÿ6 M and K3 4:2 10ÿ5. 3 In Figure 2, the solid curve was obtained using these equilibrium constants. The extinction coecient ob- tained for the NiFoOÿ complex is 6300 300 cmÿ1 Mÿ1 at 390 nm. with b=a 8:16 10ÿ3 s Mÿ1 and 1=a 9:97 10ÿ5 Mÿ1 (see Figure 4). Experiments conducted in the presence of bromo- thymol blue show that the fast process is accompanied by a pH decrease of the solutions. The rate constants measured in the presence or absence of this pH indicator are the same within experimental error. Slow process This process is detected within a few seconds. Its be- haviour is similar to that observed for the cobalt(II)± folic acid system6, i:e: the apparent rate constant, k2app, is independent of the metal ion concentration (Table 2). Experiments in the presence of bromothymol blue show a rapid pH decrease associated with the earlier stage of the reaction, followed by a pH increase in the time window where the slow process is detected. In a similar way, as mentioned previously for the fast process, the rate constants measured in the presence or absence of indicator are the same. pp pp The activation energy, measured in the linear region shown in Figure 3, is given by E1app E1 DH, where DH is associated with the enthalpy of formation of the intermediate complex. Therefore, the following values can be obtained for these magnitudes: E1 49:4 kJ molÿ1 and DH ÿ4:1 kJ molÿ1. The NiFoLOÿ com- plex, formed in this ®rst reaction stage, involves the hydroxy group and the pteridin nitrogen in the mole- cule. Complementary studies were conducted in the system 8-hydroxyquinoline±nickel(II). This ligand resembles the pteridinic moiety of folic acid. In this system, only a process in the millisecond range is observed, which can be associated with the interaction of the metal ion with the 8-hydroxyquinoline. A marked pH decrease is also detected during the reaction. The temperature dependence of the experimental rate of the slow process leads to an activation energy of 52.2 kJ mol)1. Previous studies on the cobalt(II)±folic acid system reveal only one process within the same timescale and similar apparent rate constants as measured for the slow process detected in the system reported in this paper. Several studies have been reported in the literature regarding the kinetics of nickel(II) complexation with mono-, bi- and terdentate ligands7±13. A very interest- ing review is given in Ref. 14. The eect of the chelates formation can be described through the following re- action scheme. p y p p p The following comments about the mechanism can be inferred from the previous experimental results. The slow process shows similar characteristics to those observed for the cobalt(II)±folic acid system. This process is independent of pH and metal ion concentration, and the pH behaviour during complexation is the same in both systems. The global rate constants and the activa- tion energy are similar. NiII complexation by pteroylglutamic acid NiII complexation by pteroylglutamic acid Figure 5. Arrhenius plot for k1app at dierent nickel concentrations. [NiII] = (d) 0.01; (s) 0.1 M. coordination sphere of the metal ion, an apparent rate constant, ca: k0KIP, should be expected. Here k0 means the water exchange rate in the metal ion k0 3 104 sÿ1 and KIP is the ionic pair equilibrium constant, which can be calculated using the well-known Fuoss±Bjerrum equation15. Assuming that in the co- ordination site the net charge is almost zero (the car- boxylate groups are far from this site) then KIP ca: 0:3 and the rate should be 1 103 Mÿ1 sÿ1 or higher. Values of this order have been observed in the kinetics of complexation with pyridine, bipyridine and terpyridine 4 103, 1:5 105 and 1:4 103 Mÿ1 sÿ1, respective- ly)9;10. In our case, the experimental rate constant can not be represented by this simple consideration. Therefore, the rate-determining step can not be associated with the water exchange rate in the coordination sphere of the nickel(II) ion. The complex dependence of k1 with metal ion concentration and the value for K and k1 (81 and 123, respectively) implies that, prior to chelation, a monodentate intermediate is formed and that the che- lation is controlled by deprotonation of the OH group in the pterine moiety. The values of K measured in our experiments are quite similar to those reported for monodentate ligands16 (i:e: in pyridine log K 1:87). Figure 5. Arrhenius plot for k1app at dierent nickel concentrations. [NiII] = (d) 0.01; (s) 0.1 M. In this mechanism Ni±FoL(OH) is a monodentate in- termediate complex, whereas NiFoLOÿ is a bidentate one. Therefore, the following expression for k1app can be obtained: k1app k1KNiII0=1 KNiII0. Comparison with the experimental expression gives the value of k1K a and K b. At low concentrations of metal ion, k1app k1K whereas at high concentration k1app k1. Fast process pp The fast reaction is associated with the ®rst stage of the complexation, where the pteridinic moiety of the molecule is involved, i:e:: This process is observed in the millisecond time range. It depends upon the metal ion concentration. The behav- iour of the apparent rate constant k1app with NiII con- centration is shown in Figure 3 and Table 2. NiII FoL(OH)2ÿ ! K Ni±FoL(OH) ! k1 NiFoLOÿ H Figure 4. 1=k1app versus 1/[NiII]. NiII FoL(OH)2ÿ ! K Ni±FoL(OH) ! k1 NiFoLOÿ H g The following experimental dependence can be obtained from this experimental behaviour: k1app aM2=1 bM2 or k1appÿ1 b=a 1=aM2ÿ1 k1app aM2=1 bM2 or k1appÿ1 b=a 1=aM2ÿ1 k1app aM2=1 bM2 or k1appÿ1 b=a 1=aM2ÿ1 Table 1. Experimental rate constants at dierent pH; k1app values were obtained with [NiII] = 5 ´ 10)3 M whereas k2app values were averaged over dierent NiII concentrations at the same pH Table 1. Experimental rate constants at dierent pH; k1app values were obtained with [NiII] = 5 ´ 10)3 M whereas k2app values were averaged over dierent NiII concentrations at the same pH Table 1. Experimental rate constants at dierent pH; k1app values were obtained with [NiII] = 5 ´ 10)3 M whereas k2app values were averaged over dierent NiII concentrations at the same pH pH Slow process Fast process k2app sÿ1 k1app sÿ1 5.8 0.156 35.6 6.0 0.144 36.4 6.4 0.189 37.0 6.6 0.152 ± 6.8 0.163 ± 7.0 0.141 39.0 7.2 0.146 36.5 Figure 4. 1=k1app versus 1/[NiII]. pH Slow process Fast process k2app sÿ1 k1app sÿ1 5.8 0.156 35.6 6.0 0.144 36.4 6.4 0.189 37.0 6.6 0.152 ± 6.8 0.163 ± 7.0 0.141 39.0 7.2 0.146 36.5 pH Slow process Fast process k2app sÿ1 k1app sÿ1 5.8 0.156 35.6 6.0 0.144 36.4 6.4 0.189 37.0 6.6 0.152 ± 6.8 0.163 ± 7.0 0.141 39.0 7.2 0.146 36.5 Figure 4. 1=k1app versus 1/[NiII]. Figure 4. 1=k1app versus 1/[NiII]. Transition Met. Chem., 22, 541±544 (1997) 543 References (1) R. Nayan and A. K. Dey, Z. Naturforsch., Teil B, 52, 1453 (1970). (2) (1) R. Nayan and A. K. Dey, Z. Naturforsch., Teil B, 52, 1453 y y, f , , , ( ) (2) M. Poe, J. Biol. Chem., 252, 3724 (1977). (3) y y f (2) M. Poe, J. Biol. Chem., 252, 3724 (1977). ( ) (3) M. Poe, J. Biol. Chem., 248, 7025 (1973). (4) (3) M. Poe, J. Biol. Chem., 248, 7025 (1973). (4) (4) A. Albert, Biochem J., 54, 646 (1953). (5) (5) J. T. H. Roos and D. R. Williams, J. Inorg. Nucl. Chem., 39, 367 (1977). (6) (6) A. H. Thomas, M. R. Feliz and A. L. Capparelli, Transition Met. Chem., 21, 317 (1996). (7) (7) P. K. Chattopadhyay and B. Kratochvil, Can. J. Chem., 54, 2540 (1976). ( ) ( ) (8) P. K. Chattopadhyay and B. Kratochvil, Inorg. Chem., 18, 2953 (1979). (9) (9) R. G. Wilkins, Comments Inorg. Chem., 2, 187 (1983). (10) (10) R. G. Wilkins, Acc. Chem. Res., 3, 408 (1970). (11) (11) D. M. W. Buck and P. Moore, J. Chem. Soc., Dalton Trans., 2082 (1974). (12) (12) R. B. Jordan and B. E. Erno, Inorg. Chem., 18, 2895 (1979). (13) (13) R. L. Reeves, G. S. Calabrese and S. A. Harkaway, Inorg. Chem., 22, 3076 (1983). (14) (14) R. G. Wilkins, Kinetics and Mechanism of Reactions of Transition Metal Complexes, 2nd Edit, VCH, New York, 1991 p. 219. (15) Scheme 1. (15) R. M. Fuoss, J. Am. Chem. Soc., 80, 5059 (1958). (16) (16) A. E. Mantell and R. M. Smith, Critical Stability Constants, Plenum Press, New York, 1975, Vol. 2, p. 165. (17) The second process, observed in nickel(II)±folic acid and cobalt(II)±folic acid systems, requires the transfor- mation of a bidentate into a terdentate complex, involving the nitrogen in the pterydin and benzyl rings. This terdentate complex contains two ®ve-membered rings positioned around the central ion. The intra- molecular distances involving the dierent groups are similar (dOAN 2:77 ÊA in the pteridyl moiety and dNAN 2:73 ÊA between N in the pteridyl and N in the p-aminobenzoic acid moieties), allowing the coor- dination pattern seen around the central ion in this (17) Y. Ducommun, K. E. Newman and A. E. Merbach, Inorg. Chem., 19, 3696 (1980). (18) (18) Y. Ducommun, D. Zbinden and A. E. Merbach, Helv. Chim. Slow process The principal dierences between the systems are associated with the detection of the fast process in presence of nickel(II). The rapid process observed in this system, but not detected under the same experimental conditions in the cobalt(II)±folic acid system, is probably associated with the dierence between the water exchange rate in the coordination spheres of nickel (II) 3:0 104 sÿ1 and cobalt(II) 2:0 106 sÿ117±19. NiH2O2 6 A±BH±C ! KIP NiH2O2 6 A±BH±C NiH2O2 6 A±BH±C ! k0 kÿ0 NiH2O2 5 ±A±BH±CH2O In our case A, BH and C can be identi®ed as the pter- idinic N, pteridinic hydroxy group and p-aminobenzoic amine group, respectively. This scheme can be employed to explain the ®rst stage in the complexation, involving an ionic pair and a monodentate complex. If complexation is determined (in this stage) by the release of a water molecule from the The rate constant for the slower process is very low in comparison with other complexation rate constants. However, the reactions of nickel(II) with 2-2(pyridazo)- 1-naphthol dyes (a-PAN)(13) are characterized by Transition Met. Chem., 22, 541±544 (1997) 544 Capparelli et al. abnormally low rate constants (ca. 20 s)1 at 298 K). These values have been ascribed to the formation of a terdentate chelate. In these systems the coordination pattern resembles that proposed for the nickel(II)±folic acid or cobalt(II)±folic acid complexes (see Scheme 1 and Ref. 6). complex(6,20). The activation energy obtained for the slow process is of the same order of magnitude as that observed for the cobalt(II)±folic acid system. Therefore, conformational changes during chelation involving the ligand may constitute the rate-limiting step associated with the interaction of divalent metal ions and folic acid. Therefore, this second process allows for a similar interpretation to that advanced in the cobalt(II)±folic acid system, i.e. the bidentate complex formed in the ®rst step of the complexation (NiFoLO)) undergoes a major conformational change leading to a terdendate species(6, 20). This complex is partially reprotonated in a fast step as depicted in Scheme 1. Acknowledgements This work was supported by the Consejo Nacional de Investigaciones CientõÂcas y TeÂ cnicas de la RepuÂ blica Argentina (CONICET), the ComisioÂ n de Investig- aciones de la Provincia de Buenos Aires (CICPBA) and Universidad Nacional de La Plata. One author (A.T.) thanks CONICET for a graduateship. Scheme 1. Scheme 1. References Acta, 65, 1385 (1982). (19) (19) P. Bernhard, L. Helm, I. Rapaport, A. Ludi and A. E. Merbach, Inorg. Chem., 27, 873 (1988). (20) (20) E. A. Miromov and V. S. Nabokov, Khim.-Farm. Zh., 10, 136 (1976). (Received 28 October 1996 Accepted 29 April 1997) TMC 3839 (Received 28 October 1996 Accepted 29 April 1997) TMC 3839
https://openalex.org/W2765630441	https://europepmc.org/articles/pmc5651814?pdf=render	English	null	Measurements of Rationality: Individual Differences in Information Processing, the Transitivity of Preferences and Decision Strategies	Frontiers in psychology	2,017	cc-by	10,624	ORIGINAL RESEARCH published: 18 October 2017 doi: 10.3389/fpsyg.2017.01844 Measurements of Rationality: Individual Differences in Information Processing, the Transitivity of Preferences and Decision Strategies Patrycja Sleboda* and Joanna Sokolowska Faculty of Psychology SWPS University of Social Sciences and Humanities Warsaw Poland The ﬁrst goal of this study was to validate the Rational-Experiential Inventory (REI) and the Cognitive Reﬂection Test (CRT) through checking their relation to the transitivity axiom. The second goal was to test the relation between decision strategies and cognitive style as well as the relation between decision strategies and the transitivity of preferences. The following characteristics of strategies were investigated: requirements for trade-offs, maximization vs. satisﬁcing and option-wise vs. attribute-wise information processing. Respondents were given choices between two multi-attribute options. The options were designed so that the choice indicated which strategy was applied. Both the REI-R and the CRT were found to be good predictors of the transitivity of preferences. Respondents who applied compensatory strategies and the maximization criterion scored highly on the REI-R and in the CRT, whereas those who applied the satisﬁcing rule scored highly on the REI-R but not in the CRT. Attribute-wise information processing was related to low scores in both measurements. Option-wise information processing led to a high transitivity of preferences. INTRODUCTION In research on decision-making much attention during last few decades has been devoted to personal information processing styles. This interest is related to the growing popularity of Dual- Process Theories (DPT) in which two cognitive systems are emphasized. The ﬁrst is intuitive, quick, unconscious and aﬀect-based, whereas the second is logical, conscious, slow and reason-based, also labeled referred as “rational” (e.g., Epstein, 1994; Kahneman, 2003). Various measurement techniques have been developed to diagnose the personal style of information processing. Two classes of these measurements can be distinguished: self-reported inventories and task solving tests. In the study presented here, both types of measurements were used, i.e., self-reported Rational-Experiential Inventory (the REI) proposed by Pacini and Epstein (1999) and the Cognitive Reﬂection Test (the CRT) developed by Frederick (2005). Specialty section: This article was submitted to Cognitive Science, a section of the journal Frontiers in Psychology Received: 24 July 2017 Accepted: 03 October 2017 Published: 18 October 2017 Keywords: dual-process theories, decision strategies, transitivity of preferences, bounded rationality, indexes of rationality Edited by: Eldad Yechiam, Technion – Israel Institute of Technology, Israel Reviewed by: Amos Schurr, Ben-Gurion University of the Negev, Beersheba, Israel Shahar Ayal, Interdisciplinary Center Herzliya, Israel Correspondence: Patrycja Sleboda psleboda@swps.edu.pl Edited by: Eldad Yechiam, Technion – Israel Institute of Technology, Israel Reviewed by: Amos Schurr, Ben-Gurion University of the Negev, Beersheba, Israel Shahar Ayal, Keywords: dual-process theories, decision strategies, transitivity of preferences, bounded rationality, indexes of rationality Interdisciplinary Center Herzliya, Israel Citation: The second goal of the study is to investigate the inﬂuence of individual diﬀerences in information processing on the strategies applied to selections among multi-attribute options. The following properties of strategies were investigated: requirements for trade-oﬀs (compensatory vs. non-compensatory), maximization vs. satisﬁcing and option-wise vs. attribute- wise information processing. These properties were selected because they allow diﬀerentiating among decision strategies related to the classical concept of rationality, to the concept of bounded rationality and to simple heuristics. It was expected that high scores on the Rationality subscale of the REI (the REI-R) and in the CRT would be related either to option-wise compensatory strategies and to maximization or to option-wise non-compensatory strategy and to satisﬁcing. In contrast, low scores on the REI-R and in the CRT would be related to dimension-wise non-compensatory strategies2. The third goal was to test whether the strategies based on option-wise information processing favor the transitivity of preferences, independently of the requirement of trade-oﬀs. Therefore, another important contribution from the presented research is the test of preferences’ transitivity in the light of information processing behind various decision strategies. Alternatively, Pacini and Epstein (1999) proposed a self-reported inventory, the REI (the Rational-Experiential Inventory), to identify individual diﬀerences in information processing. This inventory consists of two subscales: Experiential (the REI-E, “I like to rely on my intuitive impressions”) and Rational (the REI-R, “I enjoy problems that require hard thinking”). The REI-R has been found to be a strong predictor of performance consistent with the normative rules in tasks such as missing-a-ﬂight vignette, the thematic and abstract versions of Wason task and the jelly bean task (Witteman et al., 2009). Ayal et al. (2011; Ayal et al., 2012) reported that participants, who scored high on the REI-R less frequently chose low-diversiﬁed portfolios and less frequently had reversed preferences for normatively identical options in two diﬀerent situations. On the basis of the above ﬁndings, one may conclude that a high score on the REI-R is a good predictor of rational behavior. The distinction between two means of cognition, emphasized within DPT, is not a new idea. Immanuel Kant (1781) introduced the concept of a posteriori and a priori judgments. A posteriori judgments are derived from experience and are based on phenomena that are related to sensation. These judgments don’t lead to accurate representations of objects “as they are in themselves” and relations between them. Citation: Sleboda P and Sokolowska J (2017) Measurements of Rationality: Individual Differences in Information Processing, the Transitivity of Preferences and Decision Strategies. Front. Psychol. 8:1844. doi: 10.3389/fpsyg.2017.01844 The important contribution from the present research is that these measurements of information processing have been confronted with the fundamental assumption of rational choices accepted in utility theory (UT), i.e., the transitivity of preferences (Von Neumann and Morgenstern, 1953). Despite a high interest in personal styles of information processing in decision research, to our knowledge neither measurement of DPT has been directly confronted with the concept of October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska to mind automatically. High performance in the CRT means that System 2 “wins” with System 1 and indicates ability for analytical thinking. It was shown in number of studies that score in the CRT correlates positively with scores in IQ tests, SAT total score (Frederick, 2005; Obrecht et al., 2009; Toplak et al., 2011; Liberali et al., 2012), the ability to delay gratiﬁcations and the percentage of risky choices consistent with the normative model (Frederick, 2005). Similarly, Cokely and Kelley (2009) found that participants who scored highly in the CRT made choices in line with expected value. At the same time, a low score in the CRT positively correlated with the answers that reﬂected the conjunction and gambler’s fallacies, neglect of base rate and sample size, framing and more (Toplak et al., 2011). The CRT also measures numeracy skills, i.e., the skills that enable understanding and number use (e.g., Frederick, 2005; Reyna et al., 2009). In light of DPT, the most important property of the CRT is that the score in the CRT correlates with the Stroop eﬀect (Stroop, 1935), as shown by Toplak et al. (2011). This indicates that analytical information processing with the aid of System 2 requires conscious overcoming of automatic responses evoked by System 1. Therefore one may conclude that the CRT is a good measure of poor vs. high information processing. transitivity of preferences in UT1. So the ﬁrst goal of this study was to validate two DPT measures of rationality: the REI and the CRT in light of the transitivity of preferences, the measure of rationality in UT. 1Recently, Primi et al. (2016), investigated the relation between the CRT and transitivity of inferences, i.e., validity of conclusions in light of premises (see section Discussion). 2No hypotheses were formulated for Experiential subscale of the REI because there are many ﬁndings indicating that the REI-R is a better predictor of actual rational behavior. 1Recently, Primi et al. (2016), investigated the relation between the CRT and transitivity of inferences, i.e., validity of conclusions in light of premises (see section Discussion). 2 Citation: In contrast, a priori judgments are derived from analytical thinking, or are synthetic a priori truths based on abstract reasoning noumena, i.e., objects as they are in themselves, independently of the senses. The concept of extensional and intuitive reasoning, introduced by Tversky and Kahneman (1983), is very similar to Kant’s theory. This is also the fundamental concept in DPT proposed by Kahneman (2003; Kahneman and Frederick, 2005) who emphasized two cognitive systems: (1) intuitive, quick, unconscious, aﬀect-based and (2) logical, conscious, slow, reason-based. In light of the cited ﬁndings, it is not surprising that the scores on the REI-R and in the CRT are positively correlated (Liberali et al., 2012; Thoma et al., 2015). On the other hand, these two measurements reﬂect two diﬀerent methodological approaches: the score in the CRT results from actual performance, while the REI is a self-declarative measurement. Thus, it was interesting to check how both measurements account for transitivity of preferences and for strategies applied for selection among multi- attribute options. Frederick (2005) developed the CRT to probe individual diﬀerences in the dominant information processing system. The CRT consists of three mathematical questions (e.g., In a lake, there is a patch of lily pads. Every day, the patch doubles in size. If it takes 48 days for the patch to cover the entire lake, how long would it take for the patch to cover half of the lake?). To answer these questions correctly one has to use System 2. On the other hand, wrong answers produced by System 1 come Deviations in actual behavior from the fundamental assumptions of rational choices accepted in UT (Von Neumann and Morgenstern, 1953) have been studied for many years (e.g., Edwards, 1954; Simon, 1955, 1972, 1978, 1986; Kahneman and Tversky, 1972, 1973; Tversky and Kahneman, 1973, 1974, 1983; Shaﬁr et al., 1993) and much attention has been paid to deviations from transitivity of preferences (e.g., May, 1954; Tversky, 1969). The transitivity axiom requires a systematic order of preferences across options. This means that for any three options A, B, and C, if A is preferred to B and B is preferred to C October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 2 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska TABLE 1 \| Three options described on three attributes. Citation: Even though lexicographic models diﬀer among themselves, all of them are based on the same idea that choices are made on the basis of comparative judgments and incomplete, selective information processing. This is in contrast to MAU and CON strategies, where choices are based on global evaluations that include all the relevant information, made independently for each option. As for the trade-oﬀs’ requirement, compensatory and non- compensatory decision strategies can be distinguished. The basic compensatory strategy is the Multi-Attribute Utility (MAU) model. According to this strategy, the decision maker estimates the score (utility) on all the attributes and assigns weights to them. The utility of each attribute is multiplied by its weight. The option with the highest weighted sum should be chosen. There are also other linear models that are less sophisticated, e.g., Franklin’s Rule, Tallying (Gulliksen, 1950), but all of them require: (1) linear integration of information, (2) inter- dimensional trade-oﬀs, and (3) a separate, global evaluation of each option. Does such a model always lead to rational choices? Imagine that one has to evaluate the health of three individuals considering three essential, equally important organs, as described in Table 1. According to the linear model, Person 2 has the highest weighted sum and, therefore, should be considered the healthiest. Yet clearly we are not able to survive without any one of the above organs. From this perspective, the healthiest is Person 3, even though s/he has the lowest global evaluation. Is choosing Person 3 an irrational decision? A solution to the above problem may be the strategy that doesn’t require trade-oﬀs, as proposed by Simon (1955, 1972, 1978). It still requires option-wise evaluations, but the optimization criterion is relaxed. According to Simon’s concept of Bounded Rationality (Simon, 1957), people make decisions that may not be optimal but are still satisﬁcing. Simon’s idea is that it is suﬃcient to pick an option that satisﬁes aspirations The decision maker searches through options only until one that meets aspirations is found. So instead of the maximization of additively integrated utilities from all attributes, the comparison of scores for these attributes with the satisﬁcing threshold is the criterion for choice (conjunction rule—CON). The relation between applied strategies and information processing as well as transitivity of preferences requires explanation. Citation: Weights Option 1 Option 2 Option 3* Heart 0.33 8 1 5 Lungs 0.33 10 10 5 Liver 0.33 1 10 5 Weighted sum 6.27 6.93 4.95 Choice of Option 1, assuming heart to be the most important attribute, is in line with Lexicographic (LEX) strategy. Choice of Option 2 is in line with Multi-Attribute Utility (MAU) strategy. **Choice of Option 3, assuming 5 as a minimum cutoff, is in line with Conjunctive (CON) strategy. TABLE 1 \| Three options described on three attributes. than A has to be preferred to C. The assumption of preference transitivity is the basic requirement of rational choice (Fishburn, 1991; Müller-Trede et al., 2015). Since in DPT rationality is related to information processing with the aid of System 2, there should be a relation between measures of the dominant style in information processing and the transitivity of preferences. So in the present study we checked whether DPT measurements account for transitivity of preferences. Hypothesis 1: High scores on the REI-R and in the CRT are related to high transitivity of preferences. The second goal of the study was to investigate the inﬂuence of individual diﬀerences in information processing on strategies applied in selection among multi-attribute options. In the light of information processing, the following properties of decision strategies are important: requirements for trade- oﬀs (compensatory vs. non-compensatory), maximization vs. satisﬁcing and option-wise vs. attribute-wise information processing. high cognitive eﬀort. This can be avoided if one uses another class of non-compensatory strategies, i.e., lexicographic strategies, which are much simpler than CON. In these strategies, options are not treated separately but compared on the basis of attributes in the following way: (1) the decision maker orders attributes by importance and (2) chooses the option that is the best on the most important attribute, e.g., Person 1 from Table 1, if healthy heart is the most important attribute. If options are equally good on the most important attribute, the second most important attribute is considered, etc. There are many non-compensatory models based on attribute-wise comparisons such as Lexicographic) Rule (LEX, e.g., Luce, 1956; Tversky, 1969; Fishburn, 1970; Luce et al., 2000), Elimination by Aspects (Tversky, 1972), Minimax, Take-The-Best heuristic3 (Gigerenzer and Goldstein, 1996) or Priority Heuristic (Brandstatter et al., 2006). 3In this heuristic, the cue validity, i.e., its predictive accuracy, determines the attributes’ order. Frontiers in Psychology \| www.frontiersin.org Overview of Experimental Design Hypotheses 1–3 were veriﬁed in the study whereby respondents chose between two options constructed such that the selected option indicated the applied strategy (MAU, CON, LEX). Options were described three common attributes. To investigate the relation between individual diﬀerences in information processing and applied decision strategies, one should minimize the impact of situational factors. This can be achieved when people solve abstract problems, where there are no interferences with the previous experience, familiarity, or emotions. An abstract presentation was also used in order to diminish the inﬂuence of content on the salience/perceived importance of attributes (Gallhofer et al., 1987; Sokolowska, 1990). Another important characteristic of abstract choices is that there are no diﬀerences in JND across attributes. One may also assume that scores’ utility function for abstract attributes is monotonic. So in the present research, respondents were faced with identical choices set either in abstract or in speciﬁc context. As for transitivity, the common view is that lexicographic rules lead to intransitivity of preferences, as a result of shifting attention among various dimensions. Since some information is ignored, judgment is partial. Tversky (1969) proposed a diﬀerent explanation of intransitivity. He assumed that people diﬀer not only in the relative importance assigned to various attributes but also in Just Noticeable Diﬀerence (JND) in scores across various attributes. He gave the example of three job candidates: X, Y, Z, who diﬀered with respect to two attributes: intelligence and experience. Under the assumption that IQ is more important than experience, the candidate who scores higher in IQ than the others should be chosen. If such a diﬀerence, however, is not salient, one will look at diﬀerences in experience. This may lead to intransitive preferences, when the diﬀerence in IQ between X and Y as well as between Y and Z is not salient and then they are ordered due to the other dimension that points to X. On the other hand, the diﬀerence between X and Z in IQ scores may be noticeable—thus Z is chosen over X— resulting in intransitive preferences. This structure is called a lexicographic semi-order (LS) that results in intransitivity, “where a semi-order (LS) (Luce, 1956) or a just noticeable diﬀerence structure is imposed on a lexicographic ordering” (Tversky, 1969, p. 32). Respondents in one group were choosing one of two apartments described on attributes that had speciﬁc modalities (speciﬁc content). Overview of Experimental Design In contrast, participants in the second group were choosing between two abstract options that were described on three common dimensions that had no speciﬁc modalities (abstract content). Beside the inﬂuence of the content, decision makers may also be inﬂuenced by the goal that s/he wants to achieve. Maximizing choice accuracy is not necessarily the goal of all decision makers. Other goals might be also justiﬁed, such as minimization of eﬀort, regret or conﬂict (Einhorn and Hogarth, 1981; Tetlock, 1985). For example, if minimizing eﬀort rather than maximizing accuracy is the goal, one may use the easiest LEX strategy. In order to unify the goals of respondents, ﬁnancial rewards were introduced for those who made the most accurate choices. The important feature of Tversky’s explanation is that this can be extended to intransitivity that results from additive models. He gave an example of the Additive Diﬀerence (ADD) model. In the ADD, diﬀerences in scores for each dimension are calculated for both options and then these diﬀerences are summed. Diﬀerences in JND for various attributes can lead to the intransitivity of preferences. Note, LS can be treated as a speciﬁc case of ADD—where at least one diﬀerence in scores is described as a step function. So both models, ADD and LS, can result in intransitive preferences, when diﬀerences in scores are described by non- linear functions. MATERIALS AND METHODS Hypothesis 2: People who score highly on the REI-R and in the CRT more frequently apply strategies based on option- wise information processing, i.e., either MAU type or CON strategies. Participants Two hundred and nine respondents voluntarily participated in this study, conducted online. In Group 1 (choice of apartment) there were 105 respondents (53.3% female, M age = 27.9; SD = 5.71). 61.9% of the participants had master degree, 8.6% bachelor degree, 21% were students. In Group 2 (abstract choice) there were 104 participants (481% female, M age = 29.4; SD = 6.71). 66% of the participants had master degree, 13.5% bachelor degree and 13.5% were students. From Tversky’s reasoning (1969), it follows that the compensatory principle does not prevent the intransitivity of preferences, when sensitivity to various dimensions is described by diﬀerent non-linear functions and judgment is comparative. The important question is whether the linearity requirement is necessary, when information processing is option-wise and then each option is evaluated independently as in MAU and in CON strategies. If the utility of scores is deﬁned by a monotonic function, one may expect transitive orderings of independently formed global evaluations. For MAU it is assumed that the higher the score, the higher its utility. For CON one can assume that all utilities of scores are described by step functions. Therefore, Hypothesis 3 was formulated as follows: To motivate them, participants were told that the three participants in each group who achieve the highest number of correct choices, would get 300 PLN (80 USD) gift card to a bookstore. Respondents were told that one option was always better, however, accuracy here was not deﬁned. Financial rewards were given to those who had the highest number of choices made in line with MAU model, although participants were not aware of this. Citation: It follows from previous research that people who base their decisions predominantly on System 1 are focused on perceptual features of a task (Sloman, 1996; Kahneman, 2003) and are prone to the biases that stem from context- dependent judgments (Payne et al., 1988, 1993). Such people therefore either use simple lexicographic rules or switch between diﬀerent strategies. On the other hand, focusing on abstract aspects of a problem protects one against such judgmental biases. As mentioned earlier, Ayal et al. (2011) found that people who scored highly on the REI-R were less prone to reverse preferences for normatively identical options set in diﬀerent contexts. So one may conclude that people with dominant System 2 consider all relevant aspects and consistently apply the same criterion for similar choices. For example, a person may consistently believe that either averaging (MAU) or meeting aspirations (CON) leads to the most accurate decision. The most important diﬀerence between MAU and CON is that they imply diﬀerent criteria for decision: either averaging or meeting minimal cutoﬀs on all the relevant attributes. On the other hand, applying either strategy requires the systematic processing of all relevant information, which entails relatively October 2017 \| Volume 8 \| Article 1844 3 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska 4There are several versions of the REI (i.e., the REI-40, the REI-24 by Pacini and Epstein, 1999; 10-item version by Epstein et al., 1996). the REI-24 was used in two studies presented here, because of its high internal consistency (Ayal et al., 2012). Task Hypothesis 3: Choices based on MAU or CON strategies result in transitive preferences. Participants in Group 1 (speciﬁc content) were to imagine that they had been asked by a friend to help in choosing one of October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 4 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska two apartments. They were told that their friend had already evaluated each apartment in terms of three attributes—rent, location and neighborhood—which s/he considered the most important and of equal importance (see Figure 1). The respondents also answered the CRT questions (Frederick, 2005). 32.1% of respondents answered all the CRT questions incorrectly, while 26.3% answered all questions correctly. The diﬀerence in distributions of answers between participants who chose an apartment and those who made abstract choices, was insigniﬁcant (χ² = 2.72, df = 3, N = 209, p = 0.44). Participants in Group 2 (abstract content) made choices between two abstract options that had no content and where the attributes had no modalities. In particular, participants were given the following instruction: “You will be faced with series of choices that have no speciﬁc content. It does not matter whether you choose between two cars, two kinds of washing powder, two schools, two jobs or two candidates for the president of a country, etc. We decided to present you with abstract choices rather than with the concrete ones because we are interested in general principles of judgment and choice processes that should not depend on the object of choice. Imagine that you were asked by a friend to help choose between two options described in terms of the three most and equally important attributes......” Since the REI and the CRT are two types of techniques to distinguish the dominant information processing system, one may expect a positive correlation between the REI-R and the CRT scores. Indeed, these two scores were signiﬁcantly positively correlated for all respondents (r = 0.33, N = 209, p < 0.001) as well as for speciﬁc (r = 0.30, p = 0.002, N = 105) and for abstract (r = 0.36, p < 0.001, N = 104) content. Transitivity The corresponding sets of transitive choices were as follows: (1) if (90,20,40) > (50,50,50) and (50,50,50) > (70,30,60), then (90,20,40) > (70,30,60), (2) if (90,20,40) < (50,50,50) and (50,50,50) < (70,30,60), then (90,20,40) < (70,30,60), (3) if (90,20,40) < (50,50,50) and (90,20,40) > (70,30,60), then (50,50,50) > (70,30,60), (4) if (90,20,40) > (50,50,50) and (90,20,40) < (70,30,60), then (50,50,50) < (70,30,60), (5) if (90,20,40) > (70,30,60) and (70,30,60) > (50,50,50), then (90,20,40) > (50,50,50), (6) if (50,50,50) > (70,30,60) and (90,20,40) < (70,30,60), then (90,20,40) < (50,50,50). (1) if (90,20,40) > (50,50,50) and (50,50,50) > (70,30,60), then (90,20,40) > (70,30,60), Transitivity Seven sets of three choices, e.g., choices 4, 13, 19 (see Table 2), allowed us to test the transitivity axiom. Respondents in both groups were told that their task would be to help a decision maker choose better one of two options. Each respondent was to make 26 choices between two options, always described in terms of the three attributes. The choice options were identical in both groups and the only diﬀerence was that in Group 2, neither the options nor the attributes had concrete modalities. Stimuli were presented as histograms. The scores for each attribute were presented as bar heights; the higher the bar, the better the score. If there are three options: A, B, and C, and all three pairwise choices (A/B, A/C, and B/C) are given to participants, then there are six combinations that are consistent with the transitivity axiom. These combinations are: (1) if A>B and B>C then A>C, (2) if C>B and B>A then C>A, (3) if B>A and A>C then B>C, (4) if C>A and A>B then C>B, (5) if A>C and C>B then A>B, (6) if B>C and C>A then B>A. (1) if A>B and B>C then A>C, (2) if C>B and B>A then C>A, (3) if B>A and A>C then B>C, (4) if C>A and A>B then C>B, (5) if A>C and C>B then A>B, (6) if B>C and C>A then B>A. (1) if A>B and B>C then A>C, To assure that utility of scores on attributes would be described by a monotonic function (see p. 11), the scores on all attributes were given in numbers from 0 to 100 with assigned labels such as 0 the worst score, 50 the satisfactory score and 100 the maximum score. It was expected that in the abstract task respondents should treat scores as described either by linear or by step functions. (6) if B>C and C>A then B>A. For example, for a set of choices 4, 13 and 19, three options scored as follows on three attributes: A = 90,20,40; B = 50,50,50 and C = 70,30,60. Measurements The REI-Short 244 and the CRT The REI-Short 244 and the CRT (2) if (90,20,40) < (50,50,50) and (50,50,50) < (70,30,60), then (90,20,40) < (70,30,60), The short, 24-item REI (Pacini and Epstein, 1999), previously validated by Ayal et al. (2012; Ayal et al., 2011) and Ayal et al. (2015), was translated into Polish by Sleboda and Sokolowska. The reliability of the REI questionnaire was checked for both subscales. For the REI-R, Cronbach’s alpha coeﬃcient was α = 0.79. Cronbach’s alpha coeﬃcients were similar in both groups (α = 0.78 and α = 0.81 for speciﬁc and abstract content, respectively). For the REI-E α = 0.84 for all respondents and α = 0.85 in Group 1 and α = 0.84 in Group 2. The correlation between the two subscales was negative (for Group 1: r = −0.237; p = 0.02, N = 105; for Group 2: r = −0.014; p = 0.89, N = 104; for both studies: r = −0.12; p = 0.08, N = 209). The diﬀerence in scores on both subscales between studies was not signiﬁcant [F(1, 207) = 0.062 and 0.002, p = 0.80 and 0.97, ηp2 = 0.000 for the REI-R and the REI-E respectively]. Since the focus of the present studies is on rationality, only the REI-R was considered in further analysis. (3) if (90,20,40) < (50,50,50) and (90,20,40) > (70,30,60), then (50,50,50) > (70,30,60), (4) if (90,20,40) > (50,50,50) and (90,20,40) < (70,30,60), then (50,50,50) < (70,30,60), (5) if (90,20,40) > (70,30,60) and (70,30,60) > (50,50,50), then (90,20,40) > (50,50,50), (6) if (50,50,50) > (70,30,60) and (90,20,40) < (70,30,60), then (90,20,40) < (50,50,50). Independently of the preferred strategy, a participant had to choose one of these six combinations to be transitive. Any other choice was inconsistent with the transitivity axiom. For each transitive choice, a participant was given one point, otherwise zero. Since there were 7 sets to test transitivity, each participant could score from 0 to 7 points. A vast majority of respondents scored between 5 and 7 (the maximum) in the transitivity index. Only 1% (2 participants) scored 3, and 2% (4 participants) scored 4. They were excluded from further analyses since their groups were too small to compare with others. The observed high score in transitivity is in agreement with the expectation that people Independently of the preferred strategy, a participant had to choose one of these six combinations to be transitive. Any other choice was inconsistent with the transitivity axiom. 5In Group 2, where attributes had no modalities, the choice of an option with a score close to the maximum for either attribute was considered to be a proxy for LEX. The REI-Short 244 and the CRT For each transitive choice, a participant was given one point, otherwise zero. Since there were 7 sets to test transitivity, each participant could score from 0 to 7 points. A vast majority of respondents scored between 5 and 7 (the maximum) in the transitivity index. Only 1% (2 participants) scored 3, and 2% (4 participants) scored 4. They were excluded from further analyses since their groups were too small to compare with others. The observed high score in transitivity is in agreement with the expectation that people October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 5 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska FIGURE 1 \| Group I: An example of one out of 26 choices. FIGURE 1 \| Group I: An example of one out of 26 choices. TABLE 2 \| Options used in 26-choices design to differentiate among strategies. Choices that differentiated between: Choice no Option 1 Option 2 LEX vs. CON 4 90:20:40 50:50:50 5 90:40:20 50:50:50 6 40:90:20 50:50:50 7 20:90:40 50:50:50 8 20:40:90 50:50:50 9 40:20:90 50:50:50 MAU vs. CON 13 70:30:60 50:50:50 14 70:60:30 50:50:50 15 60:70:30 50:50:50 16 30:70:60 50:50:50 17 30:60:70 50:50:50 18 60:30:70 50:50:50 MAU vs. LEX 19 70:30:60 90:20:40 20 70:60:30 90:40:20 21 60:70:30 40:90:20 22 30:70:60 20:90:40 23 30:60:70 20:40:90 24 60:30:70 40:20:90 Additional choices 1 90:20:40 20:90:40 2 90:40:20 20:40:90 3 40:90:20 40:20:90 10 80:30:60 30:70:60 11 70:60:30 30:60:80 12 60:80:30 60:30:70 25 90:20:40 60:60:60 26 70:30:60 60:60:60 TABLE 2 \| Options used in 26-choices design to differentiate among strategies. agreement with expectations that in abstract choices JND in utility across attributes does not diﬀer, because this utility is most likely described by a linear function. Strategies Applied for the Selections Twenty six choices were constructed to determine which strategy was applied: MAU, CON (satisﬁcing), or LEX5. The choices considered by respondents are presented in Table 2. As can be seen from Table 2, 18 choices were designed speciﬁcally to diﬀerentiate among strategies and to assign either MAU or CON strategy. For example, to diﬀerentiate between CON and MAU, Choices 13–18 where used, where Option 1 always has a greater sum of weighted scores than Option 2. Another six, Choices 4–9, were used to diﬀerentiate between CON and LEX. Here, the sum of weighted scores is the same. In all 12 choices, only in Option 2 all dimensions meet the satisfactory level. To decide whether a respondent used CON, we summed up all choices in line with CON and we accepted the criterion of 10 out of 12 choices to assign this strategy to the respondent. The 10 choices out of 12 were accepted on the basis of a comparison of the probability of this sequence when a given strategy is applied with the probability of the sequence appearing randomly. The detailed description of the way in which strategies were assigned is given in Appendix 1 (Supplementary Material), accepted for assigning a strategy. To assign the MAU strategy the same analysis was done for Choices 13–18 (that distinguish MAU from CON) and Choices 19–24 (that distinguish MAU from LEX). For assigning LEX strategy, the procedure was extended to two steps. In the ﬁrst step, it was decided which attribute was the most important on the basis of three combinations of Choices 1, 2 and 3. For example, the choice of Option 1 in Choice 1 and in Choice 2 indicated that the ﬁrst attribute was most important. Next, for each attribute an additional 10 choices were analyzed (12 in total). For example, for Attribute 1, the choices were as follows: 4, 5, and 25 (Option 1), 19, 20, and 11 (Option 2), 13 and 14 (Option 1), 16 obey the transitivity axiom when the utility of scores is deﬁned by a monotonic function (see p. 11). 51.7% of participants always obeyed the transitivity axioms. More participants scored 7 in the transitivity index in abstract choices (56.7%) than in speciﬁc choices (46.7%). The average index of transitivity was also higher for abstract choices [F(1, 202) = 4.18, p = 0.04, ηp2 = 0.020]. Strategies Applied for the Selections This, again, is in 5In Group 2, where attributes had no modalities, the choice of an option with a score close to the maximum for either attribute was considered to be a proxy for LEX. October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 6 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska and 17 (Option 2). The same pattern was used for the two other attributes. and 17 (Option 2). The same pattern was used for the two other attributes. preferences for abstract choices (0.181, SE = 0.070, p = 0.011, the lower 0.043 and upper 0.320 bounds) but not for speciﬁc ones (−0.057, SE = 0.069, p = 0.41, the lower −0.192 and upper 0.079 bounds)6. Respondents were assigned to group LEX1, if they had series of 10 out of 12 choices on Attribute 1, LEX2 if they had series of 10 out of 12 choices on Attribute 2 and LEX3 if they had series of 10 out of 12 choices on Attribute 3. Finally, the respondents from these 3 groups were put to one labeled LEX. For 4 participants, it was not possible to diﬀerentiate between the LEX and MAU strategies. These respondents were excluded from further analysis. The insigniﬁcant correlations between DPT measurements and transitivity in speciﬁc content might have been caused by the diﬀerence in strategies applied in each context. This is discussed in the next section. Individual Differences in Information Processing and Applied Decision Strategies y The results of strategy assignment are given in Table 3. As can be seen from Table 3, a majority of participants was consistent in using the same criterion for choice. The most salient diﬀerence between the two groups was that 19.8% of those who chose an apartment used LEX, whereas only 8 participants (excluded from further analyses) applied an LEX-like rule for abstract choices. 25.7% of participants in speciﬁc and 36.5% in abstract content consistently chose according to the MAU strategy. 17.8 and 21.9% of participants consistently used CON in speciﬁc and abstract choices, respectively. The diﬀerences between groups were signiﬁcant (χ²= 21.56, df = 3, p < 0.001). The Applied Strategy and the Score on the REI-R As can be seen from Figure 2, there were signiﬁcant diﬀerences among users of speciﬁc strategies in the score on the REI-R [F(3, 193) = 3.40, p = 0.02, ηp2 =0.050]. LEX-users scored the lowest on the REI-R. In contrast, those using either MAU or CON strategies and the inconsistent group scored equally high on the REI-R. There was no signiﬁcant diﬀerence between MAU and CON-users (p = 0.39), while a marginally signiﬁcant diﬀerence was observed between MAU-users and the inconsistent group (p = 0.06). RESULTS Group 1: Speciﬁc content. Signiﬁcant diﬀerences in the scores on the REI-R were found [F(3, 97) = 2.92, p = 0.04, ηp2 = 0.083] for MAU, CON, LEX users and the inconsistent group. As presented in Figure 4, LEX-users scored the lowest on the REI-R and this group diﬀered signiﬁcantly from MAU-users (p = 0.02), CON-users (p = 0.02) and from the inconsistent group (p = 0.01). There were no signiﬁcant diﬀerences among the other groups. Frontiers in Psychology \| www.frontiersin.org 7Eight participants who used LEX-like strategy were omitted. Strategy and Transitivity gy y The score in transitivity was compared for users of MAU, CON and LEX strategies. As expected, there were signiﬁcant diﬀerences in the score in transitivity among users of speciﬁc strategies [F(3, 184) = 12.71, p < 0.001, ηp2 = 0.172]. Neither the interaction nor the main eﬀect of content was found in ANOVA analysis with two between-subject factors (Strategy 4 levels: MAU, CON, LEX, inconsistent, and Content 2 levels: abstract and speciﬁc). Therefore, the distribution of the transitivity index was compared for users of diﬀerent strategies for all respondents (see Table 4). As can be seen from Table 3, the majority of those who used MAU (65.6%) or CON (76.9%) was always transitive, whereas only 40% of LEX-users and 29.6% of inconsistent respondents had such preferences. These diﬀerences were statistically signiﬁcant (χ² = 35.9, df = 6, p < 0.001). In summary, participants with high scores on the REI-R consistently used strategies in which all relevant information was used. However, with speciﬁc content, the inconsistent group scored as high as CON and MAU-users. The Relation between the REI, the CRT and the Transitivity of Preferences ores on the REI-R for respondents, who applied different strategies in speciﬁc (left panel) and in abstract (right panel) content. FIGURE 3 \| The average scores on the REI-R for respondents, who applied different strategies in speciﬁc (left panel) and in abs FIGURE 4 \| The average scores in CRT for respondents who applied various strategies. diﬀered in the CRT score [F(3, 97) = 3.965, p = 0.01, ηp2 = 0.109]. The lowest mean of correct responses was observed for LEX- users, who signiﬁcantly diﬀered from MAU-users (p = 0.001). MAU-users scored higher than CON-users (p = 0.04) but not then the inconsistent group (p = 0.15) (see Figure 5). Group 2: Abstract content. Signiﬁcant diﬀerences were observed in the CRT score [F(2, 93) = 4.32 p = 0.016, ηp2 = 0.085] for MAU-users, CON-users and the inconsistent group. MAU-users scored highest in the CRT (p = 0.007). No signiﬁcant diﬀerences between CON-users and the inconsistent group in the CRT were observed (p = 0.648) (see Figure 5, right panel). In contrast to the result obtained for the REI-R in the abstract task, where users of both strategies based on global evaluations scored higher than others, only MAU-users scored highly in the CRT. This might indicate that the CRT also measures numeracy skills (see Discussion), not only the dominant style of information processing. Diﬀerent relations were observed in the speciﬁc task where, surprisingly, the inconsistent group received high results on the REI-R and in the CRT. FIGURE 4 \| The average scores in CRT for respondents who applied various strategies. ηp2 = 0.071] among users of speciﬁc strategies7. There were signiﬁcant diﬀerences between MAU-users and the inconsistent group (p = 0.009), whereas CON-users did not diﬀer signiﬁcantly from either MAU-users (p = 0.160) or from the inconsistent group (p = 0.407) (see Figure 3). The Relation between the REI, the CRT and the Transitivity of Preferences In order to check the relations between DPT measurements and transitivity, the process regression Model 1 (Preacher and Hayes, 2008; Hayes, 2009) with transitivity as the dependent variable was employed with 1,000 bootstrap samples. This technique was used because it allows one to check the conditional eﬀect of content which is a dichotomous variable. Because the REI-R and CRT were positively, signiﬁcantly correlated, this analysis was performed for each measurement technique separately. Group 2: Abstract content. The signiﬁcant diﬀerences in the scores on the REI-R were observed [F(2, 93) = 3.56, p = 0.032, FIGURE 2 \| The average scores on the REI-R for respondents who applied various strategies. The score on the REI-R (as an independent factor) was a good predictor of preference transitivity in abstract choices (0.036, SE = 0.012, p = 0.004, the lower 0.012 and upper 0.061 bounds) but not for speciﬁc ones (−0.002, SE = 0.014, p = 0.01, the lower −0.029 and upper 0.026 bounds). A similar result was obtained for the CRT as the independent factor and transitivity as the dependent variable. The score in the CRT was positively correlated with the transitivity of TABLE 3 \| Classiﬁcation of subjects on the basis of strategies applied for selection. Strategy Number of participants to whom a given strategy is attributed Group 1 Group 2 Inconsistent 37 40 LEX 20 8 CON 18 21 MAU 26 35 Total 101 104 F ti i P h l \| f ti i 7 FIGURE 2 \| The average scores on the REI-R for respondents who applied various strategies. 6The number of intuitive answers in the CRT was negatively correlated with the transitivity of preferences for abstract choices (−0.157, SE = 0.080, p = 0.05, the lower −0.314 and upper −0.0002 bounds) but not for speciﬁc ones (−0.025, SE = 0.075, p = 0.74, the lower −0.173 and upper 0.124 bounds). October 2017 \| Volume 8 \| Article 1844 7 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska FIGURE 3 \| The average scores on the REI-R for respondents, who applied different strategies in speciﬁc (left panel) and in abstract (right panel) content. FIGURE 3 \| The average scores on the REI-R for respondents, who applied different strategies in speciﬁc (left panel) and in abstract (right panel) content. DISCUSSION Even though this percentage is lower than for users of MAU and CON, it might obscure the relation between transitivity and DPT measurements with speciﬁc content. Strategy and the CRT There were signiﬁcant diﬀerences among groups of speciﬁc strategy users in the CRT score [F(3, 193) = 5.22 p = 0.002, ηp2 = 0.075]. As can be seen from Figure 4, MAU-users scored signiﬁcantly higher than the inconsistent group (p = 0.004), LEX-users (p = 0.001) and CON-users (p = 0.006). Group 1: Speciﬁc content. As can be seen from Figure 5 left panel, MAU, CON and LEX users and the inconsistent group The above results may account for the insigniﬁcant correlations between DPT measures and the index of transitivity in speciﬁc content, as reported in the previous section. 19.8% of participants used LEX in this content. Note that using the October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 8 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska FIGURE 5 \| The average scores in CRT for respondents who applied different strategies in speciﬁc (left panel) and in abstract (right panel) content. in CRT for respondents who applied different strategies in speciﬁc (left panel) and in abstract (right panel) content. DISCUSSION TABLE 4 \| The distribution of the transitivity index for users of different strategies. Index of transitivity Strategies Inconsistent (%) LEX (%) CON (%) MAU (%) 5 29.6 15 5.1 4.9 6 40.8 45 17.9 29.5 7 29.6 40 76.9 65.6 TABLE 4 \| The distribution of the transitivity index for users of different strategies. One aim of these studies was to check the relation between DPT measurements and the transitivity of preferences. It was found that people who obeyed transitivity scored highly on the REI-R and also in the CRT. This is an important and new result, which implies that both measures are good predictors of respect for transitivity. Even though both DPT measurements were very frequently used in previous studies (e.g., Ayal and Hochman, 2009; Cokely and Kelley, 2009; Pachur and Galesic, 2013; Graﬀeo et al., 2015), to our knowledge such a direct test has not been reported. One example of an indirect test is the research by Ayal et al. (2015). In Experiment 1, they found that the number of biases made could be predicted from the score on the REI and from the induced thinking mode. In Experiment 2, they found that: (1) an induced analytical thinking mode improved transitivity in analytical tasks, and (2) an induced intuitive thinking mode improved transitivity in intuitive tasks. However, in Experiment 2 the REI wasn’t used. Therefore, in neither of these studies was the direct relation between the REI and transitivity checked. In case of the CRT, Primi et al. (2016) checked the relation between scores in the CRT and the transitivity of inferences, i.e., the validity of conclusions in light of given premises. They found not signiﬁcant correlation with the CRT (r = 0.24, p = 0.67, N = 59). However, the transitivity of inferences should not be confused with the transitivity of preferences. For example, one prominent diﬀerence is that the transitivity of preferences depends on sensitivity to diﬀerences in scores on attributes (Tversky, 1969). LEX doesn’t exclude transitivity when only one attribute is used. Indeed, 40% of LEX-users always had transitive preferences. Even though this percentage is lower than for users of MAU and CON, it might obscure the relation between transitivity and DPT measurements with speciﬁc content. LEX doesn’t exclude transitivity when only one attribute is used. Indeed, 40% of LEX-users always had transitive preferences. Indexes of Rationality and Applied Decision Strategies From the results on the three indexes of rationality and applied strategies, it appears that both DPT measurements and transitivity are related to applied strategies. Those who scored highly on the REI-R used all relevant information, making speciﬁc and abstract choices based on global evaluations in both studies. This, in turn, led to transitive preferences. Furthermore, those who scored highly in the CRT used global evaluation based on additive integration of information. Even though CON- users scored lower in the CRT, they had transitive preferences. This might indicate that their the CRT score resulted from low numeracy skills rather than from information processing. It should be underlined that the positive relation between scores on the REI-R and the CRT and the transitivity of preferences was observed for abstract choices only. The key advantage of abstract frames is that they reduce conﬂict of values. Therefore, people perceive choices as logical tasks: experience, knowledge about the world, beliefs and other factors do not disturb decision processes. So, System 1 is not activated. For example, in an abstract frame one has to accept the importance of the attributes given in the instruction since one has neither experience nor knowledge about the situation or its attributes. So As mentioned earlier, exceptions were participants who switched among strategies in the speciﬁc content. They scored highly on the REI-R and in the CRT. This might indicate that when people face undesirable trade-oﬀs, they try to avoid conﬂict. To achieve this, they are more prone to use non-compensatory rules, either CON or LEX, or switching between them. In the case of LEX, they also may use more than one attribute. The poor transitivity of preferences within this group lends support to this interpretation. October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 9 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska a frame of this kind should be the best experimental condition to examine individual diﬀerences in the dominant style of information processing, what should result in the transitivity of preferences These results are in line with those reported by Toplak et al. (2011). this willingness is not the same as numeracy skills. On the other hand, CON-users had an equally high index of transitivity of preferences as MAU-users. This supports the thesis that in every-day decisions, making satisﬁcing choices is as rational as optimization. Indexes of Rationality and Applied Decision Strategies In contrast, selective and partial information processing does not guarantee transitivity. Only 40% of LEX- users respected the transitivity axiom. They also had low scores on the REI-R and in the CRT. Respondents who had transitive preferences in abstract choices, also scored highly on the REI-R and in the CRT. Lack of such relations in speciﬁc choices was most likely caused by the small fraction of LEX-users, who had transitive preferences even though they scored poorly in both DPT measurements. So for people with dominant System 2, who also meet normative criteria of rationality, content doesn’t aﬀect their rational way of decision-making. Witteman et al. (2009) obtained similar results, i.e., they found correlation between the performance in thematic and abstract Wason tasks and the score on the REI-R. As mentioned in the Introduction, the fundamental assumption in decision-making is that rational choice requires a global evaluation of each option and the additive integration of information. The alternative perspective suggested here might be that choices based on global evaluation and the Satisﬁcing Principle, instead of averaging, are also rational. Linear models lead to the optimization for the prize of trade-oﬀs, whereas a conjunctive rule that doesn’t require trade-oﬀs leads only to satisﬁcing decisions. So both have advantages and disadvantages in terms of accuracy. Thus, there are no reasons to treat linear models as the single criterion of rational choice. Another interesting result is the conﬁrmation that the three selected strategies—i.e., LEX, CON, and MAU—are good representations of three kinds of information processing: simple heuristics, bounded rationality and normative models. As might be expected, salient diﬀerences were observed between MAU and CON-users and the others, in both the index of transitivity and the score on the REI-R. MAU-users, in contrast to CON- users, also scored highly in the CRT. This may reﬂect the diﬀerence in numeracy skills between these two groups rather than a diﬀerence in rationality measured as consistency with basic logical principles. The higher transitivity and the higher DPT scores of respondents who used strategies based on global evaluation is additional support for the claim that the REI-R and CRT are good measurements of individual diﬀerences in information processing in terms of rational preferences. Our results support this view. Respondents who had transitive preferences used either averaging (31%) or the CON rule (19.8%), so they met the normative criterion of rationality. Indexes of Rationality and Applied Decision Strategies Moreover, they also scored highly on the REI-R, i.e., declared a willingness to solve problems in an analytical way. Both ﬁndings suggest that users of the CON rule met both objective and subjective criteria of rationality. However, the higher score in the CRT found for MAU-users compared to CON-users is not in agreement with this conclusion. However, the higher score in the CRT found for MAU-users compared to CON-users is not in agreement with this conclusion. This may have two explanations. First, the CRT measures not only the dominant style of information processing but also numeracy skills-CON-users had lower such skills than MAU- users. Second, the CRT may be a stronger test of analytical thinking than a self-declarative measurement such as REI-R, as suggested by Toplak et al. (2011). Therefore, the ﬁndings that users of CON had transitive preferences and scored highly on the REI-R but not in the CRT might be interpreted as the diﬀerence between the concepts of rationality and Bounded Rationality. To check this hypothesis, one should investigate which cognitive abilities are related to the use of averaging vs. the Satisﬁcing Principle as the criterion for choice. In particular, previous research was limited to a comparison between simple heuristics and linear models, forgetting Bounded Rationality idea to which they refer often. In light of the above conclusion, the high score on the REI-R and in the CRT found for respondents who didn’t consistently use the same strategy in tasks with speciﬁc content, requires additional explanation. In these tasks, respondents had to face undesirable trade-oﬀs that might cause problems with the weighting of attributes and therefore with the avoiding of conﬂict. This favors use of lexicographic rules (Beattie and Barlas, 2001) that lead to intransitivity if JND varies across attributes and more than one attribute is used (Tversky, 1969). They also might switch between CON and LEX strategies. Indeed, these respondents scored the lowest in the transitivity index. However, the intransitivity of speciﬁc choices might also result from sensitivity to conﬁgurations of values on attributes which are in disagreement with the independence axiom. These problems do not follow from processing information with the aid of System 1 and so are not measured by the REI-R or the CRT. Indexes of Rationality and Applied Decision Strategies Finally, since there is a relation between transitivity and System 2 it would be interesting to investigate whether people consider either averaging or satisﬁcing as an eﬀective tool for reaching accurate decisions and if so, what situational factors reinforce the tendency to use either strategy. Even though most authors consider simple heuristics as belonging to the class of Bounded Rationality models, they do not test a conjunctive rule that fully reﬂects Herbert Simon’s Principle of Satisﬁcing. Note that a majority of research is focused on comparison of linear models with simple heuristics, whereas CON rule is only rarely investigated. In our research, an interesting ﬁnding was that the most diﬀerentiated results were obtained for CON-users. They scored as high on the REI-R as MAU-users, but in the CRT they scored as poorly as LEX-users. 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Intransitivity, utility, and the aggregation of preference patterns. Econometrica 22, 1–13. doi: 10.2307/1909827 Müller-Trede, J., Sher, S., and McKenzie, C. R. M. (2015). FUNDING Helsinki. The protocol was approved by the Ethics Committee of University of Social Sciences and Humanities. Helsinki. The protocol was approved by the Ethics Committee of University of Social Sciences and Humanities. This study was supported by a grant from the Polish National Science Centre (Grant No. 2014/13/B/HS4/01533) awarded to JS. SUPPLEMENTARY MATERIAL PS and JS developed the experimental design, collected, analyzed and interpreted the data. PS and JS wrote the ﬁrst draft of the manuscript. Both authors participated in drafting the article and made critical revisions. Both authors approved the ﬁnal version of the paper. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2017.01844/full#supplementary-material ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Ethics Committee of University of Social Sciences and Humanities with informed consent from all subjects. The study was conducted online and all subjects gave informed consent in accordance with the Declaration of October 2017 \| Volume 8 \| Article 1844 10 System 2, Preferences’ Transitivity, Decision Strategies Sleboda and Sokolowska REFERENCES Behav. Decis. Mak. 29, 453–469. doi: 10.1002/bdm.1883 Graﬀeo, M., Polonio, L., and Bonini, N. (2015). Individual diﬀerences in competent consumer choice: the role of cognitive reﬂection and numeracy skills. Front. Psychol. 6:844. doi: 10.3389/fpsyg.2015.00844 y yg Gulliksen, H. (1950). Theory of Mental Tests. Hillsdale, NJ: Lawrence Erlbaum. Reyna, V. F., Nelson, W. L., Han, P. 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Bull. 119, 3–22. doi: 10.1037/0033-2909.119.1.3 Von Neumann, J., and Morgenstern, O. (1953). Theory of Games and Economic Behavior, 3rd Edn. Princeton: Princeton University Press. Sokolowska, J. (1990). “Attribute weighting and use of non-compensatory model as a function of perceived attribute ﬂexibility,” in Compensatory Issue in Decision Sokolowska, J. (1990). “Attribute weighting and use of non-compensatory model as a function of perceived attribute ﬂexibility,” in Compensatory Issue in Decision Making, eds K. Borcherding, O. I. Frontiers in Psychology \| www.frontiersin.org October 2017 \| Volume 8 \| Article 1844 REFERENCES Larichev, and D. M. Messick (Holland: Elsevier Science Publishers B.V.), 189–202. Witteman, C., van den Bercken, J., Claes, L., and Godoy, A. (2009). Assessing rational and intuitive thinking styles. Eur. J. Psychol. Assess. 25, 39–47. doi: 10.1027/1015-5759.25.1.39 Making, eds K. Borcherding, O. I. Larichev, and D. M. Messick (Holland: Elsevier Science Publishers B.V.), 189–202. Stroop, J. R. (1935). Studies of interference in serial verbal reactions. J. Exp. Psychol. 18, 643–661. doi: 10.1037/h0054651 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. Tetlock, P. E. (1985). Accountability: the neglected social context of judgment and choice. Res. in Organ. Behav. 7, 297–332. Thoma, V., White, E., Panigrahi, A., Strowger, V., and Anderson, I. (2015). Good thinking or gut feeling? Cognitive reﬂection and intuition in traders, bankers and ﬁnancial non-experts. PLoS ONE 10:e0123202. doi: 10.1371/journal.pone.0123202 Copyright © 2017 Sleboda and Sokolowska. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Toplak, M. E., West, R. F., and Stanovich, K. E. (2011). The Cognitive Reﬂection Test as a predictor of performance on heuristics and biases tasks. Mem. Cognit. 39, 1275–1289. doi: 10.3758/s13421-011-0104-1 October 2017 \| Volume 8 \| Article 1844 Frontiers in Psychology \| www.frontiersin.org 12
https://openalex.org/W3174033258	https://bovine-ojs-tamu.tdl.org/AABP/article/download/4669/4571	English	null	Pooling Resources to Achieve Financial, Professional and Personal Success	American Association of Bovine Practitioners Conference	2,006	cc-by	2,589	Abstract of the business management of the practice along with the work I do for our clients. I was always interested in the business side of practice, as well as the dairy farms I serve, so I completed my MBA in 2001 to help in these areas. I think we have done a pretty goodjob of manag- ing our business and meeting our clients' needs over the years, but maybe I'm biased. We certainly have our struggles, just as all of you do. Veterinarians, as well as many other people in our society, tend to lead very busy, active lives. We strive for success in our professional lives, try to reach our individual and family financial goals, and somehow try to blend all this with a successful personal and family life. Success in these three areas is necessarily unique to every person, based on their own individual goals and interests. There are many things that interfere with our success, however you may define success. Barriers to success may be time constraints, energy constraints, educational constraints or conflicts between the differ- ent foci of our lives. Pooling resources between and within veterinary practices can sometimes help to ease these various constraints and help us to achieve the success we strive for in the different facets of our lives. I bring this up to help you understand my surprise when the planning committee asked me to speak about pooling resources in veterinary practice. My first thought was that we do not pool resources and are a very self-sufficient practice. What can I possibly offer anyone concerning pooling resources? The more I thought about it however, I realized a group practice pools many resources. We just do most of it within our practice. We all have some of the same basic needs and desires. We just deal with them in different ways in different practice situations. I decided the best way to discuss this topic was to start at the basic needs level to identify what we desire for success, and then move to ways veterinarians are working together to fill these needs now and what we may be doing in the future. Pooling Resources to Achieve Financial, Professional and Personal Success Brian Reed, DVM, MBA Agricultural Veterinary Associates, P.O. Box 190, Denver, PA 17517 How do we define success? To talk about pooling resources to achieve success, we first have to consider what success is. This is going to vary for everybody based on their abilities, interests, goals and beliefs. We also can be successful in one part of our lives, but unsuccessful in other parts. This is where the idea of balance comes in. I think most people would agree that for them to feel successful, some sem- blance of balance between professional, financial and personal aspects of their lives must be struck. For many professionals who love their jobs, this balance is some- times hard to achieve. Even though we all define success differently, I will suggest most veterinarians would agree these are some of their goals in the different parts of their lives: Resume Les medecins veterinaires, comme plusieurs autres personnes dans notre societe, menent des vies tres occupees. Nous voulons avoir du succes dans notre vie professionnelle, rencontrer nos objectifs financiers de meme que ceux de notre famille, et d'une fa~on ou d'une autre melanger le tout avec une vie personnelle et familiale comblee. Le succes dans ces trois domaines est necessairement unique a chaque personne compte tenu des interets et des objectifs differents de chacun. Plusieurs choses peuvent contrecarrer notre succes peu importe comment nous le definissons. Ces obstacles au succes peuvent etre des contraintes de temps, d'energie ou de scolarite ou des conflits entre les differentes facettes de nos vies. Mettre les ressources en commun dans une pratique de meme qu'entre pratiques peut souvent aider a resoudre certaines de ces contraintes et mieux nous permettre d'avoir le succes tant recherche dans les differentes facettes de nos vies. 0 "d ('[) ~ ~ (') (') ('[) en en 8-: r:n q-[ o· p • Potential to "harvest" the business by taking in new partners and retirement options, helping fi- nancially. • Save and plan for retirement. • Have the financial means to pursue personal interests and goals. Personal goals: By no means am I saying everything goes smoothly in a group practice situation. There are numerous head- aches as well, such as having strong willed, indepen- dent thinking veterinarians and staff get along. That is one of the reasons why many veterinarians choose to work in a solo situation. • Fulfill family goals, whatever they might be. • Ability to pursue hobbies and special interests. • Ability to travel and take vacations. • Maintain physical and mental health. • Allow time and resources for spiritual growth and missions. The next step is to figure out how pooling of re- sources could allow for the advantages of a group prac- tice without being in a traditional group practice. The first item I listed, efficient use of office staff, can and has been dealt with by sharing of office staff between solo practitioners. Use of answering services accom- plishes a similar goal. Outsourcing of accounting, bill- ing and payroll functions could also be used to help the efficiencies of a small or large practice, especially if it isn't anyone's prime interest and focus within the busi- ness. This could be done between veterinary practices or done with a business of any type. These functions are fairly similar for all businesses. The advantages could include financial gains, but also could improve things on the personal side because it can free up time for overworked veterinarians and staff that have this responsibility now. These things all sound good to most people, but in reality, it is difficult to achieve and maintain balance between these different, sometimes conflicting, goals. With this in mind, it is time to take personal stock of how you are meeting your life's goals. Are you achiev- ing the success you want in all the different areas of your life? Is there a certain area or goal that is not getting met? Why isn't that goal being achieved? What is the constraint that is preventing that goal from being met? After you answer these questions, you are then ready to consider how pooling resources in practice may help you to better meet some of your goals. Introduction My background is predominantly dairy, food ani- mal practice in southeastern Pennsylvania since 1987. Our practice has grown over the years, and currently there are eight veterinarians in the practice, five of whom are partners. My responsibilities include some Professional goals: • Interesting, fulfilling work. • A sense of purpose and helping people and ani- mals. • Ability to pursue training, new challenges. THE AABP PROCEEDINGS-VOL. 39 58 • Involvement in the veterinary and/or agricul- tural community. • Allows individual doctors to pursue and focus on their particular interest areas, helping them to grow professionally and providing a higher level of service to clients. y • Respected by clients, peers and members of the community. • Partners can focus on their particular area of management, helping financially and profession- ally. Financial goals: Financial goals: • Ability to maintain the desired standard ofliv- ing. y • Built-in group of colleagues to discuss cases with, helping professionally. • Retire debt. • Retire debt. Pooling Resources One last example of pooling resources that I will discuss is the possibility of many veterinarians work- ing together under some type of corporate ownership or umbrella organization. I am not aware of any examples of this at this time in food animal practice, but they may be out there and I'm not aware of it. For the sake of discussion, let's assume there will be 10 practices in this organization. Ownership could be all under one umbrella or it could be set up as a management organi- zation that would provide certain services to the mem- ber practices on a contract basis of some type. Certain pooled resources would be provided by the central man- agement that would help the individual practices meet their financial, professional and personal goals. These might include centralized bookkeeping, communication, business management and inventory management to improve efficiencies. Continuing education and train- ing could be brought in for the group to improve the level of service provided. Permanent'"relief vet" services would be provided to allow member practices to take time for vacations, personal days and continuing edu- cation away from the practice. Perhaps human resource management functions including recruitment, hiring, training and ongoing reviews could be handled centrally. If we consider this type of practice from the basis of meeting the financial, professional and personal needs of the veterinarians and employees involved, it might make sense. p Another situation to consider would be for a larger practice to provide some on-call service for a solo practi- tioner. This happens frequently to cover personal emer- gencies and health issues. It is important to discuss this with the practice covering the duty ahead of time so they don't feel dumped on and can also make sure they have enough on-call staff to cover adequately. Some type of long-term situation could be developed, but re- alize there must be something in it for both sides. I do not know of many practices who want more emergency work to do, especially for someone else's clients. A pos- sible solution might be for the solo practitioner to cover some time occasionally for the larger practice so all the vets in the larger practice could get together for a plan- ning meeting, educational event or social gathering with- out being called away. For any of these methods of pooling resources, both parties should benefit in some way. Pooling Resources A common constraint on personal goals in a small business of any kind, and a veterinary practice in par- ticular, is time off. This includes on-call duty, vacation time, other time off during the week and time to attain continuing education. Traditionally, two or more prac- titioners might alternate some weekend duty or nights on call if they are in a close enough proximity to cover for each other. This type of arrangement can work, but it takes a commitment from both or all parties to really make it work long-term. Conflicts can occur if one party feels the other may be trying to recruit their clients or if they feel service is not being provided adequately. Re- lief veterinarians can be hired to cover for longer peri- ods of time to give a practitioner some time away for personal or professional reasons. As I said in my introduction, I am part of a group practice. A group practice is not for everyone, nor should it be. But a group practice serves as one model for pool- ing resources to help its individual members meet their goals for success. In our practice, pooling of resources helps us to better meet our financial, professional and personal success in the following ways: • Efficient use of office staff helps financially. • Shared on-call duty helps allow time for personal goals. • Adequate vacation time and days off while still serving our clients helps maintain quality of ser- vice to clients and allow time for personal goals. • Opportunities for sabbaticals and part-time work, helping to meet personal goals. The small animal side of our profession has dealt with this issue by using emergency clinics, but I'm not aware of this happening on the bovine side, although someone could potentially be hired to cover on-call duty • Continuing education opportunities help profes- sionally. • Multiple doctors allow better call efficiencies, helping financially. SEPTEMBER, 2006 59 maining practice than buying the first practice at the time of retirement. for a large practice or group of small practices in a tight geographical area. This person would be a very special person. If you find him or her, let me know! There may be some potential for a part-time employee in some lo- cations to help with on-call or extended part-time du- ties to relieve the pressure on the other doctors. Pooling Resources y Another area where pooling ofresources may help is in retirement. As a solo veterinarian nears retire- ment, they may have conflict between their personal and financial goals. On the one hand, they would like to slow down a little bit and not work so hard, maybe even enjoying their last few years in practice more at a slower or part-time pace. On the other hand, because the value of a practice is based on its profitability, they want to keep working hard to keep the value of the prac- tice high. Even then, it may be hard to sell the practice because there will be no continuity of service so clients are free to call whomever they want, and a prospective buyer would rather just hang up his or her sign and not pay anything for the practice. If the retiring veterinar- ian was counting on selling his or her practice to fund their retirement, they may be left short. Conclusion The purpose of this presentation is not to have all the answers. Rather, it is to help you realize areas of life where you may presently fall short, identify why it is happening, and figure out a way to improve the situ- ation. Every one has different needs and situations. The solutions will probably take some real creativity to find. A possible solution is to plan ahead a few years and work with another practice in the area that will ultimately fill the void of the retiring practitioner. The retiring practitioner may sell his practice to the second practice, and agree to work full or part-time for them for a number of years. This allows a smooth transition of clients, a gradual reduction in responsibilities and more personal time for the retiring practitioner, and the chance to sell the practice. Because of the continuity of service to clients, this scenario is worth more to the re- If you ask most people what is important in life, most of the answers will deal with family, relationships, purpose and enjoyment. Yet, when you look at how we spend our time, our actions say otherwise sometimes. Being in veterinary practice can be an all-consuming kind of lifestyle. It does not have to be, however. A certain balance in life is important for most people to feel they are successful. There are times when pooling resources can help veterinarians to better achieve their goals in one or more of these areas. THE AABP PROCEEDINGS-VOL. 39 60 LIGHTS INCLUDE: nual James A. Jarrett Memorial Lecture ay "Farm to Fork" Symposium sponsored day (Tuesday January 16) ete Beef and Dairy Programs Dairy Reproductive Programs Master Class ns with a four-week online course 2007 CONFERENCE HIGHLIGHTS INCLUDE: 2nd Annual James A. Jarrett Memorial Lecture Two-day "Farm to Fork" Symposium AABP-sponsored day (Tuesday January 16) Complete Beef and Dairy Programs AABP Dairy Reproductive Programs Master Class Begins with a four-week online course and is completed at the NAVC Conference with a 1.5 hour lunch discussion. VISIT US ONLINE ATWWW.TNAVC.ORGTO REGISTER 2007 CONFERENCE ORLANDO, FLORIDA JANUARY 13-17 HIGHLIGHTS INCLUDE: HIGHLIGHTS INCLUDE: 2nd Annual James A. Jarrett Memorial Lecture Two-day "Farm to Fork" Symposium AABP-sponsored day (Tuesday January 16) Complete Beef and Dairy Programs AABP Dairy Reproductive Programs Master Cla Begins with a four-week online course and is completed at the NAVC Conference with a 1.5 hour lunch discussion.
https://openalex.org/W4238040374	https://zenodo.org/record/1115993/files/66-314-2-PB.pdf	Indonesian	null	MODEL RANCANGAN PENGUKURAN KINERJA DENGAN PENDEKATAN METODE INTEGRATED PERFORMANCE MEASUREMENT SYSTEM	Teknika : engineering and sains journal	2,017	cc-by	2,531	Teknika : Engineering and Sains Journal Volume 1, Nomor 1, Juni 2017, 33-40 Teknika : Engineering and Sains Journal Volume 1, Nomor 1, Juni 2017, 33-40 ISSN 2579-5422 online ISSN 2580-4146 print Diterima: 23 Maret 2017. Disetujui : 19 Mei 2017. Dipublikasikan : 1 Juni 2017 ©2017 –TESJ Fakultas Teknik Universitas Maarif Hasyim Latif. Ini adalah artikel dengan akses terbuka di bawah lisensi CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/) Diterima: 23 Maret 2017. Disetujui : 19 Mei 2017. Dipublikasikan : 1 Juni 2017 ©2017 –TESJ Fakultas Teknik Universitas Maarif Hasyim Latif. Ini adalah artikel dengan akses terbuka di bawah lisensi CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/) ABSTRAK P2KP adalah Program penanggulangan Kemiskinan di Perkotaan atas prakarsa Departemen Pekerjaan Umum yang kemudian berganti nama menjadi PNPM Mandiri Perkotaan dimana program ini meliputi tingkat kelurahan, kecamatan, kabupaten dan propinsi. Dalam prakteknya, pelaporan kinerja PNPM Mandiri ini masih didasarkan pada laporan review Partisipatif dari masyarakat sebagai bentuk pertanggungjawabannya dimana harus dituntut transparan dan akuntabilitas. Sistem pengukuran kinerja sepeti itu tidak bisa mengukur kinerja organisasi secara pasti dan terukur nilainya. Oleh sebab itu dibuatlah rancangan sistem pengukuran kinerja organisasi dengan pendekatan Integrated Performance Measurement Systems yang mengintegrasikan semua elemen yang terlibat didalam organisasi. Berdasarkan rancangan pengukuran kinerja tersebut dan disimulasikan didapatkan hasil kinerja organisasi dan hasilnya berupa nilai pencapaian yang dapat terukur. Kata Kunci : Integrated Performance Measurement System, Pengukuran Kinerja, PNPM Mandiri Perkotaan Nurul Aziza1, Yusuf Eko Nurcahyo2 1Teknik Industri, Fakultas Teknik Universitas Maarif Hasyim Latif, Sidoarjo, Indonesia e-mail : nurul_aziza@dosen.umaha.ac.id 1Teknik Industri, Fakultas Teknik Universitas Maarif Hasyim Latif, Sidoarjo, Indonesia e-mail : nurul_aziza@dosen.umaha.ac.id 2Teknik Manufaktur Politeknik 17 Agustus 1945 Surabaya, Surabaya, Indonesia MODEL RANCANGAN PENGUKURAN KINERJA DENGAN PENDEKATAN METODE INTEGRATED PERFORMANCE MEASUREMENT SYSTEM Nurul Aziza1, Yusuf Eko Nurcahyo2 Integrated Performace Measurement Systems dari top manajemen (pemerintah desa) sampai lower manajemen (masyarakat desa) dengan pendekatan konsep Integrated Performance Measurement Systems. Model yang dikembangkan oleh Centre for Strategic Manufacturing, dari University of Strathclyde, Glasow adalah model sistem penguku- ran kinerja terintegrasi atau Integrated Performance Measurement Systems, dimana model ini memiliki karakteristik antara lain : Model yang dikembangkan oleh Centre for Strategic Manufacturing, dari University of Strathclyde, Glasow adalah model sistem penguku- ran kinerja terintegrasi atau Integrated Performance Measurement Systems, dimana model ini memiliki karakteristik antara lain : Dari hasil penelitian ini, diharapkan dapat memberikan manfaat kepada pihak lembaga sebagai studi penelitian yaitu : 1. Menyusun semua aktivitas tugas dan aktivitas organisasi sesuai dengan tujuan kritis dari top level atau tingkatan atas. 1. Adanya pengukuran kinerja lembaga yang terintegrasi dengan indikator-indikator yang telah ditetapkan oleh Lembaga PNPM Mandiri, Pemerintah Daerah serta Departemen Pekerjaan Umum. 2. Memberikan kendali bisnis pada seluruh bagian yang terlibat didalamnya searah dengan strategi bisnis yang dimiliki. 2. Mengetahui secara pasti nilai pencapaian kinerja lembaga. 3. Mengarahkan program perbaikan yang searaha dengan strategi bisnis. 4. Memaksimalkan dampak dari usaha perbaikan. METODE PENELITIAN Gambar 1. Struktur model integrated performace measurement systems PENDAHULUAN melalui proses pembelajaran dan pelembagaan nilai-nilai universal kemanusiaan (Value Based Development), prinsip-prinsip universal kemasya- rakatan, serta prinsip-prinsip pembangunan berkelanjutan (sustainable development). Program Penanggulangan Kemiskinan di Perkotaan (P2KP) dilaksanakan sejak tahun 1999 sebagai upaya pemerintah untuk membangun kemandirian masyarakat dan pemerintah daerah dalam rangka menanggulangi kemiskinan serta berusaha memberdayakan masyarakat secara berkelanjutan. Hingga pada tahun 2008 pelak- sanaan P2KP diperluas lagi menjadi Program Nasional Pemberdayaan Masyarakat Mandiri Perkotaan (PNPM Mandiri Perkotaan). PNPM dirancang sebagai gerakan bersama yang terpadu dalam penanggulangan kemiskinan melalui proses pemberdayaan masyarakat. Pemberdayaan masya- rakat memerlukan keterlibatan berbagai pihak antara lain pemerintah, kelompok ahli, dunia usaha, dan masyarakat luas. Indikator keberhasilan Program PNPM Mandiri Perkotaan mengacu pada indikator keberhasilan PNPM Mandiri yang sebagaimana ditetapkan dalam Pedoman Umum PNPM Mandiri yang diterbitkan Kantor Menko Kesra. Selain itu juga indikator keberhasilan PNPM Mandiri Perkotaan 2009 juga didasarkan pada Project Management Guidleine yang diterbitkan oleh Departemen Pekerjaan Umum. Meskipun PNPM tingkat desa adalah tingkat paling sederhana, namun pengukuran indikator keberhasilan tersebut hanya terbatas didasarkan pada dokumen masyarakat hasil review partisipatif tiap kelurahan/desa/kota. Sebagai sebuah lembaga yang dituntut harus transparan dan accountable, maka perlu adanya penilaian atau pengukuran kinerja lembaga tersebut sebagai upaya evaluasi diri dari rencana yang telah dibuat maupun yang akan dibuat yang akan memungkinkan jaminan adanya integritas aktivitas lembaga yang bermula Salah satu pemilihan lokasi sasaran PNPM Mandiri Perkotaan adalah melanjutkan kegiatan di kelurahan-kelurahan.Inti kegiatan PNPM Mandiri Perkotaan di masyarakat kelurahan/desa adalah proses menumbuhkankembangkan kemandirian dan keberlanjutan upaya-upaya penanggulangan kemiskinan dari, oleh dan untuk masyarakat, 33 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 Scoring System dengan menggunakan Objective Matrix (OMAX). 2. Performance measurement (pengukuran kinerja) yaitu suatu proses perhitungan efisiensi dan efektifitas suatu tindakan. Objective Matrix adalah suatu sistem pengukuran produktivitas parsial yang dikembang- kan untuk memantau produktivitas di tiap bagian organisasi dengan kriteria produktivitas yang se- suai dengan keberadaan bagian tersebut. Model ini melibatkan seluruh jajaran organisasi, mulai dari Sistem Pengukuran Kinerja Dalam memelihara suatu aktivitas organisasi, ada 4 aspek penting yang harus diperhatikan (Simons, 2013), yaitu : 1. Tujuan dari sistem pengukuran kinerja adalah untuk mengalirkan informasi. Yang dimaksud disini adalah data, baik itu data finansial maupun non finansial yang mempengaruhi pengambilan keputusan dan tindakan manajemen. j 2. Pengukuran kinerja merepresentasikan prose- dur rutin yang sifatnya formal dimana informasi tersebut diolah dalam sistem komputer dalam format standar, baik dalam kertas dokumen maupun dalam sistem komputer database. Gambar 1. Struktur model integrated performace measurement systems Prinsip model Integrated Performance Measurement Systems secara top down, dimana telah mengakomodasikan stakeholder require- ments dari bisnis induk, dengan cara ini diperoleh integrasi dan konsistensi dari sistem pengukuran kinerja dari semua tingkatan organisasi dapat dijaga. Prinsip didalam model ini bertujuan untuk memahami logika model itu sendiri yaitu : 3. Pengukuran kinerja didesain khusus digunakan untuk para manager yang secara langsung berhubungan dengan informasi data. 4. Para manager menggunakan sistem pengukuran kinerja untuk memantain berbagai aktivitas organisasi. Sistem pengukuran adalah metode sistema- tis untuk membentuk tujuan-tujuan bisnis bersama dengan laporan timbal balik secara berkala yang mengindikasikan kemajuan dari pencapaian tujuan-tujuan utama. 1. Kontrol manajemen. 2. Deployment. 3. Penting. 4. Jelas. 4. Jelas. Definisi pengukuran kinerja secara sistema- tis disimpulkan dan dijabarkan oleh Cambrige Research Group (Neely, 1995) sebagai berikut : Definisi pengukuran kinerja secara sistema- tis disimpulkan dan dijabarkan oleh Cambrige Research Group (Neely, 1995) sebagai berikut : 5. Resources yang digunakan. 6. Normatif planning. 7. Pengawasan aktif. 1. Performance measure (ukuran performansi) yaitu suatu metrik yang digunakan untuk menghitung efisiensi dan efektifitas suatu tindakan. 8. Klasifikasi dan ukuran kinerja unit bisnis, proses, dan aktivitas. Objective 3. Performance measurement systems (sistem pengukuran kinerja) yaitu suatu kumpulan metrik yang digunakan untuk menghitung efisiensi dan efektifitas dari suatu tindakan. 34 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 total jumlah bobit semua kriteria harus sama dengan 100% atau 1. tingkat atas sampai bawah dengan harapan aktivi- tas seluruh anggota organisasi dapat turut menilai, memperbaiki, dan mempertahankan produktivitas. d. Performance indicator, yang terdiri dari current–jumlah nilai dari semua kriteria pengukuran pada saat ini (sekarang), previous–jumlah nilai pengukuran kinerja pe- riode sebelumnya, serta indeks produktivitas (IP) yaitu perbandingan antara periode yang diukur dengan periode sebelumnya. Kelebihan-kelebihan model OMAX antara lain adalah sebagai berikut : Kelebihan-kelebihan model OMAX antara lain adalah sebagai berikut : 1. Relatif sederhana dan mudah dipahami karena indikator kinerja untuk setiap input dan output didefinisikan dengan jelas. 2. Mudah dilaksanakan dan tidak memerlukan keahlian khusus karena dapat digunakan untuk mengukur seluruh aspek kinerja yang dipertimbangkan. Struktur OMAX dapat ditabelkan dalam bentuk matrix objective seperti pada Tabel 1. 3. Datanya mudah diperoleh yang merupakan kom-binasi dari pendekatan kuantitatif dan kualitatif. 4. Lebih fleksibel dengan memasukkan pertimba- ngan pihak manajemen dalam penentuan bobot. Kekurangan-kekurangan model OMAX antara lain : 1. Memerlukan batasan-batasan yang jelas untuk objective score dari tiap indikator, karena penentuan objectives score-nya dilakukan secara objective. j 2. Dibutuhkan suatu pengukuran yang kontinyu dan terstandar untuk mendapatkan indeks kinerja yang diharapkan. Tahapan dalam pengimplementasian model OMAX yaitu : Tahapan dalam pengimplementasian model OMAX yaitu : 1. Pendefinisian yang terdiri atas : a. Penentuan kriteria produktivitas yang meru- pakan perbandingan yang menyatakan defini- si untuk kerja produktif suatu unit kerja. Kriteria-kriteria ini disesuaikan dengan apa yang akan diukur. Penentuan kriteria ini harus independen dan merupakan faktor yang dapat diukur. j p j Kriteria-kriteria ini disesuaikan dengan apa yang akan diukur. Penentuan kriteria ini harus independen dan merupakan faktor yang dapat diukur. b. Performance (nilai pencapaian sekarang), yaitu nilai tiap produktivitas berdasarkan pengukuran terakhir. 2. Pengukuran , menggunakan skala yang terdapat pada badan matriks yang disusun oleh besaran skala pencapaian tiap-tiap kriteria. Skala yang dipakai berkisar antara 0 sampai 10 untuk terbesar. Sedangkan skala 3 dipakai untuk menunjukkan nilai produktivitas yang diukur pada awal pengukuran. Peningkatan nilai produktivitas akan disesuaikan dengan cara interpolasi. Tabel 2. PENUTUP Berdasarkan hasil simulasi rancangan kinerja, maka didapatkan hasil pencapaian kinerja PNPM Mandiri untuk tingkat Kelurahan sebesar 4,926 dimana masih jauh dari target yang diharapkan sehingga perlu diadakan perbaikan- perbaikan di masa yang akan datang. Dengan rancangan pengukuran kinerja seperti di atas, maka hasil kinerja dapat diketahui nilai capaiannya dan bukan hanya sekedar bentuk laporan review yang tak bisa terukur secara pasti pencapaian indikator-indikator kinerjanya. Untuk menguji konsistensi para responden diberikan rumus persamaan (1). (1) p ( ) RI CI CR n CI n maks      1  (1) d p ( ) RI CI CR n CI n maks      1  (1) d p ( ) RI CI CR n CI n maks      1  d dengan : Berikut ini indeks random untuk matrik berukuran 3 sampai 10 (matrik berukuran 1 dan 2 mempunyai inkonsistensi 0). Objective Skala penilaian pairwise comparison Nilai Keterangan 1 elemen keputusan yang satu sama penting dibandingkan dengan elemen keputusan yang lain 3 elemen keputusan yang satu sedikit lebih penting dibandingkan dengan elemen keputusan lain 5 elemen keputusan yang satu cukup penting dibandingkan dengan elemen keputusan lain 7 elemen keputusan yang satu dinilai sangat penting dibandingkan dengan elemen keputusan lain 9 elemen keputusan yang satu dinilai mutlak sangat penting sekali dibandingkan dengan elemen keputusan lain kebalikan 1/3,1/5,...1/9 nilai ini diberikan jika aktivitas i mendapat satu angka dibandingkan dengan aktivitas j, maka j mempunyai nilai kebalikan i. p 3. Pencatatan, yang terdiri atas : p 3. Pencatatan, yang terdiri atas : a. Nilai yang merupakan hasil perkalian dari skor pada kriteria tertentu dengan bobot kriteria tersebut. b. Skor, yaitu hasil pengukuran yang diubah kedalam skor yang disesuaikan atau nilai skor yang disesuaikan dengan nilai produktivitas. c. Bobot, yaitu besarnya pengaruh kriteria yang diukur terhadap nilai produktivitas yang didasarkan pada tingkat kepentingan dimana 35 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 Berikut simulasi hasil Kinerja PNPM Mandiri untuk Tingkat Kelurahan ditampilkan dalam Lampiran Tabel 3. Analytical Hierarchy Process (AHP) AHP merupakan suatu alat bantu sistem pendukung keputusan yang digunakan untuk menyelesaikan masalah dengan multikriteria, dimana metode ini melakukan perbandingan judment pengambil keputusan secara berpasangan pada setiap level hirarki keputusannya. Metode ini juga menetapkan bobot prioritas relatif setiap elemen keputusan yang merepresentasikan intensitas prioritas keputusan itu sendiri. ukuran matrik 3 4 5 6 7 8 9 10 random Index 0,58 0,90 1,12 1,24 1,32 1,41 1,45 1,49 ukuran matrik 3 4 5 6 7 8 9 10 random Index 0,58 0,90 1,12 1,24 1,32 1,41 1,45 1,49 Bititci, U. S. (1995). Modelling of performance measurement systems in manufacturing enterprises. International journal of production economics, 42(2), 137-147. Batasan diterima tidaknya konsistensi suatu matrik sebenarnya tidak ada yang baku, hanya menurut beberapa eksperimen dan pengalaman tingkat inkonsistensi sebesar 10% kebawah adalah tingkat inkonsistensi yang masih bisa diterima. Lebih dari itu harus ada revisi penilaian dan melakukan konfirmasi kembali karena tingkat inkonsistensi yang terlalu besar dapat menjurus pada suatu kesalahan. Batasan diterima tidaknya konsistensi suatu matrik sebenarnya tidak ada yang baku, hanya menurut beberapa eksperimen dan pengalaman tingkat inkonsistensi sebesar 10% kebawah adalah tingkat inkonsistensi yang masih bisa diterima. Bititci, U. S., Suwignjo, P., & Carrie, A. S. (2001). Strategy management through quantitative modelling of performance measurement systems. International Journal of production economics, 69(1), 15-22. Lebih dari itu harus ada revisi penilaian dan melakukan konfirmasi kembali karena tingkat inkonsistensi yang terlalu besar dapat menjurus pada suatu kesalahan. Departemen Pekerjaan Umum. (2009). Pedoman Pelaksanaan PNPMMandiri Perkotaan, Jakarta. PENGUMPULAN DATA DAN SIMULASI Gambar 3. Struktur Organisasi Pengelolaan PNPM Mandiri Perkotaan Tingkat Kelurahan Keterangan : PJOK : Penanggung Jawab Operasional Kegiatan Tim Fasilitator : Tenaga Pengembangan Masyarakat P2KP Relawan : Warga setempat yang peduli membantu warga miskin di wilayahnya tanpa pamrih LKM : Lembaga Keswadayaan Masyarakat, merupakan nama generik yang dahulu dinamakan BKM (Badan Keswadayaan Masyarakat). KSM : Kelompok Swadaya Masyarakat PENGUMPULAN DATA DAN SIMULASI PENGUMPULAN DATA DAN SIMULASI PENGUMPULAN DATA DAN SIMULASI Gambar 3. Struktur Organisasi Pengelolaan PNPM Mandiri Perkotaan Tingkat Kelurahan Neely, A., Gregory, M., & Platts, K. (1995). Performance measurement system design: a literature review and research agenda. International journal of operations & production management, 15(4), 80-116. Saaty, T. L. (1990). Multicriteria Decision Making: The Analytic Hierarchy Process: Planning. Priority Setting, Ressource Allocation, Pittsburgh. Simons, R. (2013). Performance Measurement and Control Systems for Implementing Strategy Text and Cases: Pearson New International Edition. Pearson Higher Ed. Wahyuningsih, E.D.S. (2002). Perancangan Sistem Pengukuran Performansi Organisasi dengan Metode Integrated Performance Measurement Systems, ITS Surabaya. Keterangan : PJOK : Penanggung Jawab Operasional Kegiatan Tim Fasilitator : Tenaga Pengembangan Masyarakat P2KP Relawan : Warga setempat yang peduli membantu warga miskin di wilayahnya tanpa pamrih LKM : Lembaga Keswadayaan Masyarakat, merupakan nama generik yang dahulu dinamakan BKM (Badan Keswadayaan Masyarakat). KSM : Kelompok Swadaya Masyarakat Keterangan : PJOK : Penanggung Jawab Operasional Kegiatan Tim Fasilitator : Tenaga Pengembangan Masyarakat P2KP Relawan : Warga setempat yang peduli membantu warga miskin di wilayahnya tanpa pamrih LKM : Lembaga Keswadayaan Masyarakat, merupakan nama generik yang dahulu dinamakan BKM (Badan Keswadayaan Masyarakat). KSM : Kelompok Swadaya Masyarakat 36 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 Lampiran Gambar 4. Hirarki Kinerja PNPM Mandiri Perkotaan Tingkat Kelurahan N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 Lampiran Lampiran Gambar 4. Hirarki Kinerja PNPM Mandiri Perkotaan Tingkat Kelurahan Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan 37 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33 40 Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan (lanjutan 1) Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan (lanjutan 2) Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan (lanjutan 1) Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan (lanjutan 2) 38 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan (lanjutan 3) Tabel 3. Rancangan Pengukuran Kinerja PNPM Mandiri Tingkat Kelurahan (lanjutan 3) 39 N Aziza, dkk / Teknika : Engineering and Sains Journal, Vol. 1, No.1, Juni 2017, 33-40 Halaman ini sengaja dikosongkan 40
https://openalex.org/W1980639584	https://europepmc.org/articles/pmc3734216?pdf=render	English	null	Safety of TNF-α inhibitors during IBD pregnancy: a systematic review	BMC medicine	2,013	cc-by	11,309	RESEARCH ARTICLE Open Access © 2013 Nielsen 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. Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Open Access Keywords: Biologics, Crohn’s disease, IBD, Lactation, Pregnancy, Ulcerative colitis Keywords: Biologics, Crohn’s disease, IBD, Lactation, Pregnancy, Ulcerative colitis Keywords: Biologics, Crohn’s disease, IBD, Lactation, Pregnancy, Ulcerative colitis * Correspondence: ohn@dadlnet.dk 1Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark Full list of author information is available at the end of the article Safety of TNF-α inhibitors during IBD pregnancy: a systematic review Ole Haagen Nielsen1, Edward V Loftus Jr2 and Tine Jess3 Abstract Background: Tumor necrosis factor (TNF)-α inhibitors are increasingly being used in inflammatory bowel disease (IBD). Because this chronic intestinal disorder often affects women of fertile age, it is essential to assess the effect of biologics on pregnancy outcome. Methods: We performed a systematic review of the English-language literature to investigate if treatment with TNF-α blockers during pregnancy in women with IBD increases the risk of spontaneous abortions, preterm delivery, stillbirth, low birth weight, congenital malformations, or risk of infections in the offspring. Of 552 articles and abstracts reviewed, 58 articles or abstracts with unique content were identified and included in this systematic review. However, most presentations were case reports or case series supplied by a limited number of observational studies. No randomized controlled studies were available. Results: TNF-α inhibitors do not seem to affect either outcome of pregnancy in mothers with IBD, or the outcome in the offspring (congenital malformations and immunosuppression). Further, recent data have not identified any increased risk of infections in the first year of life in the offspring of mothers who received biologics, even in combination with immunomodulators (thiopurines). Conclusions: From the present systematic review, no association was found between administration of TNF inhibitors for IBD during pregnancy and adverse pregnancy outcome or congenital abnormalities. Further, no increased relative risk of infections has been reported in the first year of life in offspring of mothers who received biologics. Biologics should be discontinued during pregnancy solely if the IBD is in remission using the same stopping criteria as for patients with IBD in general, as uncontrolled activity of IBD may expose the mother and child to a risk greater than those only potentially coming from the use of TNF-α inhibitors. In such cases, inoculation of the offspring with live vaccines is contraindicated until the biologic agent is no longer detectable in the child’s circulation. Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 These four biologic agents are all classified as ‘category B’ drugs by the US Food and Drugs Administration (FDA) [9], meaning that animal reproduction studies have not shown any risk to the fetus, but there are no adequate and well-controlled studies in pregnant women. Data extraction T h ( Two authors (OHN and TJ) independently identified candidate articles from the results of the initial search, based on the title and abstract. Subsequently, both au- thors independently reviewed the full text of candidate articles to identify interventions and assess study quality. In cases of any discrepancies between the independent searchers, these were resolved in consensus with the third author (EVL). The reference lists of relevant articles were hand- searched to identify additional studies. Further, abstracts from the meetings of American Gastroenterological As- sociation (Digestive Disease Week), American College of Gastroenterology, and the European Crohn’s and Colitis Organisation from 1998 to May 2013 were searched manually for relevant abstracts based on the aforemen- tioned search terms. The aim of this study was to perform a systematic re- view of the available literature on the risk of adverse birth outcomes related to IBD after maternal exposure to IFX, ADA, or CZP, in order to improve evidence- based informed choice and clinical decision-making dur- ing pregnancy, especially in the third trimester. Data about use of GLM in IBD pregnancies are currently un- available, as it has only recently been approved for UC. Search strategy A systematic review was performed according to the guidelines established by the Meta-analysis of Observa- tional Studies and Epidemiology Group (MOOSE cri- teria) [18]. The databases searched (January 1998 to May 2013) were MedLine, EMBASE, the Cochrane Library, and the homepages of the FDA and the European Medi- cines Agency (EMA), using the combinations of the fol- lowing Medical Subject Heading (MeSH) search terms: (inflammatory bowel disease OR inflammatory bowel diseases OR Crohn’s disease OR ulcerative colitis) AND biologics OR biologic products OR infliximab OR adalimumab OR golimumab OR certolizumab. This was again combined with either pregnancy OR newborn OR lactation OR breast feeding OR infant. No authors were contacted. In the clinical scenarios of a woman with IBD on bio- logic therapy becoming pregnant, or when biologic treat- ment is being considered in a pregnant woman with active IBD, decisions are made on a case-by-case basis because the short-term and long-term implications of exposure to biologic agents have not yet been investi- gated systematically. Although no comparative effective- ness data exist to support the strategy, it has been common practice at many IBD centers to continue bio- logics through the second trimester of pregnancy [10], as the transplacental transfer of IgG begins around week 20 and increases thereafter, especially during the third trimester [11]. In accordance with this practice, IFX and ADA are discontinued around week 30 of pregnancy [12], and in patients with active IBD, it has been sug- gested to bridge therapy with glucocorticoids to control disease activity until delivery [13-16]. This approach has the benefit of minimizing the break from treatment with biologic agents and perhaps decreasing the risk of im- munogenicity [8] (that is, the risk of hypersensitivity re- actions or loss of response once the biologic is reintroduced post-partum). However, CZP may differ from the other tumor necrosis factor (TNF)-α blockers used in IBD, as it is a Fab fragment of a monoclonal antibody, which is not transported across the placenta and therefore, it may not be necessary to discontinue this drug in the third trimester [17]. Selection criteria d We wanted to include all randomized controlled trials, observational studies, case series, and case reports evaluating pregnancy outcomes of women with IBD treated with TNF-α inhibitors (IFX, ADA, GLM, or CZP) during any trimester of pregnancy, including the 90 day periconceptional period. Outcome assessment included miscarriages (spontaneous abortions), pre- term delivery, stillbirth, low birth weight, and/or con- genital malformations, and if data were available, it also included assessment of infections in the offspring. Only abstracts or articles published in English were considered. Figure 1 shows the flowchart of the study screening process. However, at present there are no definitive conclusions concerning what could be one of the most important therapeutic decision-making settings from the viewpoint of risk-benefit analysis. Thus, the decision whether to use bio- logics during pregnancy might be regarded as an example in which it is considered most prudent to err on the side of caution, especially as it is still unknown what levels in the serum of TNF inhibitors used to treat a mother with IBD will be safe or harmful for the fetus or newborn. Background In general, women with IBD are advised to conceive at quiescent stages of their disease, as this reduces the risk of obstetric complications (for example, preterm delivery and low birth weight) [1-5]. Effective control of disease activity using a wide range of medications, including bio- logics, is vitally important during pregnancy [6,7]. Biologics are effective and increasingly used in the treat- ment of inflammatory bowel disease (IBD), of which Crohn’s disease (CD) and ulcerative colitis (UC) are the two main types. As the peak incidence of IBD overlaps with the prime reproductive years, information on the effects of biologics on pregnancy outcome (including fetal harm) is essential in order to give appropriate ad- vice to women of childbearing age who require treat- ment for IBD. The most commonly used biologics in IBD are infliximab (IFX), a chimeric IgG1 monoclonal antibody; adalimumab (ADA), a human monoclonal IgG1 anti- body; and certolizumab pegol (CZP), a pegylated Fab fragment of a humanized IgG4 isotype monoclonal anti- body [8]. Recently, golimumab (GLM), also a human monoclonal IgG1 antibody, has been added to the list. Correspondence: ohn@dadlnet.dk 1Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark Full list of author information is available at the end of the article Page 2 of 13 Page 2 of 13 Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Ethics approval The search yielded a total of 58 studies (36 articles and 22 abstracts) that met the inclusion criteria (Table 1). The studies comprised 33 case reports, 21 case series, This study was an analysis of published data, which did not require ethics committee approval. Page 3 of 13 Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Studies retrieved through electronic and subsequent hand search (n=552) Excluded due to no relevance, e.g., other diseases or animals (n=49) Excluded due to non- English language (n=74) Exclusion of reviews and articles without patient cases (n=335) Studies excluded due to: -Repeated patient description (n=36) Second pass review of studies (n=429) Studies included in final analysis (n=58) Detailed evaluation of studies n=94 First pass review of studies (n=478) Figure 1 Flowchart of the study screening process. months prior to conception [12,50]. MTX is a drug clas- sified as ‘X’ by the FDA; that is, a drug for which studies in animals or humans have shown fetal abnormalities and/or for which there is positive evidence of human fetal risk, based on adverse reaction data from investiga- tional or marketing experience, and for which the risks involved in use of the drug in pregnant women clearly outweigh the potential benefits [9]. However, polydactyly is an uncommon disorder; it is not usually seen as the only complication to MTX treatment, but is more often present together with other abnormalities [50]. Studies retrieved through electronic and subsequent hand search (n=552) The first series studying intentional IFX administration throughout pregnancy examined outcomes in 10 women with active CD, and all ended in live births [32]. Of these 10 births, there were 3 cases of premature birth and 1 case with low birth weight, findings that were not unex- pected in women with CD that was sufficiently severe to require IFX treatment. No congenital abnormalities were observed 6 months post-partum [32]. Exclusion of reviews and articles without patient cases (n=335) The four largest studies providing data on the safety of IFX in an IBD population are from the Therapy Re- source Evaluation Assessment Tool (TREAT) registry [36], a pregnancy safety database maintained by the manufacturer of IFX [49], the Pregnancy in Inflamma- tory Bowel Disease and Neonatal Outcomes (PIANO) registry [51] and the Groupe d’Etude Thérapeutique des Affections Inflammatoires du Tube Digestif (GETAID) collaboration [52]. -Repeated patient description (n=36) Figure 1 Flowchart of the study screening process. Ethics approval The TREAT registry is a prospective study of North American patients with CD. As of February 2010, there were 6,273 patients in the registry, and 142 pregnancies with IFX exposure were reported. The risk of fetal malformations was no higher than in the general popu- lation [36], and a more detailed analysis of pregnancies in the TREAT registry is under way [36]. and 4 prospective studies with or without control groups. No controlled randomized studies were identi- fied. The total number of pregnant women with IBD who were exposed to anti-TNF agents was at least 1,533 (one study with 289 pregnancies included other diseases and did not specify the number of women with IBD [19]). The bias of the present study was that only non- English studies were excluded. The IFX Safety Database is a retrospective data- collection instrument set up by the manufacturer of IFX (Centocor, now Janssen Biotech) to study women with rheumatoid arthritis or CD exposed to IFX before or during pregnancy. There were 96 women with direct ex- posure to IFX who gave birth to 100 children; however, in most case, IFX treatment was discontinued in the first trimester of pregnancy. The rates of adverse outcomes were not different from that expected in the general population [49]. Infliximab In the early days of biologic treatment for IBD, IFX was reported to be associated with adverse pregnancy out- comes in some case reports [20,21], but not in others [22-27], and in case series where IFX was administered throughout pregnancy to maintain remission in IBD, no harm to the fetus/child was reported [16,19,28-34]. Fur- thermore, larger and subsequent studies did not report any increased risk for adverse events compared with un- exposed IBD pregnancies [12,17,35-49]. In a study on pregnancies exposed to IFX, Zelinkova et al. observed one adverse event (polydactyly), actually reported on two occasions [12,50]. In this particular case, the mother of the infant had also received methotrexate (MTX) 2 Preliminary data from the US national prospective PIANO registry has recently been disclosed [53]. A total of 1,232 women with IBD were enrolled as of April 1, 2013, of whom 357 women received biologics, including during the third trimester, and an additional 109 women received a combination of thiopurines and biologics [53]. Adverse pregnancy outcomes such as spontaneous abor- tions, preterm birth, low birth weight, and congenital ab- normalities were evaluated. A slightly higher relative risk blockers in IBD pregnancies e of pregnancy Study type Mode of delivery Outcome of pregnancies Case report Unknown Normal, healthy, full term (T1) Case report Unknown Premature, preterm, LBW. Adverse outcome: intracerebral and intrapulmonary bleeding followed by death 3 days later (metronidazole + azathioprine + 5-ASA was also administered in pregnancy for treatment of flare and fistulae) T2) Case report Unknown Normal, healthy, full term (correspondence with authors) Case report C-section Normal, healthy, preterm (36 weeks) 6 PC; 58/96 P(T1); unknown Case series from Infliximab Safety Database (82 CD, one UC, 10 RA, 3 unknown); 96 women, 72 births Unknown 64/96 were live births (1 preterm death at 24 week, 1 full-term with tetralogy of Fallot, 1 perinatal sepsis, 1 intestinal malrotation, 1 developmental delay); 4 pregnancies ended with twins. 14/96 were miscarriages, 18/96 were therapeutic terminations PC; 8/10 P(T2); P(T3); 7/10 PP Case series (n = 10 women) 8/10 C-section, 2/10 Vaginal 3/10 were preterm, 1/10 were LBW, 2/10 were neonatal illnesses, which resolved. Infliximab All normal at follow-up (T1-T3)/PP Case report Unknown Normal, healthy, preterm (36 weeks) (T1) Case report Vaginal Normal, healthy, full term (T1-T3) Case report Vaginal Normal, healthy, full term T3) Case series (n = 5) 4 vaginal/1 C-section Normal, healthy, full term (T1-T3) Case report Unknown Normal, healthy, preterm Case report C-section Normal, healthy, full term T3) Case report Unknown Intra-uterine fetal death due to placental abruption or intra-uterine infection p blockers in IBD pregnancies e of pregnancy Study type Mode of delivery Outcome of pregnancies Case report Unknown Normal, healthy, full term (T1) Case report Unknown Premature, preterm, LBW. Adverse outcome: intracerebral and intrapulmonary bleeding followed by death 3 days later (metronidazole + azathioprine + 5-ASA was also administered in pregnancy for treatment of flare and fistulae) -T2) Case report Unknown Normal, healthy, full term (correspondence with authors) Case report C-section Normal, healthy, preterm (36 weeks) 6 PC; 58/96 P(T1); unknown Case series from Infliximab Safety Database (82 CD, one UC, 10 RA, 3 unknown); 96 women, 72 births Unknown 64/96 were live births (1 preterm death at 24 week, 1 full-term with tetralogy of Fallot, 1 perinatal sepsis, 1 intestinal malrotation, 1 developmental delay); 4 pregnancies ended with twins. 14/96 were miscarriages, 18/96 were therapeutic terminations PC; 8/10 P(T2); P(T3); 7/10 PP Case series (n = 10 women) 8/10 C-section, 2/10 Vaginal 3/10 were preterm, 1/10 were LBW, 2/10 were neonatal illnesses, which resolved. Infliximab All normal at follow-up (T1-T3)/PP Case report Unknown Normal, healthy, preterm (36 weeks) (T1) Case report Vaginal Normal, healthy, full term (T1-T3) Case report Vaginal Normal, healthy, full term -T3) Case series (n = 5) 4 vaginal/1 C-section Normal, healthy, full term (T1-T3) Case report Unknown Normal, healthy, preterm Case report C-section Normal, healthy, full term -T3) Case report Unknown Intra-uterine fetal death due to placental abruption or intra-uterine infection (T1-T3) Case report C-section Normal, healthy, full term /P(T1-T3), 1 P(T2) Case series (3 women) 2 vaginal, 1 C-section 3 normal, healthy, full term (T1-T3) Case report Vaginal Normal, healthy, full term -T3) Case report Unknown Normal, healthy, full term (T1-T3) Case report Unknown Normal, healthy, full term (T1) Case report C-section Normal, healthy, preterm (31 weeks) (T1-T3) 2 case reports C-section Normal, healthy; 1 preterm (31 weeks) (T1-T3) Case report Unknown Normal, healthy, full term (T1-T3) Case report Vaginal Healthy, full term, no malformations Case report C-section Healthy, preterm (37 weeks) T1 T3) Case report Unknown Normal, healthy, full term T1) Case report C-section Normal, healthy, preterm (31 weeks) T1-T3) 2 case reports C-section Normal, healthy; 1 preterm (31 weeks) T1-T3) Case report Unknown Normal, healthy, full term T1-T3) Case report Vaginal Healthy, full term, no malformations Case report C-section Healthy, preterm (37 weeks) ockers in IBD pregnancies (Continued) 3) Case report Unknown Normal, healthy, full term 1-T3) Case report Unknown Normal, healthy, full term 1-T3) Case report C-section Normal, healthy, full term 1-3) Case series (3 women) Unknown All healthy, full term, 1 low birth weight, no malformations 1-T2) Case report Unknown Normal, healthy, full term 1-T3) Case series (12 women) 8 vaginal, 4 C-sections 12 healthy children ported ancies Case series (analysis of data from TREAT registry) Unknown The vast majority of babies (92.4%) were healthy with no defects or other adverse events 1-T3) Case series (556 pregnancies) Unknown There were 58 miscarriages, 38 elective abortions, 7 stillbirths, and 453 live births 1-T3)/PP Case report C-section Normal, healthy, full term 3) Case report Unknown Normal, healthy, full term 1-T3) Case report Vaginal Normal, healthy, full term 1) Case series (5 women) Unknown All full-term live births. No malformations 1-T3) Case report C-section Normal, healthy, full term 1-T3) Case-controlled study 34 women and 45 controls (OTIS) Unknown 29 live-born (1 with undescended testicle + 1 with microencephaly); 5 spontaneous abortions. Infliximab Two major structural defects in control group 1) Case report Vaginal Healthy, full term 1-3) Case report Vaginal Healthy, full term 1-T3) Case report C-section Twins, normal, healthy, full term 1-T3) Case series (5 women) 2 vaginal 3 C-sections Healthy (one infant developed pulmonary edema, but recovered fully) wn (UCB n file) Case series (16 women) Unknown 4 healthy, full term; 1 small for gestational age; and 2 lost to follow-up. 8 induced abortions amd 1 spontaneous abortion 3)/PP Case report Unknown Healthy, full term, no malformations 1; T3) Case report Vaginal Normal, healthy, full term. Adverse outcome in 1 case: bleeding 7 days post-partum, secondary to cotyledon retention 3) Case report C-section Healthy, full term, no malformations 1-T3) Case series (139 pregnancies including 17 with rheumatoid arthritis) Unknown 21 miscarriages, 15 elective abortions, and 103 live births (2 with congenital abnormalities: 1 with mild unilateral hydronephrosis and 1 with vesicouretic reflux) 1-T3) Case series (289 pregnancies including other diseases than IBD) Unknown No increased risk of adverse pregnancy outcome compared with unexposed IBD pregnancies http://www.biomedcentral.com/1741-7015/11/174 p T3) Case report Unknown Normal, healthy, full term (T1-T3) Case report Unknown Normal, healthy, full term (T1-T3) Case report C-section Normal, healthy, full term (T1-3) Case series (3 women) Unknown All healthy, full term, 1 low birth weight, no malformations (T1-T2) Case report Unknown Normal, healthy, full term (T1-T3) Case series (12 women) 8 vaginal, 4 C-sections 12 healthy children eported nancies Case series (analysis of data from TREAT registry) Unknown The vast majority of babies (92.4%) were healthy with no defects or other adverse events (T1-T3) Case series (556 pregnancies) Unknown There were 58 miscarriages, 38 elective abortions, 7 stillbirths, and 453 live births (T1-T3)/PP Case report C-section Normal, healthy, full term T3) Case report Unknown Normal, healthy, full term (T1-T3) Case report Vaginal Normal, healthy, full term (T1) Case series (5 women) Unknown All full-term live births. No malformations (T1-T3) Case report C-section Normal, healthy, full term (T1-T3) Case-controlled study 34 women and 45 controls (OTIS) Unknown 29 live-born (1 with undescended testicle + 1 with microencephaly); 5 spontaneous abortions. Infliximab Two major structural defects in control group (T1) Case report Vaginal Healthy, full term (T1-3) Case report Vaginal Healthy, full term (T1-T3) Case report C-section Twins, normal, healthy, full term (T1-T3) Case series (5 women) 2 vaginal 3 C-sections Healthy (one infant developed pulmonary edema, but recovered fully) nown (UCB on file) Case series (16 women) Unknown 4 healthy, full term; 1 small for gestational age; and 2 lost to follow-up. 8 induced abortions amd 1 spontaneous abortion T3)/PP Case report Unknown Healthy, full term, no malformations (T1; T3) Case report Vaginal Normal, healthy, full term. Adverse outcome in 1 case: bleeding 7 days post-partum, secondary to cotyledon retention T3) Case report C-section Healthy, full term, no malformations (T1-T3) Case series (139 pregnancies including 17 with rheumatoid arthritis) Unknown 21 miscarriages, 15 elective abortions, and 103 live births (2 with congenital abnormalities: 1 with mild unilateral hydronephrosis and 1 with vesicouretic reflux) (T1-T3) Case series (289 pregnancies including other diseases than IBD) Unknown No increased risk of adverse pregnancy outcome compared with unexposed IBD pregnancies α blockers in IBD pregnancies (Continued) C/P(T1-T3) 15 case series (15 women) Unknown 9 terminated pregnancies at study end. All live births were healthy, with two sets of twins C and/or P(T1) d/or P(T2) Case-controlled study (35 women with 42 pregnancies (35 IFX + 7 ADA) versus 101 pregnancies before IBD/ treatment and 56 matched pregnancies in healthy controls) Unknown 10 abortions (1 trisomy 18, 1 elective), 1 stillbirth (umbilical strangulation), 32 live births: 8/32 premature, 6/32 low birth weight. All children were born healthy but one child with necrotizing enterocolitis died at 13 days of age (mother required treatment for severe asthma and CD during T3) C/P(T1-T3) Case series (33 pregnancies in 31 women) Unknown 5/33 pregnancies ongoing. Infliximab 24/28 healthy children (4/24 preterm), 3/24 spontaneous abortions, and 1/24 provoked abortion C/P(T1-T3) Case series (29 pregnancies in 21 women) Unknown 27 normal, healthy, full term; 2 miscarriages C/P(T1-T3) Case series (14 pregnancies in 13 women) Unknown 13 normal, healthy, full term; 1 miscarriage C/P(T1-T3) 1-T2 ADA only) Case series (31 pregnancies in 28 women) Unknown 28 live births (27 normal, healthy, full term; 1 child born to a mother treated with concomitant methotrexate periconceptually had polydactyly); 3 miscarriages C/P(T1-T3) Case-controlled retrospective study (30 pregnancies in treated mothers versus 110 unexposed IBD pregnancies) Unknown Prevalence of unfavorable delivery outcome (spontaneous abortion, preterm delivery, low birth weight and malformations) were higher in the control group C/P(T1-T3) Case series (85 women) Unknown 6/85 were miscarriages, 47/85 were live births (30/85 ongoing), 8/49 were preterm, 3/49 were deaths (two in utero/one very premature),7/46 live births had neonatal complications (1 infection, 3 respiratory distress syndrome, 6 LBW <2500 g, 1 HELLP syndrome C/P(T1-T3) Case-controlled study (291 women without, and 79 with immunomodulators) Unknown No increased risk apart from higher risk of spontaneous abortion and C-section in group treated with biologics alone, and higher risk of preterm birth and infections int offspring at 12 months in combined thiopurine group verus biologics-alone group or unexposed IBD pregnancies C/P(T1-T3) Case series (66 pregnancies) Unknown 55 normal, healthy full term;, 4 preterm; 6 miscarriages; 1 congenital abnormality (several cardiac malformations) C/P(T1-T3) Case series (31 women) 15 vaginal, 16 C-section All 33 children (2 sets of twins) were normal, healthy, full term C-section cesarean section, CZP certolizumab pegol, HELLP a syndrome characterized by hemolysis, elevated liver enzymes, and low platelet count (that is, a el disease, IFX infliximab, LBW low birth weight, PC periconceptual (that is, use within 90 days before conception), PP post-partum, P(T1-T3) (pregnancy red), MTX methotrexate, TNF tumor necrosis factor, TREAT Therapy Resource Evaluation Assessment Tool. http://www.biomedcentral.com/1741 7015/11/174 p p g in healthy controls) y with necrotizing enterocolitis died at 13 days of age (mother required treatment for severe asthma and CD during T3) T3) Case series (33 pregnancies in 31 women) Unknown 5/33 pregnancies ongoing. T1) only) sarean sect IFX inflixima methotrexat Infliximab Moreover, the use of biologics in the second and third trimesters has been questioned [12]. However, Zelinkova et al. [12,50] showed that even though two patients discontinued IFX treatment as early as in week 26 of pregnancy, the in- fants still had detectable amounts of IFX in their serum samples after delivery. Low levels of IFX have been detected up to 6 months post-partum in the serum of infants whose mothers have received this drug up to de- livery [16]. IFX was undetectable in the infant at delivery only in one case, where the mother discontinued drug therapy at week 21, suggesting that discontinuation of drug therapy might be considered earlier than the begin- ning of the third trimester [50]. The French GETAID consortium prospectively recorded pregnant women with IBD during a 2 year period (2009 to 2010), and preliminary data analysis of 127 pregnancies in 120 women was reported in 2010 [52]. Of these 120 women, 54 women had received biologic therapy for their IBD. Birth outcomes of the women on biologics did not differ from those in the unexposed IBD population, suggesting an absence of any excess risk linked to anti- TNF-α therapy. Schnitzler et al. [54] conducted an observational study of 35 pregnancies under direct exposure to IFX until week 20 of pregnancy, 53 pregnancies with indirect ex- posure to IFX (women treated with IFX prior to preg- nancy), 7 pregnancies with direct exposure to ADA, and 56 matched pregnancies in healthy women. Several pa- tients experienced a flare of disease in the third trimes- ter. However, exposure to anti-TNF-α preparations was not associated with a higher incidence of adverse preg- nancy outcomes [54]. The paper concluded that preg- nancy outcomes after exposure to TNF inhibitors did not differ from those of women with IBD who were not exposed to anti-TNF treatment, but were worse than in healthy women [54]. Further, a literature review includ- ing women with rheumatic disorders or CD with direct exposure to IFX at any time during pregnancy did not find any increase in undesirable pregnancy or fetal out- come [55]. Because of this presence of IFX in the circulation after delivery, the use of live-virus vaccines (for example, varicella, measles, mumps, rubella, rotavirus, intranasal influenza, and bacillus Calmette-Guérin; BCG) is contra- indicated in patients receiving biologic therapy and their children [62]. Infliximab However, this study was subsequently criti- cized for significant methodological flaws [58,59], in- cluding selection bias, lack of a control group, and the fact that only one of the infants (the etanercept group only) had a clustering of three or more abnormalities included in the VACTERL spectrum [60]. Furthermore, the review did not take into account the total number of women exposed to anti-TNF-α agents or any confounding by severity of the underlying disease. Fi- nally, the most common defects reported in this review were cardiac defects, which are observed in the general population as well [58,59]. (RR) for preterm birth was found for women on combin- ation therapy (2.4; 95% confidence interval (CI) 1.3 to 4.3), which was, however, not found for those on bio- logics only (0.8; 95% CI, 0.5 to 1.3). No elevation in the risk of spontaneous abortion, low birth weight, or con- genital abnormalities was observed for biologics, either alone or in combination with thiopurines. Earlier reports (May 2012) had indicated that there was a higher rate of infections in the offspring [51], but the RR at month 12, adjusted for premature birth, was shown in April 2013 to be 0.9 (95% CI 0.7 to 1.1) for biologics alone, and 1.0 (95% CI 0.7 to 1.3) for those on combined therapy. When results for CZP were excluded, the values were 0.9 (95% CI 0.8 to 1.2) and 1.0 (95% CI 0.7 to 1.3), re- spectively [53]. In addition, breast feeding was not asso- ciated with any risk of infection or impairment of height or weight. The limitation on the preliminary data was that not all infants had reached the age of 12 months, and the observation will continue until the age of 4 years for all of the offspring [53]. p p [ ] Like other IgG antibodies, IFX is transferred across the placenta with the help of an Fc receptor neonatal (FcRn) molecule responsible for the transfer of immuno- globulins from the mother to the neonate [11]. The transfer occurs partly in the second, and mainly in the third trimester [31]. This finding has led many clinicians to discontinue IFX in the third trimester when the highest level of transfer occurs [61], in the belief that de- tectable serum IFX in the neonate might lead to clinic- ally relevant immunosuppression. Infliximab 24/28 healthy children (4/24 preterm), 3/24 spontaneous abortions, and 1/24 provoked abortion T3) Case series (29 pregnancies in 21 women) Unknown 27 normal, healthy, full term; 2 miscarriages T3) Case series (14 pregnancies in 13 women) Unknown 13 normal, healthy, full term; 1 miscarriage T3) DA only) Case series (31 pregnancies in 28 women) Unknown 28 live births (27 normal, healthy, full term; 1 child born to a mother treated with concomitant methotrexate periconceptually had polydactyly); 3 miscarriages T3) Case-controlled retrospective study (30 pregnancies in treated mothers versus 110 unexposed IBD pregnancies) Unknown Prevalence of unfavorable delivery outcome (spontaneous abortion, preterm delivery, low birth weight and malformations) were higher in the control group T3) Case series (85 women) Unknown 6/85 were miscarriages, 47/85 were live births (30/85 ongoing), 8/49 were preterm, 3/49 were deaths (two in utero/one very premature),7/46 live births had neonatal complications (1 infection, 3 respiratory distress syndrome, 6 LBW <2500 g, 1 HELLP syndrome T3) Case-controlled study (291 women without, and 79 with immunomodulators) Unknown No increased risk apart from higher risk of spontaneous abortion and C-section in group treated with biologics alone, and higher risk of preterm birth and infections int offspring at 12 months in combined thiopurine group verus biologics-alone group or unexposed IBD pregnancies T3) Case series (66 pregnancies) Unknown 55 normal, healthy full term;, 4 preterm; 6 miscarriages; 1 congenital abnormality (several cardiac malformations) T3) Case series (31 women) 15 vaginal, 16 C-section All 33 children (2 sets of twins) were normal, healthy, full term n cesarean section, CZP certolizumab pegol, HELLP a syndrome characterized by hemolysis, elevated liver enzymes, and low platelet count (that is, a ase, IFX infliximab, LBW low birth weight, PC periconceptual (that is, use within 90 days before conception), PP post-partum, P(T1-T3) (pregnancy TX methotrexate, TNF tumor necrosis factor, TREAT Therapy Resource Evaluation Assessment Tool. Page 7 of 13 Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 etanercept (a biologic agent not efficacious for IBD [57]), and 19 were exposed to IFX. Of the 41 children, 24 (59%) had abnormalities thought to be part of the VACTERL (vertebral abnormalities, anal atresia, cardiac defects, tracheoesophageal, renal and limb abnormal- ities) spectrum. The findings led the authors to conclude that the use of TNF-α blockers should be avoided during pregnancy. Certolizumab pegol Because CZP lacks the Fc portion of the antibody, which is crucial for the main transfer of immunoglobulins across the placenta, the transfer of CZP to the offspring is low, but a small transfer of Fab fragments is seen [31]. In a group of 10 women receiving CZP during preg- nancy, with the final dose 1 to 4 weeks before delivery, CZP was detectable at minimal levels in the infants’ cir- culation [17]. Overall, CZP is transferred across the pla- centa to a much lower degree than IFX or ADA [17]. For IBD, 21 case reports and series with more than 300 children exposed showed no increased risk of ad- verse pregnancy outcome or congenital malformations associated with ADA treatment during pregnancy com- pared with pregnancies in unexposed women with IBD [12,17,19,35,37,39,43,46,48,51,52,54,71-80], even if ADA was administered in the third trimester [17,19,35,37,39, 43,46,48,51,52,71-74,76,77,79,80]. The Organization of Teratology Information Specialists reported on a group of 34 women treated with ADA for various indications in a prospective study, and another 133 ADA-exposed women in a case series. There was no difference in pre- term deliveries, stillbirths, spontaneous abortions or congenital malformations in the group treated with ADA compared with either the general population or a control group with the same disease but not exposed to ADA [62,81]. In line with these observations, Schnitzler et al. did not find a higher incidence of adverse preg- nancy outcomes in seven women treated with ADA compared with unexposed women with IBD, although the power of that study was limited [54]. Compared with the other two TNF inhibitors ap- proved for CD, the human data on pregnancy outcomes in mothers treated with CZP during pregnancy are more limited. In three case reports where CZP injections were administered during the third trimester, healthy infants were delivered at full term [84-86], and in a case series of 16 pregnancies of CZP-treated mothers, all children were healthy [31]. In a recent case series with 139 preg- nancies, 17 were in mothers with rheumatoid arthritis; of these 139 pregnancies, 103 ended in live birth (2 with congenital abnormalities; see Table 1), 21 in spontaneous miscarriage, and 15 in elective termination [87], results that are similar to those reported in the general popula- tion [87]. Based on these findings and the low placental transfer of CZP, the drug appears to be safe to use throughout pregnancy [17]. Golimumab Because GLM was approved only recently (May 2013) by the FDA for treatment of the UC, data are not yet available for this drug in relation to IBD pregnancies. Infliximab A case report described a fatal incident of an infant who was exposed to IFX during gestation and who received a BCG vaccine at 3 months; this infant died of disseminated BCG infection [63]. Thus, the most recent recommendation from the World Congress of Gastro- enterology consensus statement on vaccinations in infants exposed to biologic therapy in utero recommend delay of However, in contrast to studies providing evidence of the safety of anti-TNF-α preparations during pregnancy, a review of more than 120,000 adverse events voluntarily reported to the FDA after drug exposure revealed 61 congenital abnormalities in 41 children of 40 mothers exposed to TNF-α blockers for various indications, in- cluding IBD [56]. Of these, 22 mothers were exposed to Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Page 8 of 13 all live-virus vaccines until after biologic molecules are no longer detectable in the child’s blood [62]. Like IFX, ADA is transferred across the placenta in the third trimester of pregnancy [31,76], and accord- ingly, it has been recommended at many IBD centers that ADA should be discontinued in the third trimester (that is, week 30), even though data, as mentioned be- fore, do not support this theory [82]. In this systematic review, identifying 17 case reports re- lated to IFX [14,16,20,22,23,29,30,33,34,40,41,45,47,63-66], 13 case series [12,17,19,28,32,37-39,42,43,46,67,68], 2 un- controlled cohort studies [19,36, and 2 controlled cohort studies [48,69] (Table 2), we found the prevalence of pregnancy complications, including preterm delivery, stillbirth, low birth weight, miscarriages, or congenital malformations in children exposed to IFX throughout pregnancy is limited, even after exposure to biologics throughout the third trimester. However, the use of IFX up to week 30 of gestation results in fetal intra-uterine ex- posure to high IFX levels (up to three-fold higher than in the maternal peripheral blood), which may raise concerns about the long-term effects of IFX on these children, in- cluding effects on their immune system [50]. Osting et al. carried out a review of TNF-α blockers (ADA and eternacept only) administered during preg- nancy from 1999 to 2009 using the Organization of Tetratology Information Specialists (OTIS) prospective registry, and concluded that 7 to 10% of children had congenital abnormalities [83]. However, the underlying disease activity might be a confounding factor with greater effect on the risk of congenital abnormalities than the biologic treatment per se. Adalimumab h l l d The clinical data for the safety of ADA during pregnancy in women with IBD are more limited than for IFX, but in animals, it appears that ADA does not increase ob- stetric risks and has no teratogenic effects [70]. In humans, data on ADA and pregnancy primarily con- cerns patients with diseases other than IBD, such as rheumatoid arthritis and psoriasis. Certolizumab pegol One of the reports observed uterine bleeding in the mother at 7 days post-partum; whether this can be ascribed to the CZP treatment re- mains to be established [86]. An ongoing cohort study evaluating ADA in pregnancy compared 80 live offspring born to 94 ADA-exposed women with CD, 53 live offspring of 58 women with CD not exposed to ADA, and 78 of 87 women from a non- diseased comparison group. The frequency of congenital abnormality in the three groups was 9.6%, 5.4%, and 5.0%, respectively [72] (P>0.05 between all groups). Evaluation of the identified studies In the studies included in this systematic review, more than 1,426 live births occurred among all the women rth outcomes in women with IBD exposed to TNF very Low birth weight Abnormalities/major b defects/malformations − 2/34 versus 2/53; OR 1.59 (0.21 to 11.9) /65; to 7.06) 6/32 versus 9/65; OR 1.44 (0.46 to 4.46) 1/42 versus 1/78; OR 1.87 (0.11 to 30.8) 13/110; to 0.77) 12/202 versus 11/110; OR 0.57 (0.24 to 1.33) 2/202 versus 0/110; (no OR or CI) to 3.31); ne:; RR 0.89 − − crosis factor. d to TN /major rmation 3; OR .9) 8; OR .8) 110; d to TN /major rmation 3; OR .9) 8; OR .8) /110; d to TN /major rmation 3; OR .9) 8; OR .8) 110; 0; rth outcom very − /65; to 7.06) 6 13/110; to 0.77) O to 3.31); e:; RR 0.89 − crosis factor. Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Page 10 of 13 Page 10 of 13 exposed to IFX, ADA, or CZP. The number of congeni- tal abnormalities associated with live births reported did not point to any pattern of specific birth defects (Table 1). There were 128 miscarriages, 81 elected abor- tions (presumably ‘under-reported’), 12 stillbirths, and 33 preterm births reported (Table 1). prevention of relapse during pregnancy should be stressed, and the patient should be monitored as a high- risk pregnancy. However, in real-world situations, this is not always the case, but it is important for the physician to discuss with each patient with IBD the risk versus benefit ratio of medical therapy, including TNF-α inhibi- tors, for disease control during pregnancy. Owing to considerable variation in study designs, se- lection of controls, and the definition and selection of outcomes in the identified studies, we were unable to conduct a meta-analysis. However, it appears clear from Table 2 that for all outcomes except congenital abnor- malities, the OR or RR did not point systematically to- wards a negative or positive effect of TNF-α inhibitors. A slightly increased risk of congenital abnormalities in women with IBD exposed to TNF-α inhibitors compared with unexposed pregnant women with IBD seemed to be suggested from the few studies with controls (Table 2), but the CIs were very broad. Evaluation of the identified studies Moreover, this observation should be weighed against the fact that the vast majority of case reports did not report congenital abnormalities, although it is thought that such ‘positive’ cases are more likely to be described and published than negative ones. Lastly, the meta-analysis by Cornish et al. [1] including 3,907 women with IBD, showed that an underlying in- creased risk of the studied outcomes already exists (still- births: OR 1.48 (95% CI 0.89 to 2.47); preterm delivery: OR 1.87 (1.52 to 2.31); low birth weight: OR 2.10 (1.38 to 3.19); and congenital abnormalities: OR 2.37 (1.47 to 3.82)) independent of treatment, compared with 320,531 pregnant non-IBD controls. Embryo-fetal perinatal developmental toxicity studies performed in cynomolgus monkeys receiving doses of TNF-α inhibitors several hundred times the recommended human dose did not reveal any evidence of teratogenic or other deleterious effects [90], such as adverse pregnancy or maternal outcomes [91]. Because IFX does not cross-react with TNF-α in species other than humans and chimpanzees, other animal reproduction studies have not been conducted with this drug. Thus, to date there is no evidence that TNF blockers are associated with embryotoxicity, teratogenicity, or increased pregnancy loss compared with pregnancies unexposed to biologics, either in rheumatology [92], or in a recent retrospective IBD mul- ticenter study [35]. The present systematic literature review reveals that many reports on biologic treatment in pregnancy lack information on the total number of women exposed to treatment, disease activity, co-morbidities, and/or con- comitant medications. In some cases, it is therefore diffi- cult to establish whether instances of fetal harm are due to medications or to the severity of the underlying illness [20,21]. Furthermore, many of the studies intending to assess the effect of TNF-α inhibitors per se were confounded by the fact that many patients are on mul- tiple medications. Finally, it should be kept in mind that biologics are more commonly used in patients with more severe disease activity, which accordingly might influence the pregnancy outcome data presented [1], es- pecially if patients are not compared with pregnant women with a similar degree of IBD disease severity treated with other medications. As an example, the un- favorable outcome described by Srinivasan [21] may po- tentially be due to the underlying severity of IBD or to other medications. Abbreviations ADA: Adalimumab; BCG: Bacillus Calmette-Guérin; CD: Crohn’s disease; CZP: Certolizumab pegol; EMA: European Medicines Agency; FcRn: Fc Discussion When summarizing the existing data in this systematic review, no association between treatment with TNF-α in- hibitors for IBD in pregnancy and adverse pregnancy out- comes such as spontaneous abortions, preterm deliveries, stillbirth, low birth weight, congenital malformations, and/ or infections, was found, even after administration of TNF-α blockers in the third trimester. In studies combin- ing biologics with thiopurines (which often occurs because of the favorable results of the Study of Immunomodulator Naïve Patients in Crohn’s disease (SONIC) [88] and UC SUCCESS (a study similar to the SONIC trial) [89] studies in CD and UC, respectively), there was no increased risk of infections in newborns compared with an unexposed control group. The aggravation of underlying disease might be more detrimental to the viability of pregnancy than the appar- ently low risk of continuing biologic therapies, and the results from this systematic review do not support the practice of stopping biologic treatment of the expectant mother after week 30 because of a theoretical risk of in- fections or congenital abnormalities in the offspring. The data presented here suggest that the rates of con- genital malformations and adverse birth events are simi- lar to the rates in the background population of pregnant women, or at least of women with IBD in gen- eral [1]. Further, there is no increased risk of infections in the offspring of mothers on biologic treatment during Ideally, any woman with IBD intending a pregnancy should have counseling with her gastroenterologist and obstetrical provider well in advance of conception, and this counseling should cover the medical treatment options and the risks associated with active disease dur- ing pregnancy. Pregnancy should if possible be planned, and the IBD should be controlled before conception. Further, the importance of adherence to treatment and Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Page 11 of 13 pregnancy. However, it should be emphasized that im- munosuppression of the offspring contraindicate the use of live vaccines until the biologic agent is no longer de- tectable in the child’s circulation, because of the risk of fatal infections [63,93,94]. receptor neonatal; FDA: Food and Drug Administration; GLM: Golimumab; IBD: Inflammatory bowel disease; IFX: Infliximab; MTX: Methotrexate; TNF: Tumor necrosis factor; UC: Ulcerative colitis. Author details 1 1Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark. 2Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN, USA. 1Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark. 2Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN, USA. 3Department of Epidemiology Research, Statens Serum Institute, National Health Surveillance and Research, Copenhagen, Denmark. 1Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark. 2Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN, USA. 3Department of Epidemiology Research, Statens Serum Institute, Nati Health Surveillance and Research, Copenhagen, Denmark. Department of Epidemiology Research, Statens Serum Institute, National Health Surveillance and Research, Copenhagen, Denmark. Health Surveillance and Research, Copenhagen, Denmark. Limitations of the study Limitations of studies identified in this systematic review included small sample sizes and paucity of studies with control groups. Future studies assessing the outcome of pregnancies exposed to IFX or other biologics during conception and pregnancy should be prospective in na- ture, and should assess comparable women with IBD with or without the relevant exposure as a control group to draw firm conclusions. Data on potential con- founders, especially maternal age, disease activity, and co-medications, should also be recorded. Furthermore, long-term follow-up studies of children exposed to bio- logic medications in the uterus should be carried out, es- pecially as no long-term data exist on outcomes in children related to the development of diseases such as asthma or autoimmune diseases. Conclusions Received: 27 November 2012 Accepted: 1 July 2013 Published: 31 July 2013 Received: 27 November 2012 Accepted: 1 July 2013 Published: 31 July 2013 There is a growing body of evidence suggesting that bio- logic agents are of low risk in pregnancy. Although it is difficult to prove absolute safety, this systematic review suggests that women who inadvertently become preg- nant while taking TNF-α inhibitors can be reassured that continuation of pregnancy does not appear to impose an increased risk to either themselves or their baby. The limited clinical results available suggest that the benefit of biologic agents in attaining response and maintaining remission in pregnant women with IBD might outweigh the risk of pregnancy complications due to flare-ups (for example, spontaneous abortions, prematurity, low birth weight or stillbirth), and the theoretical risk of drug ex- posure to the fetus [34]. However, the long-term impli- cations of fetal exposure to therapeutic monoclonal antibodies on the child’s developing immune system are still unknown. Competing interests TJ and OHN have nothing to disclose. EVL has consulted for Abbott Labs, UCB Pharma, and Janssen Biotech, and has received research support from Abbott Labs, UCB Pharma, and Janssen Biotech. However, all authors declare they have not been supported from any organization for the submitted work; have had no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and have no other relationships or activities that could appear to have influenced the submitted work. Authors’ contributions OHN conceived the study; TJ and OHN extracted the data; TJ, OHN, and EVL analysed the data, interpreted the results, and drafted the manuscript; and TJ and OHN extracted the data. EVL is the guarantor. All authors read and approved the final manuscript. 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BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Nielsen et al. 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Baker DE: Adalimumab: human recombinant immunoglobulin g1 anti- tumor necrosis factor monoclonal antibody. Rev Gastroenterol Disord 2004, 4:196–210. 71. Bosworth BP, Inra J, Eswaran S, Scherl EJ: Failed use of adalimumab in maintaining remission in Crohn’s disease during pregnancy. Am J Gastroenterol 2007, 102:S322. Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Mahadevan U, Kane S, Sandborn WJ, Cohen RD, Hanson K, Terdiman JP, Binion DG: Intentional infliximab use during pregnancy for induction or maintenance of remission in Crohn’s disease. Aliment Pharmacol Ther 2005, 21:733–738. 52. Seirafi M, Treton X, de Vroey B, Cosnes J, Roblin X, Allez M, Marteau P, Biroulet LP, Bouhnik Y: Anti-TNF therapy and pregnancy in inflammatory bowel disease: a prospective cohort study from the GETAID. Gastroenterology 2011, 140:S-175. 33. Stengel JZ, Arnold HL: Is infliximab safe to use while breastfeeding? World J Gastroenterol 2008, 14:3085–3087. 34. Tursi A: Effect of intentional infliximab use throughout pregnancy in inducing and maintaining remission in Crohn’s disease. Dig Liver Dis 2006, 38:439–440. 53. Mahadevan U: Management of your pregnant IBD patients. AGA Spring Postgraduate Course 2013, May 18–19:367–379. 54. Schnitzler F, Fidder H, Ferrante M, Ballet V, Noman M, Van AG, Spitz B, Hoffman I, Van SK, Vermeire S, Rutgeerts P: Outcome of pregnancy in 35. Casanova MJ, Chaparro M, Domenech E, Barreiro-de AM, Bermejo F, Iglesias E, Gomollon F, Rodrigo L, Calvet X, Esteve M, Garcia-Planella E, Garcia-Lopez Page 13 of 13 Nielsen et al. BMC Medicine 2013, 11:174 http://www.biomedcentral.com/1741-7015/11/174 Submit your next manuscript to BioMed Central and take full advantage of: 72. Johnson DL, Jones KL, Jimenez J, Mirrasoul BA, Salas E, Chambers CD: Pregnancy outcomes in women exposed to adalimumab: the OTIS autoimmune diseases in pregnancy project. Am J Gastroenterol 2009, 104:S410. • Convenient online submission 73. 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https://openalex.org/W3045601320	http://real.mtak.hu/122975/1/short%20long%20asfv.pdf	English	null	Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host Cells	Frontiers in genetics	2,020	cc-by	5,338	Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host Cells Ferenc Olasz 1, Dóra Tombácz 2, Gábor Torma 2, Zsolt Csabai 2, Norbert Moldován 2, Ákos Dörm ˝o 2, István Prazsák 2, István Mészáros 1, Tibor Magyar 1, Vivien Tamás 1, Zoltán Zádori 1 and Zsolt Boldogk ˝oi 2 Ferenc Olasz 1, Dóra Tombácz 2, Gábor Torma 2, Zsolt Csabai 2, Norbert Moldován 2, Ákos Dörm ˝o 2, István Prazsák 2, István Mészáros 1, Tibor Magyar 1, Vivien Tamás 1, Zoltán Zádori 1 and Zsolt Boldogk ˝oi 2 1 Institute for Veterinary Medical Research, Centre for Agricultural Research, Budapest, Hungary, 2 Department of Medical Biology, Faculty of Medicine, University of Szeged, Szeged, Hungary Keywords: African swine fever virus (ASFV), long-read sequencing, short-read sequencing, transcriptomics, direct RNA sequencing, full-length transcripts Received: 22 March 2020 Accepted: 26 June 2020 Published: 28 July 2020 Received: 22 March 2020 Accepted: 26 June 2020 Published: 28 July 2020 INTRODUCTION Methodological breakthroughs in sequencing technologies have revolutionized transcriptome proﬁling in recent years. Currently, the next-generation short-read sequencing (SRS) and third- generation long-read sequencing (LRS) platforms are widely used in genome and transcriptome research. SRS can generate large numbers of sequencing reads with unprecedented speed; however, it cannot suﬃciently cover high-complexity transcriptomes. LRS produces lower data coverage with a higher error rate, but it can overcome many of the drawbacks of SRS, including the ineﬃciency in distinguishing between transcription isoforms and identifying embedded and long transcripts. The combined use of these platforms and library preparation chemistries can generate high-quality and throughput data on full-length transcripts. LRS has been applied for the assembly of transcriptomic maps in several organisms (Sharon et al., 2013; Tilgner et al., 2015; Sessegolo et al., 2019; Yin et al., 2019; Zhao et al., 2019; Roach et al., 2020), including viruses (Tombácz et al., 2016, 2019; Balázs et al., 2017; Moldován et al., 2017; Prazsák et al., 2018; Depledge et al., 2019; O’Grady et al., 2019). Reviewed by: Xuanxuan Xing, The Ohio State University, United States Linda Kathleen Dixon, Pirbright Institute, United Kingdom Correspondence: Dóra Tombácz tombacz.dora@med.u-szeged.hu Zsolt Boldogk ˝oi boldogkoi.zsolt@med.u-szeged.hu Reviewed by: Xuanxuan Xing, The Ohio State University, United States Linda Kathleen Dixon, Pirbright Institute, United Kingdom Correspondence: Dóra Tombácz tombacz.dora@med.u-szeged.hu Zsolt Boldogk ˝oi boldogkoi.zsolt@med.u-szeged.hu African swine fever is a highly lethal animal disease aﬀecting pigs and wild boars. The causative agent of this disease is the large, double-stranded DNA virus, the African swine fever virus (ASFV), the only member of the Asfarviridae family (Mazur-Panasiuk et al., 2019). Because no eﬀective vaccination is currently available against the virus, it is unarguably the largest economic threat to the global pig industry. The ASFV genome is 190 kbp long and contains more than 190 open reading frames (ORFs), although the exact numbers of genes and proteins are unknown (Alejo et al., 2018). Approximately 20 viral genes are believed to participate in transcription and mRNA processing (Rodríguez and Salas, 2013), whereas at least 17 genes play a role in the replication, repair, and modiﬁcation of DNA (Yáñez et al., 1995; Dixon et al., 2013). Depending on the strain, ∼30–50 genes are involved in the evasion of immune surveillance and in encoding virulence and host range factors (Chapman et al., 2008). INTRODUCTION Specialty section: This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics Edited by: Youri I. Pavlov, University of Nebraska Medical Center, United States DATA REPORT published: 28 July 2020 doi: 10.3389/fgene.2020.00758 DATA REPORT Ribosomal RNA Depletion For Illumina sequencing, rRNA was eliminated from the total RNA samples using the RiboMinusTM Eukaryote System v2 (Invitrogen). Citation: Olasz F, Tombácz D, Torma G, Csabai Z, Moldován N, Dörm ˝o Á, Prazsák I, Mészáros I, Magyar T, Tamás V, Zádori Z and Boldogk ˝oi Z (2020) Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host Cells. Front. Genet. 11:758. doi: 10.3389/fgene.2020.00758 The temporal regulation of ASFV gene expression appears to be similar to that in poxviruses (Yáñez et al., 1995; Broyles, 2003; Chapman et al., 2008), in which four kinetic classes of genes have been described. The expression of immediate early and early genes precedes DNA replication, whereas the intermediate and late genes are generated subsequently to the onset of DNA replication (Rodríguez and Salas, 2013). The genome-wide ASFV transcriptome has recently been characterized by the Illumina SRS approach (Cackett et al., 2020). ASFV mRNAs have 5′ cap structures and 3′ poly(A) July 2020 \| Volume 11 \| Article 758 1 Frontiers in Genetics \| www.frontiersin.org Transcriptomes of ASFV and Host Olasz et al. tails added by the viral capping enzyme complex and the poly(A) polymerase, respectively (Salas et al., 1986). ASFV replicates relatively well in porcine primary alveolar macrophages (PAMs) in vitro, although the sensitivity of naïve PAM culture to ASFV infection varies batch by batch (Olasz et al., 2019). q g Direct RNA Sequencing—Using Samples From Mixed Time Points g Direct RNA Sequencing—Using Samples From Mixed Time Points Isolation of Total RNA A NucleoSpin R⃝RNA (Macherey-Nagel) kit was used for RNA puriﬁcation following the manufacturer’s instructions. In brief, the supernatant was removed from all wells of the 6-well plate, and 2 × 106 cells were lysed with RA1 lysis buﬀer and β- mercapthoethanol solution. Then, the lysates were transferred to NucleoSpin ﬁlters. After centrifugation (11,000 × g, 1 min), 70% ethanol were added to the lysates. The solutions were transferred to the columns, and after centrifugation, the membranes were washed with MDB buﬀer. After repeated washing, DNase reaction mixture were added, and the membranes were incubated at room temperature for 15 min. The membranes were washed with RAW2 and RA3 buﬀer, and the tubes were centrifuged at 11,000 × g for 30 s. Finally, RA3 buﬀer were added, and the tubes were centrifuged at 11,000 × g for 2 min. RNA was eluted with RNase-free H2O and centrifuged at 11,000 × g for 1 min. All buﬀers were supplied from the kit. Regarding the SRS approach, the MiSeq instrument (Illumina) was used (Supplementary Figure 1 shows the coverage depth), whereas we applied the MinION portable sequencer from Oxford Nanopore Technologies (ONT) for full-length sequencing. The random-primed SRS library was run on a single MiSeq v3 ﬂow cell, whereas three diﬀerent ONT libraries [direct RNA sequencing (dRNA-Seq), direct cDNA sequencing (dcDNA-Seq) and ampliﬁed cDNA sequencing) were sequenced on three individual ﬂow cells. Altogether the three LRS experiments resulted in 20,021,413 sequencing reads (Supplementary Table 1), of which 139,711 aligned to the viral genome (MN715134.1). The longest average read length was obtained using the dcDNA technique (1,299 bp). The average length for the ampliﬁed approach ranged between 598 and 1,017 bp, whereas the dRNA-Seq resulted in an average read length of 953 bp (Supplementary Table 1). More details about the length and quality of sequencing reads are presented in Supplementary Table 1 and Figure 1. The quality data from Illumina sequencing is presented in Supplementary Table 2. METHODS The experimental design utilized in this study is shown in Figure 2. The applied reagents are listed in Supplementary Table 3. Library Preparation for Nanopore Sequencing Library Preparation for Nanopore Sequencing Direct RNA Sequencing—Using Samples From Mixed Time Points Puriﬁcation of Polyadenylated RNA For the various Nanopore sequencing approaches, the polyA(+) RNA fraction of total RNA was isolated using the “Spin Columns method” from the Oligotex mRNA Mini Kit (Qiagen). Infection PAMs were cultivated in 6-well plates at a density of 3.3 × 105 cells and infected at a multiplicity of infection of 10 at 4 h after cell seeding. Supernatant was replaced with fresh medium at 1 h post-infection (p.i.), and infected cells were harvested at 4, 8, 12, and 20 h p.i. Mock-infected control cells were also harvested. To provide a detailed transcription map about the transcription dynamics of the virus, we performed multiplatform sequencing using both SRS and LRS techniques. The presented dataset represent a key resource for studying the ASFV transcriptome at diﬀerent time points after infection, and of the eﬀect of infection on the host gene expression. RNA Puriﬁcation Isolation of Total RNA Cells and Viruses The dRNA-Seq method using a direct RNA sequencing kit (SQK- RNA002, Version: DRS_9080_v2_revK_14Aug2019) from ONT was used for ampliﬁcation-free sequencing. This approach is highly recommended to explore special features of native RNA (e.g., modiﬁed bases) and avoid potential biases associated with reverse transcription (RT) or PCR. Total RNA from eight samples (two parallel experiments from 4, 8, 12, and 20 h p.i.) was mixed together, and then the polyA(+) fraction of RNA was puriﬁed from the sample mix. One hundred nanograms from the polyA-tailed RNA were mixed with RT Adapter (oligo dT- containing T10 adapter), RNA CS (both from the Nanopore kit; the latter was used to monitor the sequencing quality), NEBNext Quick Ligation Reaction Buﬀer, and T4 DNA ligase [both from New England Biolabs (NEB)]. The mixture was incubated for 10 min at room temperature, and then RT was conducted to generate ﬁrst-strand cDNA using dNTPs (NEB), 5× ﬁrst-strand buﬀer, DTT (both from Invitrogen SuperScript III) Fresh swine pulmonary macrophage (PAM) cells were harvested following the OIE Manual’s instructions (Oﬃce International des Epizooties (OIE), 2019). The cells were grown in RPMI 1640 containing L-glutamine (Lonza) medium supplemented with 10% fetal bovine serum (Euro Clone), 1% Na-pyruvate (Lonza), 1% antibiotic-antimycotic solution (Thermo Fisher Scientiﬁc), and 1% non-essential amino acid solution (Lonza) at 37◦C in a humidiﬁed atmosphere containing 5% CO2. The highly virulent Hungarian ASFV isolate ASFV_HU_2018 (ID Number: MN715134) was used for infection. The infectious titer of the serially diluted viral stock was calculated in PAMs using an immunoﬂuorescence (IF) assay as described previously (Olasz et al., 2019). All work with the infectious virus was conducted at the biosafety level 3 (BSL3) laboratory of the Institute for Veterinary Medical Research, Center for Agricultural Research following all current EU regulations (European Commission, 2020). July 2020 \| Volume 11 \| Article 758 Frontiers in Genetics \| www.frontiersin.org 2 Olasz et al. Transcriptomes of ASFV and Host FIGURE 1 \| Aligned read length distribution. Line chart presentation of the average of aligned read lengths obtained via Nanopore sequencing. (A) Ampliﬁed cDNA sequencing at various time points. (B) Direct cDNA sequencing and (C) direct RNA sequencing using samples from multiple time points after the infection. FIGURE 1 \| Aligned read length distribution. Line chart presentation of the average of aligned read lengths obtained via Nanopore sequencing. (A) Ampliﬁed cDNA sequencing at various time points. Cells and Viruses (B) Direct cDNA sequencing and (C) direct RNA sequencing using samples from multiple time points after the infection. direct cDNA sequencing kit (SQK-DCS109; Version: DCS_9090_v109_revJ_14Aug2019; ONT). This protocol is based on strand switching, and it is highly optimal for the generation of full-length cDNA for the identiﬁcation of potential novel transcript isoforms without potential PCR bias. The starting material was 100 ng of a poly(A)+ RNA mixture from various time points of infection (4, 8, 12, and 20 h p.i.). An oligo dT-containing VN primer (VNP) and dNTPs (10 µM) were added to the RNA. After 5 min of incubation at 65◦C, the following components were added: 5× RT buﬀer (from the Maxima H Minus Reverse Transcriptase kit, Thermo Fisher Scientiﬁc), RNaseOUTTM (Thermo Fisher Scientiﬁc), strand switching primer from the ONT kit and nuclease-free water. This mixture was pre-heated at 42◦C for 2 min, and Maxima H Minus Reverse Transcriptase was added. RT was conducted at 42◦C for 90 min, and ﬁnally, the reaction was stopped by incubation at 85◦C for 5 min. RNase Cocktail Enzyme Mix (Thermo Fisher Scientiﬁc) was used to degrade the RNA in the sample. Incubation was performed at 37◦C for 10 min. Before the second-strand synthesis, the sample was cleaned using AMPure and UltraPureTM DNase/RNase-Free distilled water (Invitrogen), and then the sample was mixed with SuperScript III enzyme (Thermo Fisher Scientiﬁc). RT was performed in a Veriti cycler (Applied Biosystems) at 50◦C for 50 min, and the reaction was subsequently terminated at 70◦C for 10 min. RNA-cDNA hybrids were puriﬁed using Agencourt RNAClean XP Beads [1.8 bead ratio (BR)]; Beckman Coulter], washed with freshly prepared 70% ethanol, and eluted in UltraPureTM nuclease-free water. The sample was then ligated to the RNA adapter (RMX from the ONT kit) at room temperature for 10 min using NEBNext Quick Ligation Reaction Buﬀer, T4 DNA ligase, and nuclease-free water. The ligation reaction was followed by a ﬁnal puriﬁcation step using XP Beads (1.0 BR). Samples were washed with wash buﬀer (ONT) and eluted in elution buﬀer (ONT). After Qubit measurement, 100 fmol from the library were loaded onto a Flow Cell. Direct cDNA-Seq—From Mixed Time Points Viral and host transcripts were also sequenced on a MinION sequencer following the instructions of the July 2020 \| Volume 11 \| Article 758 Frontiers in Genetics \| www.frontiersin.org 3 Olasz et al. Sequencing on the Illumina MiSeq Instrument The sequencing-ready library (12 pM) was loaded onto a ﬂow cell from Illumina MiSeq Reagent Kit v3 (150-cycle format, 2 × 75 bp) and sequenced on a MiSeq sequencer. Cells and Viruses Transcriptomes of ASFV and Host Flowchart diagram shows an overview of the methodological workﬂow of this study. 0.8 BR) (Beckman Coulter). Then, LongAmp Taq (NEB) was used to synthesize second-strand cDNA AMPure XP method (0.8 BR). NEBNext Ultra II End-prep reaction buﬀer and NEBNext Ultra II End-prep enzyme mix FIGURE 2 \| Flowchart diagram shows an overview of the methodological workﬂow of this study. AMPure XP method (0.8 BR). NEBNext Ultra II End-prep reaction buﬀer and NEBNext Ultra II End-prep enzyme mix (both from NEB) were added to each sample. This end repair process was performed at 20◦C for 5 min, followed by a 5-min incubation at 65◦C. Enzymes and buﬀers were removed from the reaction using the AMPure XP puriﬁcation method (1.0 BR), AMPure XP method (0.8 BR). NEBNext Ultra II End-prep reaction buﬀer and NEBNext Ultra II End-prep enzyme mix (both from NEB) were added to each sample. This end repair process was performed at 20◦C for 5 min, followed by a 5-min incubation at 65◦C. Enzymes and buﬀers were removed from the reaction using the AMPure XP puriﬁcation method (1.0 BR), XP Beads (0.8 BR) (Beckman Coulter). Then, LongAmp Taq Master Mix (NEB) was used to synthesize second-strand cDNA using the PR2 primer (ONT). Samples were incubated using the following “only one cycle protocol:” denaturation at 94◦C for min, annealing at 50◦C for 1 min, and elongation at 65◦C for 15 min. The double-stranded cDNAs were puriﬁed using July 2020 \| Volume 11 \| Article 758 Frontiers in Genetics \| www.frontiersin.org 4 Transcriptomes of ASFV and Host Olasz et al. AMPure Beads (1.8 BR). Resuspension buﬀer (NEXTﬂex R⃝Kit) was used for the ﬁnal elution. Polyadenylation of the double- stranded cDNAs was performed using NEXTﬂex R⃝Adenylation Mix at 37◦C for 30 min. The reaction was terminated by heating at 70◦C for 5 min. Molecular Index Adapters (from the Kit) were ligated to the sample at 30◦C (10 min) using the NEXTﬂex R⃝ Ligation Mix. Prior to ampliﬁcation, each sample was washed with AMPure Beads (0.8 BR). First, NEXTﬂex R⃝Uracil DNA Glycosylase was mixed with the sample, which was incubated at 37◦C for 30 min, followed by heating at 98◦C for 2 min. The sample was placed on ice, and the following components were added: PCR Master Mix, qRNA-Seq Universal forward primer, and qRNA-Seq Barcoded Primer (sequence: AACGCCAT; all from the kit). Illumina Sequencing Raw reads were trimmed using Cutadapt software (Martin, 2011), the aforementioned viral reference genome was indexed using STAR aligner v2.7.3a (Dobin et al., 2013) with the following settings: –genomeSAindexNbases 8, followed by the mapping of the reads with default options. Samtools (Li et al., 2009) was used to sort the sam ﬁles and to generate and index the BAM ﬁles. The Qualimap v2.2.1 application (García-Alcalde et al., 2012) was used to generate quality information from the Illumina dataset. Ampliﬁed cDNA-Sequencing—From Different Time Points Samples from each time point (4, 8, 12, 20 h p.i. and the mock infected cells) were sequenced by the ONT MinION device using the cDNA-PCR Barcoding protocol (SQK-PCS109 and SQK- PBK004; Version: PCSB_9086_v109_revK_14Aug2019). This protocol is recommended to identify and quantify full-length transcripts, discover novel isoforms, and splice variants and fusion transcripts from a low amount of starting material (total RNA) to generate large amounts of cDNA data. Approximately 50 ng of each of the samples were used for library preparation. VNP and dNTPs were added to the RNA and incubated at 65◦C for 5 min. The strand-switching buﬀer mixture (RT buﬀer, RNaseOUT, nuclease-free water, and SSP) was added to the samples, which were incubated at 40◦C for 2 min. RT was conducted by adding Maxima H Minus Reverse Transcriptase at 42◦C for 90 min. The enzyme was inactivated by increasing the temperature to 85◦C for 5 min. LongAmp Taq Master Mix, one of the Low Input barcode primers (LWB01-12, from the ONT’s SQK-PBK004 kit, Supplementary Table 4), and nuclease-free water were included in the RT reaction mixture. Supplementary Table 5 shows the PCR conditions. PCR products were treated with exonuclease (NEB), and the mixture was then incubated at 37◦C for 15 min, followed by 80◦C for 15 min. AMPure Beads (0.8 BR) was used for puriﬁcation, and the clean sample was eluted. Library Preparation for Illumina Sequencing q g A NEXTﬂex R⃝Rapid Directional qRNA-Seq Kit (PerkinElmer) was used to sequence the whole ASFV transcriptome via a conventional short-read approach. We used 25 ng of an rRNA- depleted RNA mixture (4, 8, 12, and 24 h p.i.) as the starting material. The ﬁrst step was enzymatic fragmentation of the RNA using NEXTﬂex R⃝RNA Fragmentation Buﬀer. The reaction was conducted at 95◦C for 10 min followed by ﬁrst-strand cDNA synthesis. First, NEXTﬂex R⃝First Strand Synthesis Primer was added to the reaction mixture, which was heated at 65◦C for 5 min and then subsequently placed on ice. NEXTﬂex R⃝ Directional First Strand Synthesis Buﬀer and Rapid Reverse Transcriptase were then added. RT was performed using the following program: incubation at 25◦C for 10 min, heating at 50◦C for 50 min, and termination at 72◦C for 15 min. This step was followed directly by second-strand cDNA synthesis via the addition of NEXTﬂex R⃝Directional Second Strand Synthesis Mix (with dUTPs) at 16◦C for 60 min. The product was cleaned using Code Availability Guppy v3.4.5: http://community.nanoporetech.com/downloads? fbclid=IwAR2IchRL4gDnfA6h996UkN4vS5pbBu6rUtKVFX3a TiBHsWFknglQ6FyvPkg minimap2: https://github.com/lh3/minimap2 STAR: https://github.com/alexdobin/STAR samtools: https://github.com/samtools/samtools SeqTools: https://github.com/moldovannorbert/seqtools. Guppy v3.4.5: http://community.nanoporetech.com/downloads? fbclid=IwAR2IchRL4gDnfA6h996UkN4vS5pbBu6rUtKVFX3a TiBHsWFknglQ6FyvPkg Guppy v3.4.5: http://community.nanoporetech.com/downloads? fbclid=IwAR2IchRL4gDnfA6h996UkN4vS5pbBu6rUtKVFX3a TiBHsWFknglQ6FyvPkg minimap2: https://github.com/lh3/minimap2 STAR: https://github.com/alexdobin/STAR samtools: https://github.com/samtools/samtools SeqTools: https://github.com/moldovannorbert/seqtools. TiBHsWFknglQ6FyvPkg minimap2: https://github.com/lh3/minimap2 STAR: https://github.com/alexdobin/STAR samtools: https://github.com/samtools/samtools SeqTools: https://github.com/moldovannorbert/seqtools SeqTools: https://github.com/moldovannorbert/seqtools. Read Processing ONT Sequencing Guppy software v3.4.5 (ONT) was used for base calling from MinION data. The raw reads were aligned to the ASFV reference genome (NCBI Nucleotide accession: MN715134.1) using the minimap2 software suite (Li, 2018) with the following options: -ax splice -Y -C5 –cs. SeqTools scripts were used to obtain the quality information. Cells and Viruses The samples were ampliﬁed according to the protocol summarized in Supplementary Table 6. The PCR products were washed with AMPure Beads (0.8 BR), and followed by a second puriﬁcation. and then the sample was subjected to adapter ligation. The ONT Adapter Mix and Blunt/TA Ligation Master Mix (NEB) were mixed with each sample, and the mixture was incubated at room temperature for 10 min. A ﬁnal AMPure XP puriﬁcation (0.4 BR) was conducted to remove any excess proteins, nucleotides, and salts from the DNA library. Adapter Bead Binding Buﬀer was used to wash the beads, and the library was eluted using elution buﬀer (both from the Nanopore kit). The samples were quantiﬁed using Qubit, and then 200 fmol from the sample were loaded on two MinION Flow Cells. Ampliﬁed cDNA-Sequencing—From Different Time Points Frontiers in Genetics \| www.frontiersin.org ETHICS STATEMENT High Sensitivity RNA, and High Sensitivity dsDNA Assay Kits. The Agilent TapeStation 4150 system was applied to detect the integrity of total RNA and perform a quality check of the Illumina libraries. In the present study, RNAs samples with RIN ≥9.4 were subjected to construct the sequencing libraries (ode Avail Supplementary Figure 2). Animals were euthanized in the animal facilities of ATK ÁOTI where all methods were performed in accordance with the relevant guidelines and regulations following the protocol approved by the Government Agency of Pest County (PE/EA/1474-7/2017). All methods were performed in accordance with the relevant guidelines and regulations following a protocol approved by the ATK ÁOTI Institutional Animal Care and Use Committee. Animals were euthanized in the animal facilities of ATK ÁOTI where all methods were performed in accordance with the relevant guidelines and regulations following the protocol approved by the Government Agency of Pest County (PE/EA/1474-7/2017). All methods were performed in accordance with the relevant guidelines and regulations following a protocol approved by the ATK ÁOTI Institutional Animal Care and Use Committee. FUNDING This study was supported by the National Research, Development, and Innovation Oﬃce grants FK 128252 to DT and K 128247 to ZB. This study was supported by the National Research, Development, and Innovation Oﬃce grants FK 128252 to DT and K 128247 to ZB. TECHNICAL VALIDATION The total RNA, polyA(+) RNA, and rRNA-depleted samples; generated cDNAs; and ﬁnal sequencing libraries were quantiﬁed by a Qubit 4 Fluorometer using Qubit RNA Broad-Range, July 2020 \| Volume 11 \| Article 758 Frontiers in Genetics \| www.frontiersin.org 5 Transcriptomes of ASFV and Host Olasz et al. DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene. 2020.00758/full#supplementary-material The datasets generated for this study can be found in the European Nucleotide Archive - accession number PRJEB36723. AUTHOR CONTRIBUTIONS FO participated in the infection experiments, the puriﬁcation of the total RNA from the virus, and with writing the manuscript. DT performed the MinION sequencing (dynamic dataset) and the Illumina sequencing, participated in data analysis, and wrote the manuscript. GT generated the ONT dRNA sequencing libraries and conducted data handling and processing. ZC conducted MinION direct cDNA sequencing and the Illumina sequencing. NM carried out bioinformatics analysis. ÁD performed the isolation of poly(A)+ RNAs and the rRNA removal from total RNAs. IP participated in the puriﬁcation of poly(A)+ RNA and data handling. IM conducted the infection experiments, puriﬁed the viral total RNA samples. TM prepared the pulmonary macrophages. VT participated in the infection experiments and the puriﬁcation of the total RNA from the virus. ZZ designed the research plan and wrote the manuscript. ZB conceived and designed the experiments, managed the study, and wrote the manuscript. All authors read and approved the ﬁnal paper. DATA RE-USE To our best knowledge, no data on the ASFV transcriptome are available; therefore, this dataset was primarily generated to characterize the RNA proﬁle of the virus. The dataset can be used to detect RNA isoforms, including length (alternative 3′ and 5′) variants, monocistronic, bicistronic, polycistronic, and complex transcripts, and to discover transcriptional overlaps and the complexity of the genetic regulation of ASFV. Nanopore dataset allows a time-course evaluation of the full-length transcriptomes of both the virus and host. The published BAM ﬁles contain the reads mapped to the MN715134.1 reference genome. BAMs [using samtools and bedtools (Quinlan and Hall, 2010)] can be converted to FastQ ﬁles, which extend the potential usage of data; e.g., they can be aligned to host genome. BAM ﬁles can be analyzed using various bioinformatics tools, such as samtools, bedtools, or the Genome Analysis Toolkit (Van der Auwera et al., 2013). The Nanopore data generated with diﬀerent library preparation approaches can be compared to analyze the diﬀerences between the sequencing chemistries, as well as the eﬀect of RT and PCR reactions on the length and quality of the reads. The provided dataset is also useful for comparing the performance of the utilized sequencing platforms. Tombo tool (Stoiber et al., 2017) can be used to identify CpG methylation patterns and base modiﬁcations (e.g., A to I editing) from raw (fast5) Nanopore sequencing data, or the EpiNano (Liu et al., 2019) algorithm can be applied to detect m6A RNA modiﬁcations. The dataset can be further analyzed using various bioinformatics program packages or visualized using softwares [e.g., Integrative Genomics Viewer (Robinson et al., 2011), Savant Genome Browser (Fiume et al., 2010), Geneious (Kearse et al., 2012)]. Depledge, D. P., Srinivas, K. P., Sadaoka, T., Bready, D., Mori, Y., Placantonakis, D. G., et al. (2019). 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Viruses. 11:E1129. doi: 10.3390/v11121129 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Prazsák, I., Moldován, N., Balázs, Z., Tombácz, D., Megyeri, K., Szucs, A., et al. (2018). Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus. BMC Genom. 19:873. doi: 10.1186/s12864-018-5267-8 Copyright © 2020 Olasz, Tombácz, Torma, Csabai, Moldován, Dörm˝o, Prazsák, Mészáros, Magyar, Tamás, Zádori and Boldogk˝oi. 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. Quinlan, A. R., and Hall, I. M. (2010). BEDTools: a ﬂexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842. doi: 10.1093/bioinformatics/btq033 Roach, N. P., Sadowski, N., Alessi, A. F., Timp, W., Taylor, J., and Kim, J. K. (2020). The full-length transcriptome of C. elegans using direct RNA sequencing. Genome Res. 30, 299–312. Frontiers in Genetics \| www.frontiersin.org REFERENCES doi: 10.1101/gr.251314.119 July 2020 \| Volume 11 \| Article 758 Frontiers in Genetics \| www.frontiersin.org 7
https://openalex.org/W4235847627	https://www.researchsquare.com/article/rs-33383/latest.pdf	English	null	Noncontact restoration of missing parts of stone Buddha statue based on three-dimensional virtual modeling and assembly simulation	Research Square (Research Square)	2,020	cc-by	8,838	Noncontact restoration of missing parts of stone Buddha statue based on three-dimensional virtual modeling and assembly simulation Young Hoon Jo (  joyh@kongju.ac.kr ) Kongju National University https://orcid.org/0000-0001-5165-3747 Seonghyuk Hong Kongju National University Seong Yeon Jo Chuncheon National Museum Yoon Mi Kwon Chuncheon National Museum Research article Keywords: Three-dimensional scanning, Three-dimensional printing, Haptic modeling system, Digital virtual restoration, Design mockup, Assembly simulation, Noncontact restoration Posted Date: October 15th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-33383/v3 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 21st, 2020. See the published version at https://doi.org/10.1186/s40494-020-00450-8. Page 1/24 Page 1/24 Abstract Three-dimensional (3D) digital technology is one of the most essential conservation methods that complements the traditional technique of the restoration of cultural artifacts. In this study, 3D scanning, virtual restoration modeling, and 3D printing were used as a non-contact approach for the restoration of a damaged stone seated Bodhisattva (stone Buddha statue). First, a three-dimensional model with an average point density of 0.2 mm was created by integrating the fixed high-precision scanning of the exterior and the handheld mid-precision scanning of the interior excavated hole. Through a 3D deterioration map of the stone Buddha statue, the area of the missing parts was measured as 400.1 cm 2 (5.5% of the total area). Moreover, 257.1 cm 2 (64.2% of the missing part area) of four parts such as the head, the surrounding area of the Baekho, the right ear, and the right eye, for which symmetry was applicable for modeling or there could be ascertainable historical evidence for the total missing parts, was selected for restoration. The virtual restoration of the missing parts of the stone Buddha statue was performed using a haptic modeling system in the following order. First, the location of the three fragments detached from the head was determined. Next, the reference model was selected, and its symmetrization and modification with respect to the original were conducted. Also, estimation modeling and outer shape description were performed through historical research and consultation with experts. The created virtual-restoration model’s (461 cm 3 ) heuristic-based assembly suitability was verified by design mock-up printing and digital–analog simulation. In particular, to address the assembly interference, the interface surface was modified and reprocessed several times. Accordingly, the final design mock-up’s volume size was decreased by 5.2% (437 cm 3 ). Photopolymerization 3D printing technology was used for the actual restoration of the stone Buddha statue and the layer thickness of the material used was set as 0.10 mm considering the surface roughness. Finally, the surface of the printed output was colored to prevent yellowing and joined to the missing part of the stone Buddha statue. This study presents a great case to shift from the traditional manual-contact method to the contactless digital method for the restoration of artifacts and is expected to largely contribute to increasing the usability of digital technologies in the restoration of cultural artifacts. Introduction Ancient monuments and artifacts are often damaged because of exposure to natural or artificial environmental factors, which can lower their historical value or significance [1–9]. Traditionally, functional repair and aesthetic restoration are undertaken to recover the original form of damaged monuments and artifacts [10,11]. The Athens Charter (1931), Venice Charter (1964), and Nara Document on Authenticity (1992) have been prepared to establish restoration principles for cultural heritages, and countries around the world have restored their cultural heritages according to their own rules following international recommendation [12]. Generally, damaged artifacts can be restored only when their historical and cultural value can be retrieved through sufficient and direct historical research. Any restoration that may distort the value or cause a controversy is restricted. Fundamentally, a traditional technique and original materials are used for restoration. However, if the traditional technique is not Page 2/24 Page 2/24 applicable and the original materials cannot be supplied, modern technologies and materials that have been scientifically and experientially established can be used. Recently, various conservation methods using advanced technologies and new materials have been used in the restoration process of cultural artifacts [13–16]. In particular, when a cultural artifact loses its historical value or changes its shape, the restoration effort focuses on recovering the entire or partial shape of a certain period via thorough historical research and consultation with experts [17–19]. The restoration of a cultural artifact is usually realized using a manual method that relies on conservators’ intuition and techniques [20,21]. Using this method, physical contact with the material is inevitable. Moreover, if the restored parts look inappropriate, it would have to be removed. In this case, the original artifact can suffer a secondary damage either from frequent physical contact or excessive force applied during the removal of the parts. With the advancement in digital technology, many approaches that complement traditional restoration methods have emerged. The most prominent technologies include three-dimensional (3D) scanning and printing. 3D scanning is used in areas of digital documentation [22–25], shape analysis [26], conservation evaluation [27], and digital restoration [28–30]. In particular, this method is actively used for digitalizing and documenting rock materials such as rock arts [31], inscribed stones [32,33], knapped stones [34], dolmen [35], and stone pagoda [36]. Moreover, 3D printing has been employed in a wide area, including the establishment of restoration planning, supplementing the lost area, replication, and exhibition. Status of the study object Wood, clay, and metal are the commonly used materials to create a small statue because they are sufficiently easy to obtain in Korea. In particular, wood and metal have been the most commonly used universal materials since a very long time; however, clay has mostly been used after the 15th century. In contrast to the commonly used materials, the study object, which is a stone-seated Bodhisattva excavated in Pyeongchang, Gangwon province in the Republic of Korea, is composed of zeolitic tuff (Fig. 1). Based on visual observation, the rock exhibits a white microcrystalline substance with a dark porphyritic texture and its rock-forming minerals include albite, biotite, and quartz [47]. This rock was traditionally used to sculpt a Buddha statue, because it is very soft and easy to handle. The statue was discovered in several fragments in 1974 and has been maintained in almost its entire form through a conservation treatment. However, four fragments that are estimated to be parts of the crown and one fragment of an ear have been separately stored without joining [48]. A huge excavated hole exists inside the statue from below the chest to the bottom, which is believed to have stored relics. Traces of coloring using mineral pigments and gold gilding are found on the exterior of the statue; however, its exact form is difficult to discern as most of them were peeled off and deteriorated. Moreover, the crown, face, and left finger are missing, and several physical damages are observed because of stone crack and exfoliation. Owing to its vulnerable conservation status, this statue has been managed in the national museum storage with stable environmental conditions for decades and has not been used for exhibition. Therefore, aesthetic and functional restoration of the damaged parts is necessary to recover its historical value and use the statue for exhibition or display. Introduction Because of the availability of a wide variety of printing materials, 3D printing is used for the actual restoration of damaged artifacts [37–45]. In this study, the restoration of a stone-seated Bodhisattva (stone Buddha statue) was performed using various digital technologies. This statue is composed of zeolitic tuff, which has rarely been reported to be used as a Buddha material around the world. Thus, its unique value as a representative style of the Buddha statue has been appreciated between the 17th and 19th century in Korea. However, this statue was mostly damaged because of the missing parts. According to the ICOMOS-ISCS glossary, the missing part is defined as the empty space, obviously located in the place of some formerly existing stone part. The protruding and particularly exposed parts of sculptures are typical locations for material loss, which result in the missing parts [46]. The missing parts of the Buddha statue are attributed to the soft and weak characteristics of the material. Therefore, noncontact restoration was realized using 3D scanning, virtual restoration modeling, and 3D printing technologies. To achieve this goal, 3D high-precision scanning was used to record the original shape of the stone Buddha statue and the missing parts were virtually restored using a haptic modeling system. Moreover, the restoration of the stone Buddha statue was completed using the 3D printed outputs of the virtual restoration model. In particular, a systematic design mockup and simulation approach as well as the convergence of digital and analog technologies were used to enhance the heuristic-based assembly suitability of the virtually restored model. Page 3/24 Page 3/24 Devices and software The digital model of the statue was created using a fixed 3D high-precision scanner and handheld mid- precision scanner. Among these, the fixed high-precision scanner (HDI Advance R3X, LMI TECHNOLOGIES, Canada) is based on triangulation and uses white structured light and two stereo cameras (2.8 MP). In this study, scanning was performed with a field of view (FOV) of 400 mm and an accuracy of 65 μm. Furthermore, the handheld mid-precision scanner (Eva, Artec3D, Luxembourg) that uses white structured light and camera (1.3 MP) has an accuracy of 0.1 mm and a maximum point resolution of 0.5 mm. Software used for operating the scanners included FlexScan3D and Artec Studio 13, and the 3D model was edited using Geomagic Design X 2019. The high-resolution digital scanning results were used to identify the manufacturing techniques and analyze the shape of the statue. Page 4/24 Page 4/24 A haptic device (Geomagic Touch X, 3D SYSTEMS, USA) and voxel-based software (Geomagic Freeform Plus, 3D SYSTEMS, USA) were used to virtually restore the missing parts. The device provides haptic feedback when a user interacts with the virtual environment. In particular, voxel-based modeling using a virtual clay material provides design flexibility. Therefore, this system was used for both 3D modeling and virtual assembly simulation, and the suitability of the 3D restoration model was evaluated. Material extrusion and photopolymerization 3D printing technologies were used to obtain the output of the virtual restoration model. A material extrusion 3D printer (Ultimaker 3 Extended, Ultimaker) was used to verify the modeling result through the output of a design mockup, and the mockup was printed using polylactic acid (PLA) material at a resolution of 0.10–0.15 mm. Moreover, the 3D printer (ProJet 6000 HD, 3D SYSTEMS, USA) used for the final restoration employed photopolymerization that provides a very high level of surface precision. The 3D model was printed using UV-hardened plastic material with a layer thickness of 10 mm. The final printed output was used to restore the statue and its exhibition after strengthening and joining. 3D scanning The missing parts were restored by a noncontact approach using digital technologies such as 3D scanning, modeling, and printing (Fig. 2). Fixed and handheld scanning was performed to record the 3D shape of the statue and its missing parts (Fig. 3). First, the exterior of the statue (scanned 101 times) and five fragments (scanned 34 times) were digitized using the fixed high-precision scanner based on stereo vision. The total 3D scans were performed by considering the structure and appearance of the statue, scope of survey, and degree of overlapping. However, the fixed high-precision scanner based on the binocular lens could not reach sufficient depths to record the shape of the excavated hole from below the chest to the bottom of the statue. Therefore, a handheld mid-precision scanner based on a single lens with depth-scanning capability was used to record the shape of the excavated hole. Each image acquired by on-site scanning provides only misaligned raw shape information. Therefore, the raw scans were post processed to complete a single model from the raw scan data. The processing was executed in the following order: filtering, aligning, registering, merging, filling holes, and RGB texture mapping. The reliability of a 3D model depends on the registering result based on the iterative closet point algorithm [49]. Therefore, registering, which is the most important stage in scan processing, was performed with an average overlap ratio of 66% and within an error vector of 45 μm. This error vector value was very similar to the distance accuracy of the 3D scanner, indicating high reliability of the registering results. Page 5/24 Moreover, high-precision scanning of the statue exterior and mid-precision scanning of the excavated hole needed to be integrated into a single model because the scanning yielded separate 3D models. Accordingly, a deviation analysis between the two scanning models to evaluate the registering error revealed that most deviations were within ±0.1 mm and the average RMS was 0.28 mm. These values are Page 5/24 similar to the accuracy of general precision scanners; thus, the convergence model was created based on an ICP algorithm. similar to the accuracy of general precision scanners; thus, the convergence model was created based on an ICP algorithm. Virtual modeling and simulation The general rule of the conservation treatment and restoration of cultural artifacts states only minimum and passive intervention under inevitable conditions. Therefore, in this study, among the damaged areas, minimum scope was selected for aesthetic restoration and exhibition. The cracks are very fine, and their depths are visually conspicuous. These cracks seemingly do not cause any severe physical and structura problems. Thus, the cracks in the statue were left unattended without conservation treatment. However, the missing parts have a critical impact on the aesthetic value of the statue. Therefore, some parts that could be recreated using symmetry modeling or assured significant evidence through historic research were restored. In particular, the entire stone fragments that were stored separately were used for the restoration. The haptic modeling system used to create a virtual model of the missing parts enables intuitive modeling because it helps recognize conflicts and interference of data. Compared with the existing method that uses a mouse, this method is considerably excellent for creating a complex shape. In particular, the system uses a voxel engine to create a model, providing a user an experience of force feedback similar to that faced in actually carving of a virtual object. This system is mainly used in the medical field, graphic design, car industry, exhibition technology, and the restoration of cultural artifacts [50–53]. In this study, the digital virtual restoration of the four selected parts was performed using a haptic modeling system. In the process of modeling, Boolean operation was used to combine several complex models into a new complete model. Boolean operation is an essential method for original-based modeling requirements such as the restoration of the cultural artifacts; therefore, the operation was mainly used for symmetrization of the missing parts. Boolean operation was performed to modify the model, matching it exactly to the original shape. Because symmetry modeling could not be applied to the center of the crown part, a statue similar to the study object was selected as a reference and estimated restoration was performed. In this study, the digital virtual restoration of the four selected parts was performed using a haptic modeling system. In the process of modeling, Boolean operation was used to combine several complex models into a new complete model. 3D documentation and shape analysis The converged 3D model comprises 36,556,604 polyfaces with an average point distance resolution of 0.2 mm. According to Fig. 4a, the 3D scanning result clearly revealed the complete shape of the statue and the polygon mesh and RGB texture-mapping models showed high reality effects. In particular, the final 3D scanning result exhibited a high resolution with millions of polygons, which well revealed the surface texture, manufacturing technique, and detailed shape of the damaged area. Based on the 3D scanning, the stone Buddha statue was measured to be 410-mm wide, 310-mm thick, and 580-mm high. Its calculated area and volume were 7,250 cm2 and 20,040 cm3, respectively. Moreover, the excavated hole was measured to be 188-mm wide, 155-mm thick, and 195-mm high, and its area and volume were 1,228 cm2 and 2,401 cm3, respectively. In particular, the cone-shaped excavated hole showed traces of chisels whose average sizes were 10.5 and 15.4 mm. Therefore, the excavated hole may have been carved using these two chisels. A 3D deterioration map was created to define the scope of the conservation treatment and restoration. According to the map, cracks and missing parts were observed around the head; the body was mainly damaged owing to the cracks (Fig. 4b). Out of the total area, the deterioration rates of the missing parts were 4.8% (348.3 cm2) in the eye and head and 0.7% (52.9 cm2) in the body. Virtual modeling and simulation Boolean operation is an essential method for original-based modeling requirements such as the restoration of the cultural artifacts; therefore, the operation was mainly used for symmetrization of the missing parts. Boolean operation was performed to modify the model, matching it exactly to the original shape. Because symmetry modeling could not be applied to the center of the crown part, a statue similar to the study object was selected as a reference and estimated restoration was performed. A design mockup helps to intuitively understand the influence of interference caused by the components and reduces the time required for completion by solving a making-related problem in advance. Preliminary verification through design mockup minimizes the unnecessary consumption of materials and time in the restoration process and maximizes the work efficiency [54]. In this study, to enhance restoration completeness using the noncontact approach, digital–analog-based assembly simulation was performed by creating a design mockup. Material extrusion 3D printing, which can minimize time, shape error, and costs, was used to create the design mockup. Page 6/24 Page 6/24 Design mockup and assembly simulation To summarize the assembly simulation process, first, an alternative model of the original statue was printed on the basis of the 3D scanning model to minimize direct physical contact with the statue. Moreover, heuristic-based assembly suitability was examined several times by printing the virtual restoration model. When an error was detected during verification, the virtual restoration model was modified and reprinted to enhance assembly completeness. However, when no error or aesthetic issue was detected, a design mockup was directly applied to the statue for further verification. After using photopolymerization 3D printing, the restoration process was completed (Fig. 7). When the printed virtual restoration model was applied to the mockup statue, the interference between the two surfaces was severe owing to the nonstructured form (Fig. 8a). This indicates the high quality of the virtual restoration model; however, it was insufficient for restoring the original artifact. Therefore, the area in the virtual model where interference occurs should be corrected so that the printed output fits the missing parts of the statue. In addition to the revision of the virtual model, assembly suitability to the 3D- printed mockup statue was intuitively checked and verified by directly modifying the mockup of the virtual restoration model (Fig. 8b). The 3D model, which was verified for digital–analog assembly suitability, was completed as the final design mockup through the outer design modification (Fig. 8c). In a quantitative analysis of the shape difference between the virtual restoration model and final design mockup of the head, the volume of the virtual restoration model was measured to be 461 cm3 and that of the design mockup assembled on the original stone Buddha statue was measured to be 437 cm3 (Fig. 8d). The volume reduction of 24 cm3 (5.2%) equals the amount of the removed interfering surface along the assembly interface. In the deviation analysis of the final design mockup against the virtual restoration model, the size of the removed joining surface was nearly 5.00 mm, with RMS and standard deviation of 0.67 and 0.56 mm, respectively. Approximately 66.6% of the virtual restoration model, whose tolerance range was within ±0.1 mm, was not modified. The interfering surface of the joining interface was removed for 33.4% of the virtual restoration model (Fig. 8e). This deviation-mapping result enabled the visualization of the modification range and deviation amount of the initial virtual restoration model, providing quantitative information about the digital–analog simulation process. Virtual restoration modeling For restoration, the big missing parts in the head, surrounding area of the Baekho, right ear, and right eye were selected, accounting for 64.2% (257.1 cm2) of the total surface area of the missing parts (400.1 cm2).During the examination of the virtual restoration modeling process of the missing parts in the head (Fig. 5), first, the original stone fragments were placed by referring to the shape and pattern of the stone Buddha statue. Then, a reference model for the missing parts was selected and copied using symmetry modeling. However, because the original statue was manually created, it lacked perfect symmetry. The restoration of the right ear has aesthetic as well as functional purposes to support the original fragment. Accordingly, for virtual restoration, the original fragment was placed in the right ear and the shape of the missing parts was acquired using the symmetry of the well-preserved left ear. Next, the angle and height from the front view were adjusted to match those in the left, and completed after partial revision (Fig. 6a, 6b). Finally, aesthetic restoration of the surrounding area of Baekho and right ear was performed by filling the inner part and retouching the outer part. In particular, their restoration was completed after several modifications in consultation with art history experts because the overall impression of the stone Buddha statue can change owing to minor shape differences (Fig. 6c). Page 7/24 Restoration of the missing parts For restoration, 3D printing technology was used to facilitate an educational effect in exhibition and enhance aesthetic and functional completeness. The material extrusion 3D printing used to create the design mockup played a critical role in analyzing the shape of the missing parts to design the assembly planning. However, this technology uses a PLA material with relatively high surface roughness and weak Page 8/24 Page 8/24 physical properties to functionally support the light-weight original stone fragments [55]. To complement this, photopolymerization 3D printing technology was used to create the final restoration model. physical properties to functionally support the light-weight original stone fragments [55]. To complement this, photopolymerization 3D printing technology was used to create the final restoration model. The model was printed using opaque UV-hardened plastic whose color was similar to that of the original statue, with a layer thickness of 0.1 mm (Fig. 9a). After approximately 15 h of printing, the supporters were removed from the printed output and the surface that was in contact with the supporter was carefully smoothed using soft sandpaper with more than 3000 mesh. Additional UV hardening was used to enhance the surface strength of the printed output (Fig. 9b). The finished 3D-printed output exhibited a smooth and glowing surface, which was disparate with that of the original statue (Fig. 9c). The initiator used in photopolymerization can cause yellowing owing to the surrounding light. It is known that the efficiency of polymerization can influence discoloration because a higher degree of conversion corresponds to a smaller amount of residual monomers available to form the colored degraded products [56]. By observing the light source as an isolated factor, it was noted that this can alter the color of the studied composite resin [57]. Therefore, to modify the surface texture of the 3D- printed output and prevent its yellowing, the surface was colored with acrylic painting and varnished to prevent discoloration of the colored layer (Fig. 9d). The missing parts of the stone Buddha statue were restored using the conservation treatment and 3D- printed output. As described, the Buddha statue is composed of zeolitic tuff. This material is very soft and has high absorption ratio owing to its porous structure. Accordingly, the interfaces of the missing parts of the original statue were strengthened using an ethyl silicate-based consolidant before joining the 3D-printed output (Fig. 9e). Restoration of the missing parts Then, the 3D-printed output was joined to the missing parts of the statue using acrylic resin and cyanoacrylate instant adhesives (Fig. 9f). In this study, various digital data and mockup of the original statue obtained using 3D scanning and printing technologies transformed the exhibition, which comprised simple display of artifacts, into an educational exhibition focusing on the restoration process. Combined with unique storytelling, the exhibition has provided a new experience to museum visitors (Fig. 10). This is an important case, which shows that established modern technologies and materials can be used for the restoration, education, and exhibition of cultural artifacts. In this study, various digital data and mockup of the original statue obtained using 3D scanning and printing technologies transformed the exhibition, which comprised simple display of artifacts, into an educational exhibition focusing on the restoration process. Combined with unique storytelling, the exhibition has provided a new experience to museum visitors (Fig. 10). This is an important case, which shows that established modern technologies and materials can be used for the restoration, education, and exhibition of cultural artifacts. Discussion The stone Buddha statue, owned by the Chuncheon museum, was largely damaged and not in its original shape because many stone fragments were stored separately. The value of the stone Buddha statue has been appreciated from the perspective of art history; accordingly, the necessity of the restoration of its missing parts based on historic research has been raised. Therefore, in this study, a noncontact restoration of the stone Buddha statue was performed by converging various digital technologies, including 3D scanning, virtual restoration modeling, assembly simulation, and printing restoration. Page 9/24 Page 9/24 Furthermore, 64.2% of the total missing parts were selected as the restoration scope, which was deemed necessary for the aesthetic and functional restoration. Moreover, the separately retained stone fragments were all used for the restoration. A digital virtual restoration of the missing parts was performed using the original-based symmetry modeling [45]. For the partial area in the head where symmetrization was difficult owing to damage to more than half of its part, restoration was performed through historical research and verification. In particular, throughout the modeling process, a haptic device and voxel software were used, facilitating an intuitive restoration process. The assembly simulation using the design mockup was the most focused stage in the whole restoration process. Generally, virtual assembly simulation is widely used in medical and industrial fields to evaluate the impact of surface interference in advance [58], and virtual assembly based on various algorithms is used in the field of cultural heritage [59–63]. However, virtual assembly has its limitations when applied to the stone Buddha statue, the study object, whose joining surface is nonstructured and complicated. Therefore, in this study, an alternative model for the original and virtual restoration models were 3D printed to minimize physical contact with the original stone statue. In particular, the 3D-printed alternative model was greatly useful for identifying the problems of the virtual restoration model by verifying the design mockup in real time, thus overcoming the physical constraints that did not allow assembly with the original statue. Moreover, the virtual restoration model was 3D printed and used for heuristic-based assembly simulation, thereby intuitively completing the restoration of the missing parts. This study serves as a remarkable case to shift toward the use of the noncontact digital method in restoration. It can also prevent secondary damage by minimizing physical contact with the original statue. Discussion In particular, the heuristic-based assembly simulation helped modify and improve the restoration plan and enhanced the restoration reliability, as the assembly result was intuitively reviewed and assessed. Recently, several solutions for digital virtual assembly have been widely explored, mainly focused on the development of automatic strategies for matching fragments [64–66], measure of the surface profile [67], fusion of multiple features [68], and alignment using bridges [69]. Thus, state-of-the-art approaches have their weaknesses and strengths according to the application aims. The stone Buddha statue was not complex and had few fragments. In particular, most assembly parts were created through Boolean operation based on the original statue. Therefore, automatic and semiautomatic approaches for assembly are not important issues in this study. However, the development of an algorithm that could be used to modify the modeling of the interference between fractured surfaces must significantly reduce the frequency of design mockup assembly, and the whole process will be accomplished in a digital virtual environment. Conclusions 1. In this study, digital recording of the original statue was performed using 3D scanning of the physically damaged stone Buddha statue and digital virtual restoration of the missing parts using a Page 10/24 haptic modeling system. Moreover, the missing parts were restored using 3D printing technology after design mockup creation and assembly simulation. 2. The 3D model of the stone Buddha statue was created by converging the fixed high-precision scanning of the exterior and the handheld mid-precision scanning of the interior excavated hole. The 3D model showed a high resolution with an average point density of 0.2 mm. In particular, the 3D scanning result of the cone-shaped excavated hole revealed that two chisels, whose sizes were 10.5 and 15.4 mm on average, were used to carve the hole. 2. The 3D model of the stone Buddha statue was created by converging the fixed high-precision scanning of the exterior and the handheld mid-precision scanning of the interior excavated hole. The 3D model showed a high resolution with an average point density of 0.2 mm. In particular, the 3D scanning result of the cone-shaped excavated hole revealed that two chisels, whose sizes were 10.5 and 15.4 mm on average, were used to carve the hole. 3. Using a 3D deterioration map of the stone Buddha statue, the area of the missing parts was measured to be 400.1 cm2 (5.5% of the total) of the total area (7,250 cm2). The restoration was performed only for 64.2% (257.1 cm2) of the total area of the missing parts, for which symmetry modeling was applicable or original forms could be estimated based on historical research. The restoration objects included four parts: the head, surrounding area of the Baekho, right ear, and right eye. 4. The virtual restoration of the missing parts of the stone Buddha statue was performed using a haptic modeling system: first, the location of the original fragments was determined; second, a reference model was selected and symmetry modeling was conducted; finally, the virtual restoration model was modified and improved, and estimation modeling through historical research and description of the outer shape was also used. 5. The created virtual restoration model was verified for its heuristic-based assembly suitability by design mockup printing and digital–analog simulation. Accordingly, the initial virtual restoration model decreased in volume by 5.2%. Abbreviations 3D: Three-dimensional; PLA: Polylactic acid; FOV: field of view Conclusions Moreover, the removed surface owing to the interference effect between the virtual restoration model and final design mockup was 33.4%, and their RMS was calculated to be 0.67 mm. 6. Photopolymerization 3D printing technology was used for the actual restoration of the stone Buddha statue, and considering the surface roughness, the material layer thickness was set at 0.10 mm. In addition, the surface of the printed output was colored to prevent yellowing, and the final restoration was completed by joining the missing parts of the statue. 7. This study serves as a remarkable case to shift from the traditional manual-contact method to the noncontact digital method in the restoration of artifacts, and heuristic-based assembly simulation helps in modifying and improving the restoration plan and enhances the restoration reliability. In the future, it is assumed that digital–analog-based restoration technology will be extensively used in the conservation of cultural artifacts. 7. This study serves as a remarkable case to shift from the traditional manual-contact method to the noncontact digital method in the restoration of artifacts, and heuristic-based assembly simulation helps in modifying and improving the restoration plan and enhances the restoration reliability. In the future, it is assumed that digital–analog-based restoration technology will be extensively used in the conservation of cultural artifacts. Declarations Page 11/24 Page 11/24 Funding This work was financially supported by the Chuncheon National Museum and the Ministry of Science, ICT, and Future Planning. Availability of data and materials The datasets used and/or analysis results obtained in the current study are available from the corresponding author on request. Author details 1Department of Cultural Heritage Conservation Sciences, Kongju National University, Gongju, 32588, Republic of Korea. 2Conservation Science Laboratory, Chuncheon National Museum, 24325, Republic of Korea. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF- 2016R1C1B2010883). The authors would like to thank Chuncheon National Museum for their support of this paper. Competing interests The authors declare that they have no competing interests. Author’s contributions All authors contributed to the planning and design of this article. YHJ and SH performed the data acquisition and data analysis. SYJ and YMK performed the conservation treatment. YHJ wrote the manuscript and all authors revised it critically. All authors read and approved the final manuscript. References Page 12/24 Page 12/24 1. Fitzner B. 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Zhang Y, Li K, Chen X, Zhang S, Geng G, A multi feature fusion method for reassembly of 3D cultural heritage artifacts. J Cult Herit. 2018;33:191–200. References 69. Arbace L, Sonnino E, Callieri M, Dellepiane M, Fabbri M, Idelson AI, Scopigno R, Innovative uses of 3D digital technologies to assist the restoration of a fragmented terracotta statue. J Cult Herit. 2013;14:332–45. 69. Arbace L, Sonnino E, Callieri M, Dellepiane M, Fabbri M, Idelson AI, Scopigno R, Innovative uses of 3D digital technologies to assist the restoration of a fragmented terracotta statue. J Cult Herit. 2013;14:332–45. 66. Papaodysseus C, Arabadjis D, Exarhos M, Rousopoulos P, Zannos S, Panagopoulos M, Papazoglou- Manioudaki L, Efficient solution to the 3D problem of automatic wall paintings reassembly. Comput Math Appl. 2012;64:2712–34. Figures Figure 1 Figure 1 Figure 1 Status of the Buddha statue. Page 17/24 Page 17/24 Figure 2 Methods and data processing. Figure 2 Figure 2 Methods and data processing. Methods and data processing. Page 18/24 Figure 3 3D scanning process and the result of convergence modeling of the statue. Figure 3 D scanning process and the result of convergence modeling of the statue. 3D scanning process and the result of convergence modeling of the statue. Page 19/24 Figure 4 Result of 3D scanning and shape analysis of the statue. (a) The 3D polygon mesh and RGB texture mapping model. (b) A 3D deterioration map of the stone Buddha statue, and the mapping of the i Figure 4 Result of 3D scanning and shape analysis of the statue. (a) The 3D polygon mesh and RGB texture- mapping model. (b) A 3D deterioration map of the stone Buddha statue, and the mapping of the restoration ranges. Result of 3D scanning and shape analysis of the statue. (a) The 3D polygon mesh and RGB texture- mapping model. (b) A 3D deterioration map of the stone Buddha statue, and the mapping of the restoration ranges. Page 20/24 Figure 5 Virtual restoration modeling process of the head of the statue. Figure 6 Result of the virtual restoration modeling of the face of the stone Buddha statue. (a and b) The right ear. (c) The surrounding area of Baekho and right eye. Figure 5 Figure 5 Figure 5 Figure 5 Virtual restoration modeling process of the head of the statue. Figure 6 Page 21/24 Result of the virtual restoration modeling of the face of the stone Buddha statue. (a and b) The right ear. (c) The surrounding area of Baekho and right eye. Figure 7 Result of the virtual restoration modeling of the face of the stone Buddha statue. (a and b) The right ear. (c) The surrounding area of Baekho and right eye. Result of the virtual restoration modeling of the face of the stone Buddha statue. (a and b) The right ear. (c) The surrounding area of Baekho and right eye. Page 21/24 (c) The surrounding area of Baekho and right eye. Figure 7 Figure 7 Page 21/24 Heuristic-based design mockup and assembly simulation process. Figure 8 Simulation process using the design mockup. (a) Verifying assembly suitability using a 3D-printed alternative model. (b) Direct modification of the design mockup printing output. (c) Final design mockup after revising the joining surface. (d) The modification result of interface modeling. (b) Deviation analysis and mapping result of the virtual restoration model and final design mockup model. Heuristic-based design mockup and assembly simulation process. Heuristic-based design mockup and assembly simulation process. Figure 8 Simulation process using the design mockup. (a) Verifying assembly suitability using a 3D-printed alternative model. (b) Direct modification of the design mockup printing output. (c) Final design mockup after revising the joining surface. (d) The modification result of interface modeling. (b) Deviation analysis and mapping result of the virtual restoration model and final design mockup model. Simulation process using the design mockup. (a) Verifying assembly suitability using a 3D-printed alternative model. (b) Direct modification of the design mockup printing output. (c) Final design mockup after revising the joining surface. (d) The modification result of interface modeling. (b) Deviation analysis and mapping result of the virtual restoration model and final design mockup model. Page 22/24 Page 22/24 Page 22/24 Figure 9 3D printing and conservation treatment processes for the restoration of the stone Buddha statue. (a) 3D- printed outputs using photopolymerization. (b) UV hardening to enhance surface strength. (c) 3D-printed outputs of the four selected parts, including the head, surrounding area of the Baekho, and right ear and eye. (d) 3D printing output colored with acrylic painting to modify the surface texture and prevent yellowing. (e) The interfaces of the missing parts of the original stature strengthened using an ethyl silicate-based consolidant. (f) Joining of the missing parts of the statue using acrylic resin and cyanoacrylate instant adhesives. Figure 9 3D printing and conservation treatment processes for the restoration of the stone Buddha statue. (a) 3D- printed outputs using photopolymerization. (b) UV hardening to enhance surface strength. (c) 3D-printed outputs of the four selected parts, including the head, surrounding area of the Baekho, and right ear and eye. (d) 3D printing output colored with acrylic painting to modify the surface texture and prevent yellowing. (e) The interfaces of the missing parts of the original stature strengthened using an ethyl silicate-based consolidant. (f) Joining of the missing parts of the statue using acrylic resin and cyanoacrylate instant adhesives. y y Page 23/24 Figure 10 Restoration of the stone Buddha statue and its use in an exhibition. (a) The restoration result of the missing parts. (b) A special exhibition on the restoration process of the stone Buddha statue. Figure 10 Restoration of the stone Buddha statue and its use in an exhibition. (a) The restoration result of the missing parts. (b) A special exhibition on the restoration process of the stone Buddha statue. Page 24/24
https://openalex.org/W2781719558	https://europepmc.org/articles/pmc5754074?pdf=render	English	null	Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants	PloS one	2,018	cc-by	12,141	RESEARCH ARTICLE Tjasˇa Lukan1,2, Fabian Machens3¤, Anna Coll1, Sˇ pela Baebler1, Katrin Messerschmidt3, Kristina Gruden1 Tjasˇa Lukan1,2, Fabian Machens3¤, Anna Coll1, Sˇ pela Baebler1, Katrin Messerschmidt3, Kristina Gruden1 1 National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia, 2 International Postgraduate School, Ljubljana, Slovenia, 3 University of Potsdam, Cell2Fab Research Unit, Potsdam, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: University of Potsdam, Department Molecular Biology, Potsdam, Germany * tjasa.lukan@nib.si Abstract Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite sev- eral modular assembly methods developed in recent years, the plant biotechnology commu- nity has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar- free and sequence-independent multigene assembly strategy AssemblX, based on overlap- depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection mark- ers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science. Introduction One of the main technological obstacles in plant biotechnology is cloning for delivery of multi- ple DNA fragments into the plant genome [1]. Therefore, there is an increased demand for the assembly, introduction and expression of DNA encoding multiple genes, which could cope with ongoing needs, such as reconstruction of complex biochemical pathways [2,3], engineer- ing synthetic signal transduction systems [4] and other high-impact goals in plant synthetic biology [5]. Competing interests: Patent is pending for AssemblX cloning method and vectors (Patent number EP16188155.2, inventors: Katrin Messerschmidt, Fabian Machens, Lena Hochrein, Bernd Mueller-Roeber), which are used as a part of Plant X-tender. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. Two crucial factors are required for the development of an efficient cloning strategy in plant synthetic biology: first, a method by which the foreign coding sequence (CDS) and regu- latory elements, i.e. promoter and terminator sequences, can be assembled and inserted into the plant expression vector in a highly efficient and versatile manner and second, the availabil- ity of plant expression vectors capable of holding and introducing multiple expression cassettes into the plant genome. Although a remarkable variety of plant expression plasmids has already been developed, the basic design of many of these vectors is quite restrictive and rarely permits the cloning and transfer of more than a single expression cassette into plant cells [6,7]. Due to the increasing requirement for high throughput approaches to assemble complex designs, classical restriction-based cloning became limited. Critical points are low cloning efficiency, high expenditure of time, the introduction of unwanted sequences at the junction sites and the occurrence of restriction enzyme recognition sites within expression cassettes. Although the latter drawback was overcome by exploiting the power of rare-cutters for the assembly of multiple expression cassettes by designing the pAUX series of plasmids [8] and their variations pSAT [9,10], the system is still limited by the number of different commercially available rare-cutting restriction enzymes. To overcome the limitations of classical restriction-based cloning methods, the emergence of different recombination-dependent methods was exploited for multigene cloning applica- tions. Since the invention of the first recombination methods based on the Cre/loxP recombi- nation system (see review [11]), the number of elements that can be assembled was increased by combining this system with two rare-cutter endonucleases [12]. Plant X-tender: An extension of the AssemblX system for plant synthetic biology funding No. P4-0165 and projects N4-0026, J4- 7636 and 1000-15-0105. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Citation: Lukan T, Machens F, Coll A, Baebler Sˇ, Messerschmidt K, Gruden K (2018) Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants. PLoS ONE 13(1): e0190526. https://doi. org/10.1371/journal.pone.0190526 Editor: Paulo Lee Ho, Instituto Butantan, BRAZIL Received: June 19, 2017 Accepted: December 15, 2017 Published: January 4, 2018 Copyright: © 2018 Lukan 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: Paulo Lee Ho, Instituto Butantan, BRAZIL Received: June 19, 2017 Accepted: December 15, 2017 Published: January 4, 2018 Copyright: © 2018 Lukan 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: The nucleotide sequences of constructed Plant X-tender vectors are deposited in GenBank under accession numbers MG561370-MG561373. The Plant X- tender expression vectors and their maps are available from Addgene (Addgene IDs 98888- 98891). Funding: The research was financially supported by the Federal Ministry of Education and Research of Germany (https://www.bmbf.de/en/index.html): FKZ 031A172; and by the Slovenian Research Agency (https://www.arrs.gov.si/): research core 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Introduction Another recombination- based cloning method, Invitrogen’s Gateway technology, which is based on the bacteriophage λ site-specific recombination system [13] overcomes the shortcoming of classical Cre/loxP recombination system, since it allows delivery of DNA fragments from entry into multiple des- tination vectors with high specificity and efficiency [13,14,15]. Although the upgrade of the technique into Multisite Gateway enables delivery of up to three independent transcription units into plant cells [16,17], it is still limited in its flexibility due to a small number of available att sites. However, with MultiRound Gateway cloning, based on multiple rounds of LR recom- bination reactions, it is possible to assemble up to seven expression cassettes [18,19]. Despite many benefits of Gateway-based systems, there are still several drawbacks such as the intro- duction of unwanted cloning scars at the junction sites and high cost, especially for large scale projects [20]. Another vector set that overcomes the drawback of type II restriction enzymes was designed by Ghareeb et al., 2016 [21]. The system, named COLORFUL-Circuit, is based on the rare-cutter SfiI. Since the overhang sequences generated by this restriction enzyme can be freely modified to generate unique non-palindromic ends, this system has the potential for a high throughput approach. To date, COLORFUL-Circuit assembly enables the introduction of up to five expression cassettes and is specifically designed for studies involving fusions with fluorescent proteins. In recent years, significant efforts were dedicated to the development of simple and efficient cloning systems for standardized assembly of genetic modules, using defined rules [22,23]. 2 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology High throughput modular ligation-dependent methods, efficient for multipart assemblies for expression in plants, are Golden Gate [24] and derived strategies i.e. MoClo [25], GoldenBraid [26] and GreenGate [27]. However, disadvantages of those methods are common to conven- tional cloning strategies, as they are based on the use of type IIS restriction endonucleases. For those, the main disadvantage is the high occurrence of recognition sites within expression cas- settes, which becomes increasingly relevant in the case of large multigene constructs. Introduction In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [28], uracil-specific excision reagent cloning (USER) [29], Gibson assembly [30], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [31], transformation-associated recombination (TAR) cloning [32] and seamless ligation cloning extract (SLiCE) [33], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [34]). Moreover, they do not require premade standardised biological parts and can thus be used to easily clone and characterize novel functional parts. Therefore, the overlap-based assembly methods and Gibson assembly, in particular, are the most accepted methods within the syn- thetic biology community [35]. However, so far they have not been widely adopted in plant sciences, most probably due to the lack of software tools to facilitate the design of complex constructs as well as the lack of cloning parts and vectors, depended on highly efficient over- lap-based cloning methods, which would build the basis for the assembly and delivery of mul- tiple-gene cassettes into plants cells. Recently, Hochrein et al. (2017) [36] developed the AssemblX toolkit, a new cloning strat- egy supported by the AssemblX web tool. AssemblX enables the assembly of subunits into a multigene structure. The predefined parts order is based on overlaps between adjacent assem- bly units. Specific overhang sequences first need to be introduced into subunits (e.g. promoter, CDS, terminator) by PCR using custom-designed primers. These primers contain overlaps to the neighbouring fragment in their 5’ region to allow the assembly into entry Level 0 vectors by using overlap-dependent cloning methods. The so-assembled gene cassettes can be com- bined into the multigene structure in a Level 1 AssemblX vector. To facilitate the transfer of assembly units between different assembly Levels without relying on PCR amplification, rare- cutting restriction enzymes are employed. A multitude of Level 0 and Level 1 vectors is avail- able to allow the assembly of currently up to 25 expression cassettes in a user-defined order. Although the AssemblX toolbox is designed to support easy transfer to any organism, the Level 2 AssemblX vectors are currently available only for bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) [36]. Recently, we developed a design strategy for plant expression vectors and we implemented it, as a grammar, in the Computer-Aided Design (CAD) software GenoCAD [37]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 In vitro DNA assembly cloning reactions SLiCE extract was prepared as described elsewhere [39] with some modifications. Cells were grown in 100 ml 2 x YT medium in 250 ml baffled flasks to OD600 = 2. SLiCE reactions were performed as described by Zhang (2012) [32] with 50–500 ng of linear vector and an appropri- ate amount of insert DNA ranging from 1: 2 to 1: 20 vector to insert molar ratio. Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB). Assembly reaction con- tained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix. Assembly reactions were incubated at 50˚C for 1h. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E. coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. Transformed cells were spread on plates con- taining appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml). Design of AssemblX constructs for plant expression When needed to be assembled from different subunits, the expression cassettes for Level 0 were designed in silico using the extended plant grammar implemented in GenoCAD (http:// www.genocad.com/). Sequences were exported as GenBank format and imported into AssemblX web tool (www.assemblx.org) for the design and virtual assembly of multigene con- structs and the primer design. Introduction This soft- ware tool allows the user to quickly design genetic constructs based on the notion of genetic parts, thereby laying a foundation for the set-up of overlap-dependent assembly methods in plants [38]. The grammar includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs thus minimizing the risk of introducing errors [37]. The aim of our work was to develop tools allowing full flexibility and fast and efficient assembly cloning for multiple protein expression in plants and thus contribute to filling the knowledge gap that impedes the burst of plant synthetic biology. We here present Plant X-ten- der, an extension of the AssemblX system for the assembly and expression of multigene con- structs in plants. Plant X-tender consists of a set of plant expression vectors. We additionally developed tools to support researchers in plant synthetic biology with the extended GenoCAD plant grammar and protocols for most efficient cloning into the novel vector set. The Plant X- tender enables transfer of multigene constructs from AssemblX vectors to plant expression PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 3 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology vectors and delivery of multiple-gene cassettes into plant cells in an easy and scalable manner. Our proof of principle experiments pave the way for more complex and increasingly flexible approaches for large-scale engineering in plant synthetic biology. In vivo DNA assembly cloning reactions in S. cerevisiae(TAR) In vivo DNA assembly cloning was performed as described by Hochrein et al. (2017) [36]. Sac- charomyces cerevisiae transformation was performed according to the LiAc/SS carrier DNA/ PEG method [40] using strains YPH500 (ATCC1 76626™) or BY4741 (ATCC1 201388™). The plasmids were isolated from positive colonies using Zymoprep Yeast Miniprep II kit (Zymo Research Corporation) and transformed into electrocompetent ElectroMAX™DH5α-E™E. coli (Thermo Fisher Scientific). Construction of Plant X-tender expression vectors Vector pCAMBIA1300 (Marker Gene Technologies, M1591) was digested with BamHI and HindIII (NEB) and purified from the gel (Fig 1A). Vectors pK7WG, pB7WG and pH7WG (Karimi et al., 2002) were digested with XbaI and SacI (NEB), allowing the release of T35S– AttR2–ccdB–AttR1 cassette from the vector backbone and purified from the gel (Fig 1B). The ccdB region was amplified from AssemblX pL0A_0–1 Level 0 plasmid [36] using KG15/KG16 primers, purified from the gel and amplified with KG15/KG18 for pCAMBIA1300 and KG19/ KG21 primers for the other three plasmid backbones to add homology regions with flanking restriction enzyme recognition sites (see S1 Table for the list of primers). The gel-isolated I- SceI–A0–HindIII–ccdB–HindIII–B0–I-SceI cassette was assembled into the purified plasmid backbones using different assembly methods and transformed into commercial or homemade PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 4 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology Fig 1. Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I-SceI–A0–HindIII–ccdB–HindIII–B0–I-SceI cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with BamHI and HindIII to obtain pCAMBIA_ASX. (B) T35S–AttR2–ccdB–AttR1 cassette was released from the Gateway plasmid backbone by digestion with XbaI and SacI and replaced with a I-SceI–A0–HindIII–ccdB–HindIII–B0–I-SceI cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, Spec: selection marker conferring spectinomycin resistance in E. coli and A. tumefaciens, Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccdB: bacterial suicide gene, HindIII, I-SceI, BamHI, XbaI, SacI: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. https://doi.org/10.1371/journal.pone.0190526.g001 Fig 1. Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I-SceI–A0–HindIII–ccdB–HindIII–B0–I-SceI cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with BamHI and HindIII to obtain pCAMBIA_ASX. Construction of Plant X-tender expression vectors (B) T35S–AttR2–ccdB–AttR1 cassette was released from the Gateway plasmid backbone by digestion with XbaI and SacI and replaced with a I-SceI–A0–HindIII–ccdB–HindIII–B0–I-SceI cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, Spec: selection marker conferring spectinomycin resistance in E. coli and A. tumefaciens, Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccdB: bacterial suicide gene, HindIII, I-SceI, BamHI, XbaI, SacI: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. https://doi.org/10.1371/journal.pone.0190526.g001 https://doi.org/10.1371/journal.pone.0190526.g001 https://doi.org/10.1371/journal.pone.0190526.g001 One Shot1 ccdB Survival™2 T1R Competent Cells (Thermo Fisher Scientific). Transformation efficiencies of homemade competent E. coli are listed in S2 Table. We confirmed the function- ality of ccdB gene by transformation of ccdB Survival E. coli strain which allowed propagation of plasmids containing the ccdB gene. Transformation of ccdB-sensitive E. coli strain which precludes propagation of plasmids containing the ccdB gene with purified plasmids was per- formed as a control. We verified the correct constructions of the Plant X-tender expression vectors by sequencing the homology regions A0 and B0, I-SceI and HindIII recognition sites and the ccdB gene. We deposited the nucleotide sequences of constructed vectors pCAM- BIA_ASX, pK7WG_ASX, pH7WG_ASX and pB7WG_ASX in GenBank under accession numbers MG561370-MG561373. The Plant X-tender expression vectors and their maps are available from Addgene (Addgene IDs 98888-98891). Colony PCR and sequencing Positive assemblies were confirmed by colony PCR using KAPA Taq PCR Kits (Kapa Biosys- tems) or Maxima Hot Start Green PCR Master Mix (Thermo Fisher Scientific) following the manufacturer’s protocols. For further verification, plasmids were isolated from positive colonies using GeneElute Plasmid Miniprep Kit (Sigma) or NucleoSpin Plasmid Easy Pure (Macherey-Nagel) and analysed by sequencing. DNA concentration and purity were evaluated using NanoDrop ND1000 spectrophotometer (Nanodrop technologies). Plasmid DNA and oligonucleotides were prepared according to service provider requirements (LGC Genomics or GATC service provider). Sequences were analysed with CLC Main Workbench (Qiagen). Primers used for colony PCR and sequencing are listed in S4 and S5 Tables. Nicotiana benthamiana transient transformation N. benthamiana seeds (obtained from prof. Van der Vlugt, Wageningen University and Research Centre) were soaked in gibberellic acid overnight to induce germination. Next day the seeds were rinsed three times with sterile water, transferred to soil and grown under con- trolled environmental conditions as previously described [41]. Five weeks old plants were used for transient transformation. Constructs were introduced into homemade electrocompetent Agrobacterium tumefaciens GV3101 by electroporation (Eppendorf Electroporator 2510) fol- lowing manufacturer’s procedure at 2000 V and confirmed by colony PCR. The transformed cells were cultured to an OD600 = 0.5, harvested by centrifugation, resuspended in 0.2 mM acet- osyringone water solution (prepared from 200 mM acetosyringone in DMSO) to OD600 = 0.5 and mixed with A. tumefaciens transformed with silencing suppressor p19 (kindly provided by prof. Jacek Hennig) in the ratio 1: 1. The mixture was infiltrated into the second fully developed bottom leaf of N. benthamiana plants. Empty A. tumefaciens GV3101 was used as a control. Construction of assembly cassettes and cloning into Plant X-tender expression vectors pL0A_0–1, pL0A_0-R and pL0A_1-R Level 0 vectors [36] were linearized with HindIII as described in [36]. Inserts were amplified from templates using Phusion1 High-Fidelity DNA 5 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology Polymerase (NEB) and primers with flanking homologous sequences (S3 Table) according to manufacturer’s protocol. Following agarose gel electrophoresis, inserts were purified from the gel using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel) kits, assembled into Level 0 vectors using NEBuilder HiFi assembly master mix (NEB) and transformed into E. coli NEB 5α (NEB). pL1A-hc / pL1A-lc (A0/AR) Level 1 vectors [36] were linearized with PacI (NEB). Expres- sion cassettes were released from Level 0 vectors using PmeI (NEB), separated by agarose gel electrophoresis and purified from the gel as described above. Multiple cassettes were assem- bled into Level 1 vectors using TAR or NEBuilder HiFi. Plasmids were isolated from positive yeast colonies and transformed into electrocompetent ElectroMAX™DH5α-E™Cells (Thermo Fisher Scientific). Multigene constructs were released from Level 1 vector with I-SceI (NEB). For inserts with similar length to the backbone, the plasmid was additionally digested with NheI (NEB) to allow separation of the insert and the backbone by gel electrophoresis. Follow- ing isolation from the gel using Wizard1 SV Gel and PCR Clean-Up System (Promega), inserts were assembled into HindIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method. Constructs were transformed into One Shot1 TOP10 Chem- ically Competent E. coli (Thermo Fischer Scientific), homemade TOP10 chemically competent E. coli or homemade TOP10 electrocompetent E. coli. Incorrect assemblies were selected against by the expression of a suicide gene and by antibiotic selection. The Plant X-tender toolbox for the assembly of multigene constructs Moreover, it includes a library of plant genetic parts classified in structural categories which constitute a repository of biological parts sharable with the plant community and easily accessible for all the users. The newly developed grammar is available at Figshare (https://doi.org/10.6084/m9.figshare.4977464) and can be imported into the GenoCAD. We used newly developed grammar to virtual assemble the selected biological parts into the expression cassette (Fig 2, GenoCAD). Thus, the GenoCAD plant grammar complements the AssemblX web tool by supporting the user throughout the design of Level 0 constructs. In the next step, we exported the sequences of the designed expression cassettes from GenoCAD and imported them into the AssemblX web tool to virtually assemble the expression cassettes in Level 1 AssemblX vectors in order to combine them in a multigene construct (Fig 2, AssemblX web tool). Finally, we generated the plasmid maps of the intended final product by cutting and pasting the assembly module from Level 1, flanked by A0 –B0 homology regions, into the final destination plant expression vector. In general, specific overlapping homology regions (A0 and B0 in this study) are incorporated into the assembly module by restriction digestion of Level 1 AssemblX vector using rare-cutting restriction enzyme. Introduced homology regions will later allow the scar-less assembly into Plant X-tender expression vector containing the same homology regions. We used newly developed grammar to virtual assemble the selected biological parts into the expression cassette (Fig 2, GenoCAD). Thus, the GenoCAD plant grammar complements the AssemblX web tool by supporting the user throughout the design of Level 0 constructs. In the next step, we exported the sequences of the designed expression cassettes from GenoCAD and imported them into the AssemblX web tool to virtually assemble the expression cassettes in Level 1 AssemblX vectors in order to combine them in a multigene construct (Fig 2, AssemblX web tool). Finally, we generated the plasmid maps of the intended final product by cutting and pasting the assembly module from Level 1, flanked by A0 –B0 homology regions, into the final destination plant expression vector. In general, specific overlapping homology regions (A0 and B0 in this study) are incorporated into the assembly module by restriction digestion of Level 1 AssemblX vector using rare-cutting restriction enzyme. Introduced homology regions will later allow the scar-less assembly into Plant X-tender expression vector containing the same homology regions. The Plant X-tender toolbox for the assembly of multigene constructs The AssemblX toolkit contains vectors of three different levels and an accompanying online tool [36]. We expanded the toolkit by developing the Plant X-tender toolbox. The toolbox consists of four newly constructed Plant X-tender plant expression vectors. To facili- tate its use in synthetic biology approaches in plant science, the developed vector series is com- plemented by the in silico design tool GenoCAD and protocols for most efficient cloning into the novel vector set. We first customized the plant grammar implemented in GenoCAD [37] by adding sequences of nopaline synthase promoter (pNOS) and cauliflower mosaic virus CaMV 35S ter- minator (t35S) to the GenoCAD plant library. We constructed an additional plant grammar rule to facilitate virtual assembly of the selected biological parts into the expression cassette. The new rule allows the design of a simple expression cassette segment without the whole vector. The user is guided through the design of an expression cassette that, as a minimum requirement, includes promoter, CDS and terminator. However, it offers flexibility with regard to the CDS, which can represent a plant gene, a fluorescent protein or both, and can be fused to epitope tags and linkers, if needed. The user can also decide to add one or two promoters and terminators. Moreover, it includes a library of plant genetic parts classified in structural categories which constitute a repository of biological parts sharable with the plant community and easily accessible for all the users. The newly developed grammar is available at Figshare (https://doi.org/10.6084/m9.figshare.4977464) and can be imported into the GenoCAD. We first customized the plant grammar implemented in GenoCAD [37] by adding sequences of nopaline synthase promoter (pNOS) and cauliflower mosaic virus CaMV 35S ter- minator (t35S) to the GenoCAD plant library. We constructed an additional plant grammar rule to facilitate virtual assembly of the selected biological parts into the expression cassette. The new rule allows the design of a simple expression cassette segment without the whole vector. The user is guided through the design of an expression cassette that, as a minimum requirement, includes promoter, CDS and terminator. However, it offers flexibility with regard to the CDS, which can represent a plant gene, a fluorescent protein or both, and can be fused to epitope tags and linkers, if needed. The user can also decide to add one or two promoters and terminators. Confocal microscopy Expression of fluorescent proteins was followed six days after agroinfiltration using Leica TCS SP5 laser scanning confocal microscope mounted on a Leica DMI 6000 CS inverted PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 6 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology microscope (Leica Microsystems) with an HC PL FLUOTAR 10x objective with zoom factor 1 or 3.05. The 405 nm and 543 nm laser lines were used for the excitation of the enhanced cyan fluorescent protein (ECFP) and monomeric red fluorescent protein 1 (mRFP1). The ECFP emission was measured in the window from 450 to 530, while the mRFP1 was measured in the window from 570 to 630 nm. Three regions of interest per one agroinfiltrated area were scanned bidirectionally with a resolution of 512 x 512 pixels, line average 3 and scan speed 400 Hz. Image merging of brightfield with maximum projections from Z-stacks was performed using Leica LAS AF Lite software (Leica Microsystems). PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 The Plant X-tender toolbox for the assembly of multigene constructs The Plant X-tender cloning procedure follows the AssemblX strategy (Fig 2). The strategy relies on overlap-based cloning methods which utilize various overlapping homology regions flanking the DNA parts to allow the scar-less assembly of multiple parts into a single DNA con- struct. Specific overhang sequences first need to be introduced into subunits (e.g. promoter, CDS, terminator) by PCR using custom-designed primers overlapping the neighbouring 7 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites (AscI, SbfI, SwaI, FsaI, PmeI) flanking the homology regions. (D) Assembly of expression cassettes into PacI digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I-SceI allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) HindIII digestion enables the linearization of Plant X-tender expression vector and the release of ccdB cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker (URA3) flanked by homology regions into Plant X- tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, HindIII, I-SceI, PacI, AscI, SbfI, SwaI, FseI, PmeI: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E. coli and A. tumefaciens, Amp: selection marker conferring ampicillin resistance in E. coli and A. tumefaciens, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, URA3: yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccdB: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector. https://doi.org/10.1371/journal.pone.0190526.g002 https://doi.org/10.1371/journal.pone.0190526.g002 fragment in their 5’ region to allow assembly based on overlap-dependent cloning methods and to determine the orientation and order of DNA fragments in AssemblX Level 0 vectors (Fig 2A and 2B). Level 0 vectors differ in two variable homology regions that will later define the position of the assembly unit within the final construct. The so-assembled gene cassettes are combined into a multigene structure in a Level 1 AssemblX vector (Fig 2C and 2D). Differ- ent Level 1 vectors are compatible with different Level 0 vector sets depending on the homol- ogy regions they contain. In the last step, the construct is transferred into newly developed Plant X-tender expression vectors exploiting homologous recombination between the homol- ogy regions of the expression vector and the homology regions of the construct, thus allowing introduction of up to five expression cassettes into a single plasmid (Fig 2E–2G). Although it is highly recommended to use the plant grammar implemented in GenoCAD and AssemblX web tool for the design to decrease the risk of introducing errors, one or both design tools could be omitted to apply cut-and-paste approach. PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology Plant X-tender: An extension of the AssemblX system for plant synthetic biology Fig 2. Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into HindIII digested Level 0 vectors via overlap-based assembly methods. Only Fig 2. Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into HindIII digested Level 0 vectors via overlap-based assembly methods. Only PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 8 / 19 Validating the Plant X-tender expression vectors We tested the developed Plant X-tender toolbox by cloning the expression cassette p35S:: H2BRFP_tNOS into newly developed Plant X-tender expression vectors. We further con- firmed the functionality of the system by transient expression of H2BRFP in N. benthamiana (Fig 3). This expression cassette, which consists of a histon sequence fused to red fluorescent protein mRFP1, was already confirmed to be functional [42], therefore we used it as the proof of concept. We amplified the expression cassette from a template plasmid using primers with appropriate 5’ and 3’ extensions, giving homology to A0 and AR homology regions and assem- bled it in pL0A_0-R Level 0 vector [36] by NEBuilder HiFi assembly (Fig 3A and 3B). We con- firmed that eight out of eight colonies contained the plasmid with DNA insert by colony PCR. Additionally, the correct junction sites in NEBuilder HiFi assembled plasmid were confirmed by sequencing. Subsequently, following the AssemblX procedure, we assembled the resulting Level 0 unit into pL1A-hc / pL1A-lc (A0/AR) Level 1 vector [36] by TAR and NEBuilder HiFi assembly to compare the efficiency of both (Fig 3C and 3D). For TAR, all clones analysed by colony PCR were confirmed to contain the correct insert length, while NEBuilder HiFi assem- bly resulted in less than 60% of clones with the correct insert length. The correct junction sites of TAR assembled plasmid were confirmed by sequencing. In the next step, we tested several cloning and transformation parameters for transferring the assembled expression cassette (Level 1 module) from the AssemblX Level 1 vector to the Plant X-tender expression vector pCAMBIA_ASX (Fig 3E and 3F). Transfer parameters tested were the cloning method, amount of the plasmid backbone, the molar ratio between the plasmid backbone and the insert, transformation method, E. coli strain, transformation efficiency of E. coli and the PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 9 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology Fig 3. Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in HindIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. Validating the Plant X-tender expression vectors (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by PmeI digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into PacI digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I-SceI digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N. benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E. coli and A. tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E. coli and A. tumefaciens, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, LB: left border of T-DNA, RB: right border of T-DNA, HindIII, I-SceI, PacI, PmeI: restriction enzyme recognition sites, URA3: yeast selection marker, ccdB: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector. Fig 3 Functional evaluation of constructed vectors by cloning express Fig 3. Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in HindIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. https://doi.org/10.1371/journal.pone.0190526.g003 Efficient multigene cassettes cloning with Plant X-tender Finally, we tested the usability of Plant X-tender for multigene cloning by introducing two expression cassettes, containing two different reporter genes (i.e. H2BRFP and ECFP), into newly developed Plant X-tender expression vectors. We confirmed the functionality of the sys- tem by transient expression of H2BRFP and ECFP in N. benthamiana (Fig 4). We amplified the p35S::H2BRFP_tNOS expression cassette from the template using primers with appropri- ate 5’ and 3’ extensions to add A0 and A1 homology regions and assembled it in Level 0 vector pL0A_0–1 [36] by NEBuilder HiFi assembly. In the case of the pNOS::ECFP_t35S expression cassette, we amplified each Level 0 subunit from separate templates using primers with over- laps between adjacent subunits and destination plasmid. We assembled the three subunits in Level 0 vector pL0A_1-R [36] by NEBuilder HiFi assembly (Fig 4A and 4B). We used Geno- CAD and the AssemblX webtool to design the construct and the cloning primers. We con- firmed that eight out of eight colonies contained the insert for both Level 0 assemblies by colony PCR. The correct junction sites in the assembled plasmids were confirmed by sequenc- ing. We subsequently assembled both expression cassettes in Level 1 vector pL1A-hc / pL1A-lc (A0/AR) [36] by TAR and NEBuilder HiFi assembly (Fig 4C and 4D). According to the colony PCR results, TAR assembly was confirmed to be more efficient in comparison to NEBuilder HiFi assembly in the conditions tested here. For TAR assembly, all clones were confirmed to contain the correct insert length, while NEBuilder HiFi assembly resulted in less than 70% of clones with the correct insert length in the conditions tested here. The multigene construct consisting of both expression cassettes with flanking homology regions A0/B0 was released from Level 1 vector and cloned into pCAMBIA_ASX vector (Fig 4E and 4F). We evaluated dif- ferent cloning and transformation methods for this step. The lower molar ratio of insert to plasmid was determined to be more efficient. In the tested conditions, NEBuilder HiFi cloning was determined as not applicable in combination with electroporation, while SLiCE could be used in combination with electroporation or heat shock. SLiCE cloning was confirmed to be applicable even in combination with low efficient homemade chemically competent E. coli cells (S6 Table). Validating the Plant X-tender expression vectors We obtained a higher number of colonies when the higher amount of the plasmid was used (150 ng in comparison to 50 ng). Likewise, a higher molar ratio of insert to the plasmid (10: 1 in comparison to 2: 1) resulted in higher transformation efficiency. Our results showed that SLiCE cloning method is applicable even in combination with homemade chemically competent or electrocompetent E. coli with lower transformation efficiency in comparison to expensive commercial ones (S2 and S6 Tables). We further transferred the expression cassette p35S::H2BRFP_tNOS into pK7WG_ASX, pH7WG_ASX and pB7WG_ASX vectors using the most favourable assembly method determined by the pCAMBIA_ASX transfer, SLiCE (Fig 3C–3F). We confirmed the functionality of the constructs by the confocal imaging of H2BRFP upon agroinfiltration of N. benthamiana leaves (Fig 3G–3I). volume of cloning mixture used for transformation (S6 Table). Our results proved SLiCE assembly to be more efficient compared to NEBuilder HiFi assembly when 50 ng of the plas- mid and a 1: 2 molar ratio between the plasmid and the insert were used. We obtained a higher number of colonies when the higher amount of the plasmid was used (150 ng in comparison to 50 ng). Likewise, a higher molar ratio of insert to the plasmid (10: 1 in comparison to 2: 1) resulted in higher transformation efficiency. Our results showed that SLiCE cloning method is applicable even in combination with homemade chemically competent or electrocompetent E. coli with lower transformation efficiency in comparison to expensive commercial ones (S2 and S6 Tables). We further transferred the expression cassette p35S::H2BRFP_tNOS into pK7WG_ASX, pH7WG_ASX and pB7WG_ASX vectors using the most favourable assembly method determined by the pCAMBIA_ASX transfer, SLiCE (Fig 3C–3F). We confirmed the functionality of the constructs by the confocal imaging of H2BRFP upon agroinfiltration of N. benthamiana leaves (Fig 3G–3I). Efficient multigene cassettes cloning with Plant X-tender We confirmed the functionality of the construct by confocal imaging of H2BRFP for the p35S::H2BRFP_tNOS expression cassette and ECFP for the pNOS:: ECFP t35S expression cassette (Fig 4G–4J), upon agroinfiltration of N. benthamiana leaves. ECFP_t35S expression cassette (Fig 4G–4J), upon agroinfiltration of N. benthamiana leaves. Validating the Plant X-tender expression vectors (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by PmeI digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into PacI digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I-SceI digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N. benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E. coli and A. tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E. coli and A. tumefaciens, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, LB: left border of T-DNA, RB: right border of T-DNA, HindIII, I-SceI, PacI, PmeI: restriction enzyme recognition sites, URA3: yeast selection marker, ccdB: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector. Fig 3. Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plan https://doi.org/10.1371/journal.pone.0190526.g003 https://doi.org/10.1371/journal.pone.0190526.g003 10 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology volume of cloning mixture used for transformation (S6 Table). Our results proved SLiCE assembly to be more efficient compared to NEBuilder HiFi assembly when 50 ng of the plas- mid and a 1: 2 molar ratio between the plasmid and the insert were used. Discussion (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, Amp: selection marker conferring ampicillin resistance in E. coli and A. tumefaciens, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, LB: left border of T-DNA, RB: right border of T-DNA, HindIII, I-SceI, PacI, AscI, SbfI, SwaI, FseI, PmeI: restriction enzyme recognition sites, URA3: yeast selection marker, ccdB: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector. https://doi.org/10.1371/journal.pone.0190526.g004 Plant X-tender, a set of tools that complements the AssemblX toolkit [36] and thus enables easier development of plant synthetic biology applications. The Plant X-tender integrates and expands already existing tools, instead of providing a new but isolated cloning procedure. This is especially beneficial for researchers working with different organisms in parallel, as it will enable the use of a single cloning scheme for different organisms, including plants. When designing Plant X-tender expression vectors, attention was focused on providing full flexibility regarding the design of the expression cassettes. The selection of appropriate regula- tory elements is crucial since the repetitive use of the same promoter for expression of multiple genes was shown to be associated with transgene silencing [44,45,46]. In addition, in the AssemblX system, the cloning steps for each assembly reaction are designed in a user-oriented workflow, allowing the user to freely choose the final orientation of modules and thus avoid gene silencing driven by inverted repeats [21,44,47,48]. Moreover, this planning strategy allows the use of linkers between expression cassettes in order to improve expression, folding and/or stability of the proteins when expressing recombinant fusion proteins [49]. Although a few plant expression plasmids for multigene cloning have already been developed, they are rarely fully flexible regarding regulatory elements, orientation of expression cassettes and addition of linkers between them, if required. Discussion (F) Assembly of two expression cassettes and yeast selection marker (URA3) into HindIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N. benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into HindIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using PmeI. (D) Assembly of expression cassettes into PacI digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I-SceI homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker (URA3) into HindIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N. benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A. tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, Amp: selection marker conferring ampicillin resistance in E. coli and A. tumefaciens, Kan: selection marker conferring kanamycin resistance in E. coli and A. tumefaciens, LB: left border of T-DNA, RB: right border of T-DNA, HindIII, I-SceI, PacI, AscI, SbfI, SwaI, FseI, PmeI: restriction enzyme recognition sites, URA3: yeast selection marker, ccdB: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector. https://doi.org/10.1371/journal.pone.0190526.g004 pCAMBIA_ASX_multigene (upper panel) or with empty A. tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. Discussion There is a growing need for simple but flexible cloning strategies, which will allow high throughput approaches in plant synthetic biology [1] and biotechnology [43]. We developed PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 11 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology Fig 4. Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cl pCAMBIA_ASX and introduced into N. benthamiana. (A-F) Scheme of cloning procedure. (A) Amp expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homol p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen L Fig 4. Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N. benthamiana. (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in Fig 4. Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N. benthamiana. (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in 12 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into HindIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using PmeI. (D) Assembly of expression cassettes into PacI digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I-SceI homing endonuclease, cutting outside the homology regions A0 and B0. Discussion Moreover, since our cloning approach is based on rare-cutting restriction enzymes in combination with overlap-based methods, it is sequence independent and thus avoids elimination of unwanted restriction sites. However, the issue of introducing sequence errors and time-consuming parts domestication could nowadays be avoided by DNA synthesis. To avoid possible confusion, one should note different nomenclature used for the naming of the assembly levels in AssemblX and Type IIS-mediated assembly methods. In AssemblX, the subunits refer to basic elements below the transcriptional unit level (e.g. promoter, CDS, terminator) which are assembled in Level 0 vectors. Therefore, Level 0 refers to single gene cas- settes, while Level 1 refers to multigene constructs. On the other hand, Level 0 in MoClo and GoldenBraid [25, 26] usually refers to basic elements, single gene cassettes are Level 1 and mul- tigene constructs are Level 2 assemblies. Another advantage of the AssemblX-based approach presented here, lies in the flexibility to select the most appropriate method among several overlap-based assembly methods according to the user requirements. To determine the most efficient assembly method and cloning condi- tions, we evaluated different overlap-based assembly methods for cloning in various condi- tions. Cloning efficiency of 100% in the case of Level 0 assembly by NEBuilder HiFi for all tested expression cassettes could be attributed to optimized homology regions present in Level 0 vectors and ccdB counterselection. In contrast, using TAR assembly method for Level 1 assembly resulted in higher cloning efficiency, if compared to NEBuilder HiFi assembly method in tested conditions and for the selected inserts. Our results are consistent with the results of de Kok et al. (2014) [54], who obtained higher cloning efficiency by TAR assembly in comparison to a highly promoted commercial assembly kit (Gibson assembly). In the last step of the cloning strategy followed here, we assembled the constructs into Plant X-tender expres- sion vectors. Results from the assembly of p35S::H2BRFP_tNOS expression cassette (Fig 2) are in agreement with the results from Zhang et al. (2012) [33], who claimed that increasing the amount of the insert at the same vector amount as well as increasing the amount of the vector and the insert yield higher cloning efficiencies. In contrast, results obtained by the assembly of p35S::H2BRFP_tNOS + pNOS::ECFP_t35S multigene construct (Fig 3) speak in favour of lower molar ratio. The transformation of highly efficient chemically competent E. Discussion With these problems in mind, Plant X-tender expression vectors are available as empty backbones. Another advantage of the Plant X-tender vector system presented here is the flexibility in the choice of a selection marker. Since there are reports on interactions between selection markers and gelling agents [50] as well as connections between selection markers and regener- ability [51], our set of Plant X-tender expression vectors, including three expression vectors with the same backbone (pK7WG_ASX, pH7WG_ASX and pB7WG_ASX) but different selec- tion markers, enables the selection of the most appropriate selection marker matching the tar- get tissue as well as transformation media. According to our experience, the backbone can affect the expression pattern as well as transformation efficiency. Therefore, we constructed an additional Plant X-tender expression vector, pCAMBIA_ASX, with the intention of providing flexibility in terms of backbones. One major advantage of the novel vector set is the inclusion of the ccdB gene (Fig 1), which precludes growth of non-recombinant clones [52] and makes the screening easier and more efficient. The other is the ability to assemble scar-free constructs, which is especially important PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 13 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology since the introduced sequences can affect transgene function [27] and was also implemented in recently developed COLORFUL-circuit vectors [21]. Although restriction enzyme-based methods derived from the Golden Gate strategy share a number of characteristics that encourage their adoption by the scientific community, they have one disadvantage in common. All parts are required to be devoid of any restriction sites used in the assembly. This could pose a problem, as Ghareeb et al. (2016) [21] noticed a high occurrence of restriction enzyme recognition sites used in Golden Gate technology in several plant genomes. To overcome this downside of restriction enzyme-based methods, Ghareeb et al. (2016) [21] exploited the use of a newly discovered rare-cutting restriction enzyme. Despite the utility and advantages of the system, annealing of short sticky ends may have limited affinity and specificity when assembling multiple DNA parts in one pot (reviewed in [53]). Since the cloning approach presented here is based on overlap-based cloning methods which utilize longer overlapping homology regions the affinity and speci- ficity issue is overcome. PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Discussion coli with a SLiCE assembly mixture containing 150 ng of plasmid and a 2-fold molar excess of insert resulted in higher number of colonies in comparison to the higher molar ratio (1: 5) of vector and insert (S6 Table). Our results from the multigene assembly are consistent with the results from Okegawa and Motohashi (2015) [55] and Motohashi (2015) [56], who observed a signifi- cant decrease of cloning efficiency by increasing molar ratio of vector to insert when using SLiCE assembly. 14 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Plant X-tender: An extension of the AssemblX system for plant synthetic biology High cloning efficiency could be attributed to optimized homology regions [36] introduced in the Plant X-tender expression vectors and to ccdB counterselection. Although SLiCE has originally been used in combination with commercially available highly competent E. coli cells [33], our results showed that SLiCE is applicable even in combination with low efficient home- made chemically competent or electrocompetent E. coli (S6 Table), which is in agreement with published data from Motohashi (2015) [56] and Messerschmidt et al. (2016) [39]. However, in the case of more demanding cloning settings or low plasmid concentrations, highly competent E. coli cells are recommended. The cloning strategy presented here is designed in three successive cloning levels, allowing multiple parallel assemblies resulting in increasingly complex structure. Although Plant X-ten- der for AssemblX enables the introduction of large constructs into plant expression vectors, the stability of expression in the plant genome is still highly unpredictable and low. However, as plant genome engineering using synthetic biology is developing fast [57], it is most likely that this problem will be reduced, for example in combination with CRISPR/Cas9 guided insertion of constructs into the plant genome. Taken together, we here present Plant X-tender that enables easy cloning of multigene cas- settes and their expression in plant cells as an extension of the AssemblX toolbox [36]. The sys- tem is highly flexible and fast thus allowing easier introduction of synthetic biology into plant science. S2 Table. Transformation efficiencies of homemade chemically competent and electro- competent E. coli. (PDF) S3 Table. Oligonucleotides used for the amplification of Level 0 subunits by PCR. A) Oligo- nucleotides used for the amplification of Level 0 subunit for the assembly of expression cassette p35S::H2BRFP_tNOS. B) Oligonucleotides used for the amplification of Level 0 subunits for the assembly of multigene construct p35S::H2BRFP_tNOS + pNOS::ECFP_t35S. Nucleotides in bold represent overlaps between adjacent parts, e.g. homology regions of destination plas- mid (Level 0 AssemblX vector) or sequence of adjacent modules. (PDF) S3 Table. Oligonucleotides used for the amplification of Level 0 subunits by PCR. A) Oligo- nucleotides used for the amplification of Level 0 subunit for the assembly of expression cassette p35S::H2BRFP_tNOS. B) Oligonucleotides used for the amplification of Level 0 subunits for the assembly of multigene construct p35S::H2BRFP_tNOS + pNOS::ECFP_t35S. Nucleotides in bold represent overlaps between adjacent parts, e.g. homology regions of destination plas- mid (Level 0 AssemblX vector) or sequence of adjacent modules. (PDF) S4 Table. List of primers for sequencing and colony PCR. (PDF) Supporting information S1 Table. Primers used for the construction of Plant X-tender expression vectors. (PDF) S1 Table. Primers used for the construction of Plant X-tender expression vectors. (PDF) S2 Table. Transformation efficiencies of homemade chemically competent and electro- competent E. coli. (PDF) PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 Acknowledgments The authors thank Prof. Dr. Bernd Mueller-Roeber (University of Potsdam, Germany) for critical reading of the manuscript, Prof. Dr. Jim Haseloff and Dr. Ferna´n Federici (University of Cambridge, UK) for providing the plasmid containing H2BRFP, Prof. Dr. Jacek Henning (Polish Academy of Sciences, Poland) for providing the plasmid containing p19 silencing suppressor, Dr. Maria Pla and Dr. Anna Nadal (University of Girona, Spain) for providing pCAMBIA1300. The authors are grateful to Dr. David Dobnik for the assistance with image analysis, Vid Puzˇ for the assistance with figures preparation and Barbara Dusˇak and Lena Hochrein for the technical support. S5 Table. Primers for sequencing and colony PCR. (PDF) S6 Table. Optimization of assembly and transformation methods for the insert cloning into Plant X-tender expression vectors. Expression cassette p35S::H2BRFP_tNOS (insert 1) and multigene construct p35S::H2BRFP_tNOS + pNOS::CFP_t35S (insert 2) were separately assembled into pCAMBIA_ASX to determine optimal conditions for insert cloning into the expression vectors. Bottom two rows represent a negative control (the plasmid backbone with- out the insert). m (ng): amount of pCAMBIA_ASX expression vector, AM: assembly method, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, MR: molar ratio between the plasmid backbone and the insert, TM: transformation method, E. coli: competent E. coli used for transformation, HM: homemade TOP10 chemically competent PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 15 / 19 Plant X-tender: An extension of the AssemblX system for plant synthetic biology E.coli, HM: homemade TOP10 electrocompetent E.coli, C: commercial TOP10 chemically competent E.coli, T (μl): volume of assembly mixture used for transformation, number of colo- nies: the number of colonies grown after overnight incubation, cloning efficiency: the ratio between the number of clones with the correct insert length confirmed by colony PCR and the number of colonies subjected to colony PCR. (PDF) Data curation: Tjasˇa Lukan, Fabian Machens. Funding acquisition: Tjasˇa Lukan, Katrin Messerschmidt, Kristina Gruden. Funding acquisition: Tjasˇa Lukan, Katrin Messerschmidt, Kristina Gruden. Funding acquisition: Tjasˇa Lukan, Katrin Messerschmidt, Kristina Gruden. Investigation: Tjasˇa Lukan, Fabian Machens, Katrin Messerschmidt. Investigation: Tjasˇa Lukan, Fabian Machens, Katrin Messerschmidt. Methodology: Tjasˇa Lukan, Fabian Machens, Katrin Messerschmidt, Kristina Gruden. Resources: Katrin Messerschmidt, Kristina Gruden. Methodology: Tjasˇa Lukan, Fabian Machens, Katrin Messerschmidt, Kristina Gruden. Resources: Katrin Messerschmidt, Kristina Gruden. Methodology: Tjasˇa Lukan, Fabian Machens, Katrin Messerschmidt, Kristina Gruden. Resources: Katrin Messerschmidt Kristina Gruden Software: Tjasˇa Lukan, Fabian Machens, Anna Coll. Software: Tjasˇa Lukan, Fabian Machens, Anna Coll. Supervision: Katrin Messerschmidt, Kristina Gruden. Validation: Tjasˇa Lukan, Fabian Machens. Validation: Tjasˇa Lukan, Fabian Machens. Validation: Tjasˇa Lukan, Fabian Machens. Visualization: Tjasˇa Lukan, Fabian Machens, Anna Coll, Sˇpela Baebler. Writing – original draft: Tjasˇa Lukan, Anna Coll, Kristina Gruden. Writing – review & editing: Tjasˇa Lukan, Fabian Machens, Anna Coll, Sˇpela Baebler, Katrin Messerschmidt, Kristina Gruden. Author Contributions Conceptualization: Tjasˇa Lukan, Fabian Machens, Anna Coll, Katrin Messerschmidt, Kristina Gruden. Data curation: Tjasˇa Lukan, Fabian Machens. PLOS ONE \| https://doi.org/10.1371/journal.pone.0190526 January 4, 2018 References References 1. Liu W, Stewart CN. Plant synthetic biology. Trends Plant Sci. 2015; 20: 309–317. https://doi.org/10. 1016/j.tplants.2015.02.004 PMID: 25825364 2. Giuliano G. Plant carotenoids: Genomics meets multi-gene engineering. Curr Opin Plant Biol. 2014; 19 111–117. https://doi.org/10.1016/j.pbi.2014.05.006 PMID: 24912125 3. Lu Y, Rijzaani H, Karcher D, Ruf S, Bock R. 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https://openalex.org/W3135181042	https://www.researchsquare.com/article/rs-223105/latest.pdf	English	null	Unsteady hybrid-nanofluid flow comprising ferrousoxide and CNTs through porous horizontal channel with dilating/squeezing walls	Scientific reports	2,021	cc-by	10,134	Muhammad Bilal (  m.bilal@math.uol.edu.pk ) Muhammad Bilal (  m.bilal@math.uol.edu.pk ) The University of Lahore, Gujrat Campus, Gujrat, Pakistan Hamna Arshad The University of Lahore, Gujrat Campus, Gujrat, Pakistan Hamna Arshad The University of Lahore, Gujrat Campus, Gujrat, Pakistan Hamna Arshad The University of Lahore, Gujrat Campus, Gujrat, Pakistan Muhammad Ramzan Research Article Posted Date: February 24th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-223105/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 1 a. Corresponding author e-mail address: m.bilal@math.uol.edu.pk Abstract The key objective of the present research is to examine the hybrid magnetohydrodynamics (MHD) nanoﬂuid (Carbon-nanotubes and ferrous oxide-water)CNT −Fe −3O −4/H −2O ﬂow into a horizontal parallel channel with thermal radiation through squeezing and dilating porous walls. The parting motion is triggered by the porous walls of the channel. The ﬂuid ﬂow is time-dependent and lam- inar. The channel is asymmetric and the upper and lower walls are distinct in temperature and porosity. With the combination of nanoparticles of Fe3O4 and single and multi-wall carbon nanotubes, the hybrid nanoﬂuid principle is exploited. By using the similarity transformation, the set of partial diﬀerential equations (PDEs) of this mathematical model, governed by momentum and energy equations, is reduced to corresponding ordinary diﬀerential equations (ODEs). A very simple numerical approach called the Runge-Kutta system of order four along with the shooting technique is used to achieve the solutions for regulating ODEs. MATLAB computing software is used to create temperature and velocity proﬁle graphs for various emerging parameters. At the end of the manuscript, the main conclusions are summarized. Keywords: Hybrid-nanoﬂuid; carbon nanotubes; Horizontal porous channel, magnetohydro- dynamics; thermal radiation Unsteady hybrid-nanoﬂuid ﬂow comprising ferrousoxide and CNTs through porous horizontal channel with dilating/squeezing walls Muhammad Bilal1,a, Hamna Arshad1, Muhammad Ramzan2 1. Department of Mathematics, The University of Lahore, Gujrat Campus, Gujrat, Pakistan. 2. Bahria University, Islamabad, Pakistan, a. Corresponding author e-mail address: m.bilal@math.uol.edu.pk Muhammad Bilal1,a, Hamna Arshad1, Muhammad Ramzan2 1. Department of Mathematics, The University of Lahore, Gujrat Campus, Gujrat, Pakistan. 2. Bahria University, Islamabad, Pakistan, a. Corresponding author e-mail address: m.bilal@math.uol.edu.pk a. Corresponding author e-mail address: m.bilal@math.uol.edu.pk 1 Introduction We are living in the age of machines. To improve the mechanism and functioning of the cooling system of these machines, a new technology of insertion of the nano-sized particles in the base ﬂuid was introduced few years ago. Nanoﬂuids are used to enhance the heat transfer rate and thermal conductivity of the base ﬂuid. Normally base ﬂuids are tri-ethylene-glycol, water, refriger- ants, ethylene, lubricants and oils, polymeric solutions, and bio-ﬂuids. The common nanoparticle such as copper, gold, silver, alumina, titania, zirconia, Al2O3, CuO, metal carbides SiC, metal nitride AIN, SiN, the carbon in the forms of graphite, diamond, carbon nanotubes, and some other functionalized nanoparticles are used. Nanoﬂuids have unique properties like homogeneity, high thermal conductivity at lower nanoparticle aggregation, stability for a long period, and very less clogging in ﬂow passages. That’s why these ﬂuids have large number of applications in the electrical appliances such as micro-electromechanical systems, cooling of microchips, microreac- tors and ﬂuidic digital display devices, etc. Nanoﬂuids are also useful in heating and cooling of buildings, heat interchangers, transportation industry, sensing, microﬂuidics, lubricant systems, pharmaceutical processes, nano cryosurgery, refrigeration of electronic apparatus, cancer thera- peutics, nano-drug delivery, cryopreservation, and imaging. Ferroﬂuids are a famous kind of nanoﬂuid formed by the insertion of nanoparticles containing iron like Cobalt ferrite, Hematite, and magnetite having a size range of 5 −15nm coated with a layer of surfactant in the base ﬂuid. Ferroﬂuids have dual properties, as it acts as liquid and mag- netic solids at the same time. If the magnetic ﬁeld applied across the ferroﬂuids, we can control the ﬂow of ﬂuid and the rate of heat transfer. Ferroﬂuids have vast applications in the industrial area such as vacuum chambers in the semiconductor industry, in gravity gradient satellites as viscous dampers, rotating X −ray anode generators, accelerometer, energy conversion devices, to remove the dust particles in high-speed computers and to eliminate the other impurities from biomedical industries. Zaheer and Mariam [1] presented the stagnation point ﬂow of ferroﬂuids having het- erogeneous and homogeneous reaction along with non-linear slip condition. They reported that the velocity component is higher for ferroﬂuid as compared to the pure base ﬂuid. Abid et al. [2] scrutinized the inertial and microstructure properties of ferroﬂuids in the presence of thermal con- ductivity. They also observed that the velocity of ferroﬂuids is much greater than the simple base ﬂuid. 1 2 2 1 Introduction The MHD ﬂow of ferroﬂuids with heat ﬂux along with the stretching cylinder was studied by Qasim et al. [3]. During their work, they found that in the presence of a magnetic ﬁeld, the heat transfer rate of magnetic nanoparticles is lower than the nonmagnetic nanoparticles Al2O3. The electrically conducting ferroﬂuid ﬂow in the magnetic ﬁeld was studied by Maria et al. [4] over a curved stretching disk. They assume that ferroﬂuid velocity is a decreasing function of the volume fraction of the nanoparticle. Khan et al. [5] experimented together with heat ﬂux and heat viscos- ity on heat transfer and magnetohydrodynamic ferroﬂuid ﬂow. They gave the results that with the increasing volume fraction of nanoparticles, the rate of heat transfer and skin friction improves. The most eﬃcient nanoparticles are carbon nanotubes. Carbon nanotubes (CNT) are the tubular structures of carbons, also known as graphene sheets. Single Wall carbon nanotubes 3 (SWCNT’s) structure is a cylindrical shape which composed of a single layer of graphene particles holding all the atoms at one place and has 0.5 −1.5nm diameter. MWCNT is a collection of graphene layer interconnected tubes of exponentially rising diameter. CNT are highly eﬃcient nanotubes as they have six times better mechanical, physicochemical, and thermal properties as compared to other nanoparticles/nanotubes. CNTs play an important role in the ﬁeld of optics, engineering, chemical production, material science, and microelectronic cooling. Hayat et al. [6] discussed the DarcyForchheimer ﬂow of water-based SWCNT and MWCNT over a rotating disk which is convectively heated. They reported that the velocity parameter of the ﬂuid is higher with the increase in SWCNT and MWCNT volume fraction. The electromagnetohydrodynamic (EMHD) ﬂow of kerosene oil and water base nanoﬂuid ﬂow over a stretching layer in the presence of thermal radiation was demonstrated by Zahir et al. [7]. They provided the results that a high density of CNTs in nanoﬂuid possesses an increase in the rate of heat transfer. The eﬀect of MHD, suction/injection, and chemical reaction for the mass and heat transfer ﬂow of water-based MWCNT and SWCNT over a porous vertical conic segment was explored by Sreedevi et al. [8]. They presented the knowledge that with an improvement in the volume fraction, the rate of heat transfer increases and that the increase in heat transfer rate in water-based MWCNT nanoﬂuid is greater than that of SWCNT. 1 Introduction Owing to the advantages of the nanoﬂuids, there is still some limitations/drawback of nanoﬂu- ids, i.e. a single type of nanoparticles are not able to enhance all required properties of the base ﬂuid. To overcome this problem, hybrid nanoﬂuids have been introduced. Hybrid nanoﬂuids are obtained by the insertion of two or more nanoparticles in the base ﬂuid. In hybrid nanoﬂuid, the physical and chemical properties of diﬀerent materials combine simultaneously and provide a homogeneous mixture. These nanoﬂuids have improved the impact of thermal conductivity and heat transfer rate which reduced the cost in industrial areas. Ghadikolaei and Gholinia [9] worked on heat transfer and natural convection ﬂow of hybrid nanoﬂuid near a vertical porous stretching sheet. They showed the direct relation of nanoﬂuid velocity with Grashof number and inverse relation with the magnetic ﬁeld parameter. Iqbal et al. [10] examined the collective im- pacts of thermal radiation and Hall current through a rotating vertical channel in the existence of transverse constant magnetic ﬁeld and hybrid-nanoﬂuid. Sajjad et al. [11] developed the Lattice Boltzmann (LBM) program for multi-wall carbon nanotubes to study the heat transfer impact of hybrid-nanoﬂuid (MWCNT −Fe3O4 water). Their results demonstrate that by increasing the magnetic ﬁeld, the heat transfer rate is reduced and this reduction is due to the Darcy number and increased because of the porosity parameter. An LBM numerical analysis used by Yuan Ma et al. [12] to analyze the hybrid nanoﬂuid ﬂow of the MHD Ag −MgO/water for heat transfer analysis across the tube. They noticed that applying Ag −MgO to water raises the rate of heat transfer and that the rate of heat transfer decreases by strengthening the Hartmanns number. During this analysis, they found that the production of entropy in the nanoﬂuid Al2O3/H2O is smaller than the hybrid nanoﬂuid Cu −Al2O3/H2O. Hayat and Nadeem [14] were evaluated the ﬂow of hybrid nanoﬂuid (Ag −CuO/water) in the presence of thermal radiation over a linear stretching layer. They concluded that the hybrid nanoﬂuid improves the heat transfer rate and 4 distribution of temperature. The normal convection heat transfer ﬂow of hybrid nanoﬂuid was analyzed by Mehryan et al. [15] in a complex porous T-shaped cavity with a cold upper and a hot bottom wall using a numerical technique. They argue that the magnetic ﬁeld viscosity param- eter decreases the rate of heat transfer inside the solid and liquid phases. 1 Introduction The two-dimensional MHD ﬂow and heat transfer of Hybrid nanoparticles Cu−Al2O3/H2O suspended in a micropolar dusty ﬂuid were reviewed by Ghadikolaei et al. [16] using the numerical technique RK of order 5. Zouhaier and Aﬁf [17] reported under the prompt of magnetic ﬁeld on the phenomena of induced convection ﬂow of hybrid nanoﬂuid (Cu −Al −2O −3/H −2O). They showed that with the low Hartmann number, the increase in heat transfer is visible. The emission of electromagnetic waves from a heated surface to the surroundings in every direction is known as thermal radiation. Thermal radiations travel towards the absorption point with the speed of light. The thermal radiation and Hall current eﬀect on mixed convection MHD rotating ﬂuid ﬂow in a porous vertical channel were combinedly studied by Singh and Pathak [18]. In an asymmetric channel that is tapered, Kothandapania and Prakash [19] technically scruti- nized the thermal radiation eﬀect as well as the magnetic ﬁeld on the peristaltic motion of the Williamson ﬂuid. Theoretically, Satya [20] studied the thermal radiation and heat generation eﬀect on mixed convection unstable ﬂow through a porous and wavy vertical channel of the electrically conducting incompressible viscous ﬂuid. Theoretically, Saswati and Rita [21] calculate the thermal radiation and Hall current eﬀect in a porous revolving channel on the MHD rotating ﬂow of the elastic-viscous ﬂuid.The eﬀect of thermal radiation on the ﬂow of viscous ﬂuid in an asymmetric deformable horizontal porous channel was studied by Naveed et al. [22]. There are many practical examples of ﬂuid ﬂow in the parallel deformable channels such as res- piratory systems, coolant circulation, interbody ﬂuid transportation, aerospace engineering, and industrial cleaning procedures, etc. Yang at al. [23] worked on MHD and heat transfer electroos- motic ﬂow of a ﬂuid in a microchannel of rectangular shape. Guillermo at al. [24] combined the eﬀects of entropy generation, thermal radiation, hydrodynamic slip, and MHD ﬂow of a nanoﬂuid in a porous horizontal microchannel. Zhao and Yang [25] scrutinized the electroosmotic ﬂow of the power-law (non-Newtonian) ﬂuid in a cylindrical microchannel. Khan and Naz [26] investigated the mass and transfer ﬂow of three-dimensional second-grade ﬂuid in a porous channel. They reported that the velocity of the ﬂuid increases when there is no injection of ﬂuid but at some point, velocity has an inverse relation with the suction parameter of the ﬂuid. Rauf at al. 1 Introduction [27] discussed the thermally radiative mix convective nanoﬂuid ﬂow in a stretchable porous channel. Idowu at al. [28] illustrated the thermal conductivity along with mass and heat transfer on the oscillatory MHD ﬂow of the Jeﬀery ﬂuid in a porous channel. Xinhui at al. [29] proposed research on the incompressible viscous ﬂow of Newtonian ﬂuid in an asymmetric porous channel. They investigate the rate of mass and heat transfer. Sheikholeslami [30] studied the ﬂow of water- based CuO nanoﬂuid in a porous horizontal channel in the presence of a magnetic ﬁeld. Aksoy and Pakdemirli [31] obtained the approximate analytical solution of third-grade ﬂuid ﬂow in a parallel-plate porous channel. Bataineh at al. [32] analyzed the rate of heat transfer and MHD 5 ﬂow of second-grade ﬂuid in a channel having porous walls. They used the famous numerical tech- nique RK4 and homotopy analysis method to get the graphical results of velocity and temperature parameters. Inspiring from the above-referred literature review, it is noticed that the study of Hybrid nanoﬂuid with Fe2O3, and SWCNT, MWCNTs through a horizontal parallel porous channel with the mag- netic ﬁeld is still missing and owning to its importance in many engineering and industrial appli- cations, it is addressed in this article. Due to the high-temperature phenomenon, the impact of thermal radiation is also assumed. Governing equations are modeled under diﬀerent assumptions and are solved numerically by the shooting method. Runge-Kutta method of order four with New- ton’s method is used eﬀectively for the graphical results. We believe that is study will uniquely contribute to predicting the signiﬁcance of hybrid nanoﬂuid through the horizontal porous channel. 2 Mathematical formulation − 1 (ρCp)hnf ∂ ∂ex + ∂ ∂ey qrad, (4) where where qrad = 4 ∗σ eT 3 3ak ∂eT ∂ey qrad = 4 ∗σ eT 3 3ak ∂eT ∂ey qrad = 4 ∗σ eT 3 3ak ∂eT ∂ey subject to the boundary conditions at ey = −a et : eT −eTl = 0, ev = −evl = −Al ·a, eu = 0, at ey = a et : eT −eTu = 0, ev = −evu = −Au ·a, eu = 0,      (5) (5) where eu and ev are the velocity components in the ex and ey direction respectively, ep the pressure, µhnf, σhnf ρhnf the dynamic viscosity, electric conductivity, density of hybrid-nanoﬂuid, B◦, qrad, eT, Cp, are the magnetic ﬁeld strength, thermal radiation, the temperature of the ﬂuid and the speciﬁc heat at constant pressure, σ∗is Boltzmann constant, eTl and eTu are the temperature of the lower and upper wall respectively, ak is for the mean absorbtion constant, Al, Au the permeability of lower and upper wall respectively. Taking the partial derivatives of Eqs. (2) and (3) with respect to ey and ex respectively and eliminating the pressure gradient term, We get: µhnf ∂3eu ∂ex2∂ey + ∂3eu ∂ey3 −ρhnf ∂2eu ∂et∂ey + eu ∂2eu ∂ex∂ey + ∂eu ∂ex ∂eu ∂ey + ev ∂2eu ∂ey2 + ∂eu ∂ey ∂ev ∂ey −σhnfB2 ◦ ∂eu ∂ey = µhnf ∂3ev ∂ey2∂ex + ∂3ev ∂ex3 −ρhnf ∂2ev ∂et∂ex + eu∂2ev ∂ex2 + ∂ev ∂ex ∂eu ∂ex + ev ∂2ev ∂ex∂ey + ∂ev ∂ey ∂ev ∂ex . 2 Mathematical formulation We considered a rectangular-shaped channel that is semi-inﬁnite and protected by a dense pliable sheet at the leading edge. An electrically conducting unsteady ﬂuid ﬂows between these plates. Both walls have diﬀerent porosity factors and squeeze or dilate with uniform rate. At the origin of the channel, the middle portion of the cylinder is set as shown in the Fig. 1. Tl and Tu are symbolized as the lower and upper wall temperature. Hybrid nanoﬂuid (CNT −Fe3O4/H2O) ﬂows through the channel which is viscid and incompressible. The physical ﬂow properties of hybrid-nanoﬂuid (CNT −Fe3O4/H2O) depends on time. Initially, the ferrous oxide nanoparti- cles (Fe3O4) of volume fraction (ϕ1 = 0.1) dissolved in the carrier ﬂuid (H2O) which form the water-based Ferro-nanoﬂuid (Fe3O4/H2O). After that, hybrid nanoﬂuid (CNT −Fe3O4/H2O) is formed by adding a diﬀerent volume fraction of CNT (ϕ2) into the initially formed Ferro-ﬂuid (Fe3O4/H2O). Also assumed that the walls of the rectangular channel are porous and ﬂuid in- jection and suction take place due to dilation and squeezing of the walls. The porosity of the upper and lower walls was diﬀerent and the middle is at the origin position of the channel. The Figure 1: Geometry of the Problem. Figure 1: Geometry of the Problem. Figure 1: Geometry of the Problem. mathematical representation of the mass, momentum and energy conservation of above explained mathematical representation of the mass, momentum and energy conservation of above explained 6 model are determined as follows: ∂ev ∂ey + ∂eu ∂ex = 0, (1) ∂ep ∂ex = µhnf ∂2eu ∂ex2 + ∂2eu ∂ey2 −ρhnf ∂eu ∂et + ∂eu ∂ex eu + ∂eu ∂ey ev −σhnfB2 ◦eu, (2) ∂ep ∂ey = µhnf ∂2ev ∂ex2 + ∂2ev ∂ey2 −ρhnf ∂ev ∂et + ∂ev ∂ex eu + ∂ev ∂ey ev , (3) ∂eT ∂et + ∂eT ∂ex eu + ∂eT ∂ey ev = δhnf ∂2 eT ∂ex2 + ∂2 eT ∂ey2 ! − 1 (ρCp)hnf ∂ ∂ex + ∂ ∂ey qrad, (4) ∂eT ∂et + ∂eT ∂ex eu + ∂eT ∂ey ev = δhnf ∂2 eT ∂ex2 + ∂2 eT ∂ey2 ! 2 Mathematical formulation (6) (6) In above equations, volumetric heat capacity (ρCp)hnf, thermal diﬀusicity δhnf = khnf (ρCp)hnf , ther- mal conductivity khnf and electric conductivity σhnf of the hybrid nanoﬂuid are deﬁned as: In above equations, volumetric heat capacity (ρCp)hnf, thermal diﬀusicity δhnf = khnf (ρCp)hnf , ther- mal conductivity khnf and electric conductivity σhnf of the hybrid nanoﬂuid are deﬁned as: ρhnf µhnf = 1 νhnf , σhnf σbf = σCNT + 2σbf −2ϕ2(σbf −σCNT ) σCNT + 2σbf + ϕ2(σbf −σCNT ) , δhnf = khnf (ρCp)hnf , σbf σf = σMS + 2σf −2ϕ1(σf −σMS) σMS + 2σf + ϕ1(σf −σMS) , µhnf = µf (1 −ϕ1)5/2 (1 −ϕ2)5/2 , ρhnf = ρf (1 −ϕ2) 1 −(1 −ρMS ρf )ϕ1 + ϕ2 ρCNT ρf , (ρCp)hnf (ρCp)f = (1 −ϕ2) " 1 − 1 −(ρCp)MS (ρCp)f ! ϕ1 # + (ρCp)CNT (ρCp)f ϕ2, khnf kbf = 1 −ϕ2 + 2ϕ2 kCNT (kCNT −kbf ) ln kCNT +kbf 2kbf 1 −ϕ2 + 2ϕ2 kbf (kCNT −kbf ) ln kCNT +kbf 2kbf , kbf kf = kMS + (m −1) kf −(m −1) ϕ1 (kf −kMS) kMS + (m −1) kf + ϕ1 (kf −kMS) ,                                                      (7) (7) 7 In the above expression (7), kf and kbf are respectively the thermal conductivity of base ﬂuid H2O and Fe2O3-nanoﬂuid. Here, m must be chosen 3 for the spherical nanoparticles. Also, kMS and kCNT signiﬁes the thermal conductivity of Fe2O3 and CNTs respectively. ϕ1, ϕ2, (CP )CNT , ρCNT , (Cp)f, ρf, µf are respectively indicates the volume fraction of Fe2O3, volume fraction of CNTs, speciﬁc heat at constant press of CNTs, density of CNTs, speciﬁc heat at constant pressure of base ﬂuid, density of base ﬂuid, and dynamic viscosity of the carrier ﬂuid. The electric conductivity of Fe2O3-nanoﬂuid and base ﬂuid are represented by σbf and σf respectively. Table 1 shows the thermal and physical properties of carrier ﬂuid (H2O), nanoparticles Al2O3 and Carbon nanotubes. 2 Mathematical formulation Applying the similarity transformation ξ = ey a et , eu = νf exF ξ ξ, et a2 et , ev = −νfF ξ, et a et , T(ξ) = eT −eTu eTl −eTu . (8) (8) The similarity transformation satisﬁes the continuity Eq. (1) identically, while the momentum Eq. (6) and energy Eq. (4) got the following non-dimensional form; F ξξξξ + νf νhnf α(3F ξξ + ξF ξξξ) −F ξF ξξ + FF ξξξ −F ξξMD1D2 − a2 νhnf F ξξet = 0 (9) −khnf kf T ′′ −Pr(ρCp)hnf (ρCp)f (αξ + FR) T ′ + Rd " (1 + (Tr −1)T(ξ))3T ′′ +3(1 + (Tr −1)T(ξ))2T ′2 (Tr −1) # = 0 (10) F ξξξξ + νf νhnf α(3F ξξ + ξF ξξξ) −F ξF ξξ + FF ξξξ −F ξξMD1D2 − a2 νhnf F ξξet = 0 (9) (9) −khnf kf T ′′ −Pr(ρCp)hnf (ρCp)f (αξ + FR) T ′ + Rd " (1 + (Tr −1)T(ξ))3T ′′ +3(1 + (Tr −1)T(ξ))2T ′2 (Tr −1) # = 0 (10) In the above equations, the wall’s deformation rate is α = a ·a/νf and its value is considered to be positive for the dilating channel. Xinhui et al. [29] suggested that for the uniformity of α in time, a similar solution w.r.t. both time and space can be accomplished by choosing α a constant and it leads F ξξet = 0. To ﬁgure out this condition, the wall’s deformation rate (expansion ratio) α must be prescribed by the channel’s initial hight. F ξξξξ + νf νhnf α(3F ξξ + ξF ξξξ) −F ξF ξξ + FF ξξξ −F ξξMD1D2 = 0 (11) (11) The boundary conditions are: The boundary conditions are: at ey = −a et : F ξ, et \|ξ=−1 = Rl, F ξ ξ, et \|ξ=−1 = 0, T a−1 et ey \|ξ=−1 = 1, at ey = a et : T a−1 et ey ξ=1 = 0, F ξ, et \|ξ=1 = R, F ξ ξ, et \|ξ=1 = 0   (12) (12) In Eq. (12), Rl = eνla νf and R = eνua νf are respectively the Reynolds number with reference to the top and bottom wall of the horizontal channel. These are negative for suction case whereas positive for the injection case. 2 Mathematical formulation The local Nusselt number, (rate of heat transfer) is deﬁned as: The local Nusselt number, (rate of heat transfer) is deﬁned as: The local Nusselt number, (rate of heat transfer) is deﬁned as: The local Nusselt number, (rate of heat transfer) is deﬁned as: Nu = − ak−1 f eTl −f Tu khnf ∂eT ∂y ! y=0 (18) (18) The deﬁned transformation can convert the above expression into dimensionless form. The local Nusselt number for the upper and lower wall of the channel are: The deﬁned transformation can convert the above expression into dimensionless form. The local Nusselt number for the upper and lower wall of the channel are: Nulower = −T ′ (−1) khnf kf , (19) Nuupper = −T ′ (1) khnf kf . (20) (19) (20) 2 Mathematical formulation Implementing the following scale variable for the further simpliﬁcation of the governing Eq. (11) and boundary conditions (12). u = ·a −1 u, v = ·a −1 v, x = ·a −1 x, F = FR (13) (13) ransformation (13) yields the following equation This transformation (13) yields the following equation This transformation (13) yields the following equation This transformation (13) yields the following equation F (iv) + λ1{α(3F ′′ + ξF ′′′) −R(F ′F ′′ −FF ′′′)} −F ′′RMD1D2 = 0, (14) (14) 8 and the temperature Eq. (10) becomes; λ3T ′′ + Prλ2 (αξ + FR) T ′ −Rd " (1 + (Tr −1)T(ξ))3T ′′ +3(1 + (Tr −1)T(ξ))2T ′2 (Tr −1) , # = 0 (15) (15) where, where, where, M = σfB2 ◦a2 ρfυf , D2 = [(1 −ϕ1)5/2(1 −ϕ2)5/2], Tr = eTl eTr , Pr = δf υf , Rd = 4 ∗σ eT 3 u 3akkf , D1 = σCNT + 2σbf −2ϕ2(σbf −σCNT ) σCNT + 2σbf + ϕ2(σbf −σCNT ) × σMS + 2σf −2ϕ1(σf −σMS) σMS + 2σf + ϕ1(σf −σMS) , λ1 = υf υhnf = (1 −ϕ1)5/2 (1 −ϕ2)5/2 {1 − 1 −ρs ρf ϕ1} (1 −ϕ2) + ρCNT ρf ϕ2 , λ3 = khnf kf , λ2 = (ρCp)hnf (ρCp)f ,                              (16)  (16) (16) The transformed form of auxiliary conditions is: ( ) The transformed form of auxiliary conditions is: at ey = −a et : F a−1 et ey = A , F ′ a−1 et ey = 0 , T a−1 et ey = 1, at y = a et : F a−1 et ey = 1 , F ′ a−1 et ey = 0 , T a−1 et ey = 0,    (17) (17) here, the permeability parameter is symbolized as A = νl/νu. The local Nusselt number, (rate of heat transfer) is deﬁned as: here, the permeability parameter is symbolized as A = νl/νu. Table 1: Experimental upshots of traits of H2O,Fe3O4,SWCNT and MWCNT. Table 1: Experimental upshots of traits of H2O,Fe3O4,SWCNT and MWCNT. Table 1: Experimental upshots of traits of H2O,Fe3O4,SWCNT and MWCNT. Material H2O(f) Fe3O4(MS) SWCNT MWCNT ρ(kgm−3) 997.1 5200 2600 1600 Cp(Jkg−1K−1) 4179 670 425 796 k(Wm−1K−1) 0.613 6 6600 3000 σ(sm−1) 5.5×10−6 0.74×106 106 107 So the transformed ﬁrst order equations are: So the transformed ﬁrst order equations are: Q′ 1 = Q2, Q′ 2 = Q3, Q′ 3 = Q4, Q′ 4 = λ1{R(Q2Q3 −Q1Q4) −α(3Q3 + ξQ4)} + Q3RMD[(1 −ϕ1)5/2(1 −ϕ2)5/2], Q′ 5 = Q6, Q′ 6 = Pr λ2 (αξ + Q1R) Q6 −3Rd{1 + (Tr −1)Q5}2Q2 6 (Tr −1) Rd{(1 + (Tr −1)Q5}3 −khnf kf ,                                (21) (21) Q′ 6 = Pr λ2 (αξ + Q1R) Q6 −3Rd{1 + (Tr −1)Q5}2Q2 6 (Tr −1) Rd{(1 + (Tr −1)Q5}3 −khnf kf , and the initial conditions are Q1 = A , Q2 = 0 , Q5 = 1, (22) Q3 = E , Q4 = G , Q6 = H, (23) (22) (23) (22) (23) here, E, G and H are the assumed initial guesses. The above system of equations are numeri- cally integrated by RK-4 method and initial guesses are modiﬁed by utilizing Newton’s method. Programming is done on MATLAB. All the results are computed with the tolerance of 10−5. 3 Solution methodology To solve the governed ODEs in above section, a well-known numerical technique Runge-Kutta method of order four (RK4) along with shooting technique has been used. In ﬁrst step, highly coupled and nonlinear ODEs equation (14) and (15) has been reduced to non-linear ﬁrst order ODEs by assuming: Q1 = F , Q2 = F ′ , Q3 = F ′′ , Q4 = F ′′′, Q5 = T, Q6 = T ′ 9 4 Results and discussions This segment’s main concern is to precisely show up the physical importance of the graphi- cal simulations. The ﬂuid ﬂow with the transfer of heat characteristics of a hybrid nanoﬂuid (CNT −Fe3O4/H2O) was observed through a porous permeable channel whose walls often dis- play partitioning motion. In addition, the main objective is to envision the eﬀect on velocity and temperature distribution of various physical parameters such as Reynolds number R, rate of wall deformation α, solid volume fraction (0.005 ≤φ2 ≤0.06), magnetic parameter M, temperature ratio parameter Tr, thermal radiation parameter Rd and porosity parameter A. The physical and thermal characteristics of base ﬂuid (H2O), Ferro-oxide (Fe3O4), and CNTs have been displayed in Table 1. Therefore, since this analysis includes water as a carrier ﬂuid, it is presumed that the Prandtl number is equal to 6.2. 10 Figs. 2 and 3 are designed to see the velocity conduct against the rising wall deformation rate α while dilating and contracting channel accompanied by injection R > 0. Fig. 2 indicates that when the walls undergo injection and are allowed to expand (α > 0), a vacant region is created near the walls, primarily because of the dilation of the channel. The neighboring layers of ﬂuid are then moved inwards to ﬁll this area and, as a result, a decrement in the speed of the ﬂuid is observed, which slows down the rate of ﬂow along the walls. On contrary, the velocity of the ﬂuid increases with the absolute values of α in the middle of the channel, and hence, the conservation of momentum has been maintained. It is also noted that decrement in the velocity of the ﬂuid is more prominent in the case of hybrid-nanoﬂuid (Fe2O3 −SWCNTMWCNT −H2O) as com- pared to simple nanoﬂuid (Fe2O3 −H2O). Furthermore, while contracting channel accomplished by injection. Fig. 3 shows the neighboring ﬂuid layers suppress near the channel’s walls, and hence, an increment in velocity takes place. which speeds up the ﬂow rate along the walls but the speed of ﬂuid slows down in the middle of the channel. Here, the hybrid-nanoﬂuid moves more quickly than the nanoﬂuid. Figs. 4 and 5 were painted to investigate the velocity actions when the walls are embraced or parti- tioned with injection or suction together. Fig.4 shows the velocity action when the ﬂuid is injected at the walls along with the channel’s expanding motion. 4 Results and discussions The subjacent area of the channel shows that the velocity of the ﬂuid slows down strongly, with the increasing value of absolute R. Fig. 4 also indicates that the velocity of (CNT −Fe3O4/H2O) hybrid nanoﬂuid in the channel’s top portion is dominant as compared to the (Fe3O4/H2O) nanoﬂuid. MWCNTs also hold superiority in comparison with SWCNTs, just in the top portion of the channel. Also, Fig. 5 is represented to explain the situation when the dilating action of the walls occurs along with suction. The channel’s subjacent area shows an increase in velocity of the ﬂuid, with the increasing absolute R likely because of the porosity of the walls A = evl/evu. Accordingly, the term A = 0.5 suggests that the suction regulates the ﬂow activity in the channel’s top portion and that increased ﬂuid ﬂow was observed. It was clearly shown from Fig. 5 that the velocity of (Fe3O4/H2O) nanoﬂuid in the channel’s top portion is dominant as compared to the (CNT −Fe3O4/H2O) hybrid nanoﬂuid. Single wall carbon nanotubes (SWCNTs) also hold superiority in comparison with MWCNTs, just in the channel’s top portion. Figs. 6 and 7 are drawn to describe the temperature behavior when the walls are squeezing with injection or suction together. Fig. 6 shows the temperature behavior of ﬂuid when the ﬂuid’s injection at the walls along with the channel’s squeezing motion. It is found that the tempera- ture demolished for the higher injection rate R. Due to more injection of nanoﬂuid, the collision between the particles increases which ultimately enhances the temperature of the ﬂuid. Inter- estingly, the gap between the hybrid-nanoﬂuid and simple nanoﬂuid is too wide, which indicates that a decline in the temperature of nanoﬂuid (Fe2O3) is eminent. While Fig. 7 represents the situation when the dilating (α > 0) action of the walls occurs along with suction (R < 0). We noticed a reverse relation as compared to injection case (R > 0). When the walls are allowed to contract (α < 0) and ﬂuid is sucked from the walls, the temperature of the ﬂuid is enhanced. Here, nanoﬂuid (Fe2O3) gains more temperature over the hybrid-nanoﬂuid. mpact of the magnetic parameter M over the velocity proﬁle F ′(ξ) when both α and R are 11 positive, i.e. channel’s walls are expanding with the injection of ﬂuid, is displayed in Fig. 8. 4 Results and discussions By rising the magnetic ﬁeld strength M, the velocity of the ﬂuid increases. It is also observed that the Ferroﬂuid (Fe3O4/H2O) shows primacy in speed as compared to the (CNT −Fe3O4/H2O) hybrid-nanoﬂuid. The parameter thermal radiation (Rd) is very inﬂuential for the increment of temperature as illustrated in Fig. 9. It is clear that the temperature, for the injection and contrac- tion of the walls of the channel, rises signiﬁcantly. The behavior close to the upper wall is the same for injecting and expanding cases, i.e. a higher temperature with rising Rd. With the increas- ing values of the thermal radiation parameter (Rd), the mean coeﬃcient of absorption decreases, and as a result, a rise in the ﬂuid temperature is expected. Here again, in comparison, a rise in temperature is more prominent in hybrid-nanoﬂuid, especially for SWCNTs. Fig. 10 assists in visualizing temperature variations under the power of porosity variable A with the simultaneous squeezing or injection scenario. The graph represents the fact that an increase in the porosity parameter A of the channel causes a clear decrement in the temperature proﬁle. It is also clear from Fig. 10 that the hybrid-nanoﬂuid poses dominant behavior in temperature relative to the ferroﬂuid. Fig. 11 is sketched to assist in visualizing the temperature variations under the eﬀect of the absolute parameter of porosity A along with the squeezing/injection scenario. The graph carries higher values of temperature with an increase in the value of absolute A. This is because of the predominance of temperature at the channel’s lower wall, so very less amount of temperature diﬀerence was observed near the upper wall in the region. Also, it was recorded that the region, located near the channel’s bottom wall, shows an increase in temperature proﬁle, which is because of the ﬂuid with higher thermal energy induced from the channel’s lower extremity. When it goes on, this injected ﬂuid slowly experiences a loss of energy and eventually exits, the upper part of the channel, with the least possible temperature. In addition, Fig.11 noted that the hybrid nanoﬂuid poses a dominance in temperature relative to Ferroﬂuid. Figs. 12 and 13 are plotted to test the eﬀect of volume fraction φ2) of nano-meter-sized particles of SWCNTs and MWCNTs. By rising the number of CNTs φ2 in the base ﬂuid, the subjacent part of the channel shows higher values of velocity and temperature respectively in Figs. 4 Results and discussions -1 -0.5 0 0.5 1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.430.440.450.460.47 0.33 0.34 0.35 0.36 0.37 R = -0.5, -1.0, -1.5, -2.0 1 = 0.1, 2 = 0.04, A = 0.5, = 1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 5: Inﬂuence of −R on F ′(ξ). 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, A = 0.5, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 R = 10, 12, 14, 16 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, A = 0.5, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 R = -10, -12, -14, -16 Solid Lines: Fe O & SWCNT -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.58 0.59 0.6 0.16 0.17 0.18 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 = 0.0, 0.5, 1.0, 1.5 1 = 0.1, 2 = 0.04, R = 2.5, A = 0.5, Rd = 0.4 Pr = 6.2, M = 0.3, Tr = 1.7, -1 -0.5 0 0.5 1 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 F ( ) 0.66 0.67 0.155 0.16 0.165 = 0.0, -0.5, -1.0, -1.5 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 1 = 0.1, 2 = 0.04, R = 2.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4, A = 0.5 Figure 2: Inﬂuence of α on F ′(ξ). Figure 3: Inﬂuence of −α on F ′(ξ). 4 Results and discussions 12 and 13. In 12 Hybrid-nanoﬂuid’s (CNT −Fe3O4/H2O) velocity tends to have lower values as compared to the Ferroﬂuids whereas in 13 Hybrid-nanoﬂuid’s (CNT −Fe3O4/H2O) temperature is more prominent. The impact of temperature ratio parameter Tr (it is the ratio between the temperature at the wall and the reference temperature) is displayed in Fig. 14 for the variation in temperature distribution against the injection/squeezing channel. For the higher values of Tr, the temperature diﬀerence between the walls of the channel is high. A thinner thermal boundary layer is being seen as we push towards the upper wall. A considerable diﬀerence between the hybrid-nanoﬂuid and Ferro-nanoﬂuid is observed, as the hybrid shows a high temperature rise then the Ferro-nanoﬂuid. The ratio between the momentum diﬀusivity to thermal diﬀusivity is famous as Prandtl number Pr. The eﬀect of Prandtl number Pr on temperature proﬁle has been displayed in Fig. 15. By rising the Prandtl number is the falling thermal diﬀusivity of the ﬂuid which ultimately reduces the temperature of the ﬂuid. Reduction in temperature is more conspicuous in Ferro-nanoﬂuid ﬂuid. 12 -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.58 0.59 0.6 0.16 0.17 0.18 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 = 0.0, 0.5, 1.0, 1.5 1 = 0.1, 2 = 0.04, R = 2.5, A = 0.5, Rd = 0.4 Pr = 6.2, M = 0.3, Tr = 1.7, Figure 2: Inﬂuence of α on F ′(ξ). -1 -0.5 0 0.5 1 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 F ( ) 0.66 0.67 0.155 0.16 0.165 = 0.0, -0.5, -1.0, -1.5 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 1 = 0.1, 2 = 0.04, R = 2.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4, A = 0.5 Figure 3: Inﬂuence of −α on F ′(ξ). -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.54 0.55 0.56 0.19 0.2 0.21 R = 0.5, 1.0, 1.5, 2.0 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 1 = 0.1, 2 = 0.04, A = 0.5, = 1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 Figure 4: Inﬂuence of R on F ′(ξ). 4 Results and discussions -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 A = 0.1, 0.3, 0.5, 0.7 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 10: Inﬂuence of A on T(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 A = -0.1, -0.3, -0.5, -0.7 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 11: Inﬂuence of −A on T(ξ). 0.4 0.5 0.16 0.17 0.18 0.8 1 0.4 0.42 0.44 0.46 0.48 -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.5 0.51 0.52 0.53 0.17 0.18 0.19 M = 0.0, 0.4, 0.8, 1.2 1 = 0.1, 2 = 0.04, R = 2.5, A = 0.5, Rd = 0.4, Pr = 6.2, = 1.5, Tr = 1.7, Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) Rd = 0.2, 0.4, 0.6, 0.8 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, A = 0.5 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 9: Inﬂuence of Rd on T(ξ). Figure 8: Inﬂuence of M on F ′(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 A = -0.1, -0.3, -0.5, -0.7 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 A = 0.1, 0.3, 0.5, 0.7 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 11: Inﬂuence of −A on T(ξ). Figure 10: Inﬂuence of A on T(ξ). 4 Results and discussions -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.54 0.55 0.56 0.19 0.2 0.21 R = 0.5, 1.0, 1.5, 2.0 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 1 = 0.1, 2 = 0.04, A = 0.5, = 1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 -1 -0.5 0 0.5 1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.430.440.450.460.47 0.33 0.34 0.35 0.36 0.37 R = -0.5, -1.0, -1.5, -2.0 1 = 0.1, 2 = 0.04, A = 0.5, = 1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 5: Inﬂuence of −R on F ′(ξ). Figure 4: Inﬂuence of R on F ′(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, A = 0.5, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 R = -10, -12, -14, -16 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, A = 0.5, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 R = 10, 12, 14, 16 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 7: Inﬂuence of −R on T(ξ). Figure 6: Inﬂuence of R on T(ξ). 13 -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.5 0.51 0.52 0.53 0.17 0.18 0.19 M = 0.0, 0.4, 0.8, 1.2 1 = 0.1, 2 = 0.04, R = 2.5, A = 0.5, Rd = 0.4, Pr = 6.2, = 1.5, Tr = 1.7, Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 8: Inﬂuence of M on F ′(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) Rd = 0.2, 0.4, 0.6, 0.8 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, A = 0.5 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 9: Inﬂuence of Rd on T(ξ). 4 Results and discussions -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) -0.62 -0.6 -0.58 -0.56 0.38 0.4 0.42 0.44 0.46 0.48 2 = 0.0, 0.04, 0.08, 0.12 1 = 0.1, A = 0.5, R = 15, = -1.5, Pr = 6.2, M = 0.3, Tr = 1.7, Rd = 0.4 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 -1 -0.5 0 0.5 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 F ( ) 0.56 0.58 0.6 0.15 0.16 0.17 0.18 2 = 0.0, 0.07, 0.14, 0.20 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 1 = 0.1, = 1.5, R = 2.5, A = 0.5, Rd = 0.4 Pr = 6.2, M = 0.3, Tr = 1.7, Figure 12: Inﬂuence of φ2 on F ′(ξ). Figure 13: Inﬂuence of φ2 on T(ξ). 14 -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) Tr = 1.0, 1.3, 1.6, 2.0 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Rd = 0.4, A = 0.5 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 14: Inﬂuence of Tr on T(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, R = 15, = -1.5, A = 0.5, M = 0.3, Tr = 1.7, Rd = 0.4 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Pr = 2.0, 4.0, 6.0, 8.0 Figure 15: Inﬂuence of Pr on T(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) Tr = 1.0, 1.3, 1.6, 2.0 1 = 0.1, 2 = 0.04, R = 15, = -1.5, Pr = 6.2, M = 0.3, Rd = 0.4, A = 0.5 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Figure 14: Inﬂuence of Tr on T(ξ). -1 -0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 T( ) 1 = 0.1, 2 = 0.04, R = 15, = -1.5, A = 0.5, M = 0.3, Tr = 1.7, Rd = 0.4 Solid Lines: Fe2O3 & SWCNT Dashed lines: Fe2O3 & MWCNT Dashed dotted: Fe2O3 Pr = 2.0, 4.0, 6.0, 8.0 Figure 14: Inﬂuence of Tr on T(ξ). Figure 15: Inﬂuence of Pr on T(ξ). 4 Results and discussions The rate of local heat transfer is measured numerically for the diﬀerent parameters. Tables are arranged for both hybrid-nanoﬂuid (Fe2O3, SWCNTs, MWCNTs −H2O) and Ferro-nanoﬂuid (Fe2O3 −H2O). In table 2, for the variation of Prandtl number, temperature ratio parameter, thermal radiation parameter, and porosity parameter, the numerical values are arranged against the local Nusselt number. Porosity parameter A and Prandtl number Pr are the main factors that enhance the rate of heat transfer ﬂow in the dilation channel when ﬂuid is injected through the channel’s wall. This increment is much more dominant at the lower wall when compared with the upper wall. It is also clear from the table that Ferro-nanoﬂuid is higher then hybrid- nanoﬂuid. The same table represents the opposite inﬂuence of temperature ratio parameter Tr and thermal radiation parameter Rd against Nusselt number. Heat transfer rate on the channel wall decreases for the increasing values of Tr and Rd. Again this decrement is eminent at the lower wall. However, the rate of heat transfer is now reduced quickly for the hybrid-nanoﬂuid when compared with Ferro-nanoﬂuid. In table 3, we arranged the numerical values of local Nusselt number for volume fraction of nanoparticles (φ1, φ2), local Reynolds number (suction/injection parameter) R and channel’s wall deformation α. It is observed that by increasing the amount of Ferricoxide nanoparticles in the base ﬂuid, and if the injection rate is enhanced, the rate of heat transfer is signiﬁcantly boosted. The lower wall exhibits more heat transfer when compared with the upper wall. Ferro-nanoﬂuid has the least Nusselt number while MWCNTs have the most. On the other hand, the volume fraction of CNTs and the channel’s deformation rate are responsible for lowering the rate of heat transfer at the walls of the channel. similar behavior is noted for the upper and lower walls. Decreasing the Nusselt number is more eﬃcient in the case of hybrid-nanoﬂuid. Table 3: Numerical values of Nusselt number for various parameters, whereas Pr = 6.2, Tr = 1.7, Rd = 0.4, A = 0.5 Table 2: Numerical values of Nusselt number for various parameters, whereas φ1 = 0.1, φ2 = 0.04, R = 15, α = 0.5, M = 0.3 3: Numerical values of Nusselt number for various parameters, whereas Pr = 6.2, Tr = 1.7 A 3: Numerical values of Nusselt number for various parameters, whereas Pr = 6.2, Tr = 1.7 0.4, A = 0.5 4 Results and discussions 15 Pr Tr Rd A Nulower Nuupper Nulower Nuupper Nulower Nuupper Fe2O3 −SWCNT Fe2O3 −MWCNT Fe2O3 −H2O 6.2 1.7 0.4 0.5 1.517729 0.014100 1.560450 0.011172 1.662829 0.002404 4.0 1.097992 0.070470 1.124646 0.061734 1.182060 0.026963 5.0 1.288579 0.034531 1.322851 0.028914 1.402397 0.009175 6.0 1.479635 0.016409 1.520990 0.013123 1.619758 0.003013 1.0 2.354463 0.009130 2.452406 0.007117 3.136879 0.001187 1.2 2.129890 0.010075 2.210584 0.007884 2.676347 0.001394 1.4 1.882794 0.011339 1.946602 0.008912 2.230198 0.001689 0.5 1.378682 0.018459 1.414348 0.014925 1.469865 0.004162 0.6 1.266438 0.023622 1.296848 0.019445 1.322189 0.006732 0.7 1.173897 0.029619 1.200254 0.024776 1.205356 0.010287 0.6 1.667940 0.011950 1.718973 0.009349 1.847373 0.001849 0.7 1.823725 0.010131 1.883504 0.007826 2.039437 0.001423 0.8 1.984810 0.008592 2.053704 0.006553 2.238419 0.001096 Table 2: Numerical values of Nusselt number for various parameters, whereas φ1 = 0.1, φ2 = 0.04, R = 15, α = 0.5, M = 0.3 Table 2: Numerical values of Nusselt number for various parameters, whereas φ1 = 0.1, φ2 = 0.04, R = 15, α = 0.5, M = 0.3 φ1 φ2 R α Nulower Nuupper Nulower Nuupper Nulower Nuupper Fe2O3 −SWCNT Fe2O3 −MWCNT Fe2O3 −H2O 0.1 0.04 15 0.5 1.517729 0.014100 1.560450 0.011172 1.662829 0.002404 0.04 0.797639 0.105171 0.824031 0.091282 0.749715 0.087370 0.06 1.024559 0.052765 1.056644 0.044296 1.037788 0.025860 0.08 1.265556 0.026861 1.302985 0.021876 1.343539 0.007685 0.05 1.448629 0.017786 1.497790 0.013743 1.618192 0.002262 0.07 1.323761 0.025699 1.382308 0.019226 1.531724 0.002015 0.1 1.163902 0.037905 1.230074 0.027745 1.408744 0.001720 10 1.105311 0.064197 1.131874 0.055941 1.188414 0.023514 15 1.517729 0.014100 1.560450 0.011172 1.662829 0.002404 20 1.933315 0.002799 1.990987 0.002013 2.133490 0.000219 0.7 1.504068 0.013856 1.545868 0.010972 1.645167 0.002348 0.9 1.490515 0.013616 1.531406 0.010774 1.627669 0.002294 1.1 1.477071 0.013379 1.517063 0.010579 1.610335 0.002241 16 Conﬂict in Interest The author declares no conﬂict of interest regarding the publication of this article. The author declares no conﬂict of interest regarding the publication of this article. Authors ContributionsH.A. wrote the manuscript; M.B. supervised and conceived the idea; M.R. did the software work. Data Availability Statement: The data that support the ﬁndings of this study are available from the corresponding author 5 Concluding remarks The leading target is to contemplate the heat and ﬂow characteristics of hybrid-nanoﬂuid (CNTs− Fe2O3) with water H2O as carrier ﬂuid through the rectangular asymmetric permeable horizontal parallel channel with an external applied magnetic ﬁeld. The thermal radiation impact is also investigated. By varying the diﬀerent physical parameters, ﬂow and heat transfer features have been demonstrated with the help of graphs. The local rate of heat transfer is numerically calculated and arranged in the form of tables. A good comparative analysis is done for simple and hybrid nanoﬂuid. The main features are as follows: • Higher magnetic ﬁeld shows in increment in ﬂow speed between the expanding walls of the channel along with the injection of ﬂuid. The ﬂuid even gains more speed when we use Ferro-nanoﬂuid instead of hybrid-nanoﬂuid. • Higher magnetic ﬁeld shows in increment in ﬂow speed between the expanding walls of the channel along with the injection of ﬂuid. The ﬂuid even gains more speed when we use Ferro-nanoﬂuid instead of hybrid-nanoﬂuid. • For the squeezing walls of the channels, the temperature of the ﬂuid rises when the thermal radiation parameter is enhanced. This enhancement is even more prominent in the case of hybrid-nanoﬂuid (CNTs −Fe2O3, H2O). • The inﬂuence of injection and suction is quite opposite for the temperature distribution. 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Bernstein method for the MHD ﬂow and heat transfer of a second grade ﬂuid in a channel with porous wall. Alexandria Engineering Journal, 55:2149–2156, 2016. Figures Figure 1 Figures Figures Figure 1 See the Manuscript Files section for the complete ¦gure caption. Figure 1 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 2 Figure 2 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 3 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 4 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 5 See the Manuscript Files section for the complete ¦gure caption. Figure 5 See the Manuscript Files section for the complete ¦gure caption. Figure 6 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 7 See the Manuscript Files section for the complete ¦gure caption. Figure 7 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 8 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 9 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 10 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 11 See the Manuscript Files section for the complete ¦gure caption. Figure 11 See the Manuscript Files section for the complete ¦gure caption. Figure 12 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption. Figure 13 See the Manuscript Files section for the complete ¦gure caption. Figure 13 See the Manuscript Files section for the complete ¦gure caption. Figure 14 See the Manuscript Files section for the complete ¦gure caption. Figure 14 See the Manuscript Files section for the complete ¦gure caption. Figure 15 See the Manuscript Files section for the complete ¦gure caption. See the Manuscript Files section for the complete ¦gure caption.
https://openalex.org/W4393133567	https://jurnal.lp2msasbabel.ac.id/index.php/sus/article/download/4214/1703	Indonesian	null	Implementasi Manajemen Pembelajaran Aqidah Akhlak Dalam Meningkatkan Prestasi Belajar Siswa	Jurnal Sustainable	2,023	cc-by	2,281	Abstrak Penerapan manajemen pembelajaran penting dilakukan guna tercapainya tujuan pembelelajaran. Dalam implementasi manajemen pembelajaran aqidah akhlak di MAN 2 Takengon, dilakuakn tahapan perencaan, pelaksanaan dan evaluasi dalam. Penelitian ini bertujuan untuk mengetahui pengaruh manajemen pembelajaran aqidah akhlak dalam meningkatkan prestasi belajar siswa kelas IX MIPA. Penelitian ini menggunakan jenis penelitian kuantitatif, pengumpulan data dilakukan dengan wawancara dan tes. Analisa data dilakukan dengan uji normalitas data, uji homogenitas data, uji validitas data, uji realibilat data, dan uji t-test. Hasil penelitian menunjukkan bahwa uji paired T-test pada variabel Prestasi belajar didapatkan nilai T-hitung 6,384 sedangkan nilai T-tabel sebesar 2,036. yang artinya Nilai THitung > Ttabel sehingga H1 diterima serta H0 ditolak, yang berarti manajemen pembelajaran aqidah akhlak berpengaruh terhadap prestasi belajar siswa Kata kunci: Implementasi, Manajemen, Akqidah Akhlak, Prestasi Kata kunci: Implementasi, Manajemen, Akqidah Akhlak, Prestasi History: Received : 26 Nov 2023 Revised : 28 Nov 2023 Accepted : 30 Nov 2023 Published : 31 Dec 2023 1IAIN Takengon Author Correspondent: irwan123@gmail.com Publishers: LPM IAIN Syaikh Abdurrahman Siddik Bangka Belitung, Indonesia Licensed: This work is licensed under aCreative Commons Attribution 4.0 International License. History: Received : 26 Nov 2023 Revised : 28 Nov 2023 Accepted : 30 Nov 2023 Published : 31 Dec 2023 1IAIN Takengon Author Correspondent: irwan123@gmail.com Publishers: LPM IAIN Syaikh Abdurrahman Siddik Bangka Belitung, Indonesia Licensed: This work is licensed under aCreative Commons Attribution 4.0 International License. Implementasi Manajemen Pembelajaran Aqidah Akhlak Dalam Meningkatkan Prestasi Belajar Siswa g Irwansyah1* Volume 6 Number 2 2023, 784-789 E-ISSN: 2655-0695 DOI:https://doi.org/ 10.32923/kjmp.v6i2.4214 Pendahuluan Pada dasarnya, pendidikan adalah hal penting yang tidak bisa luput dari kehidupan manusia dikarenakan pendidikan sendiri merupakan salah satu unsur membentuk akhlak juga kepribadian yang baik pada diri manusia (Ula & Suwarno, 2023). Adapun pendidikan memiliki 2 jenis yaitu pendidikan formal dan pendidikan informal. Pendidikan non formal mampu didapatkan manusia dari keluarga, lingkungan atau dengan memaknai setiap pengalaman hidup yang disebut juga pengalaman, sedangkan pendidikan formal merupakan metode pembelajaran terstruktur yang diselenggarakan di institusi pendidikan (Masang, 2021). Kedua jenis pendidikan tersebut sama- sama bertujuan untuk mengembangkan kemampuan yang dimiliki oleh individu, karena sejatinya manusia punya potensi bawaan yang dibawa sejak lahir sebagai khalifah di bumi (Rodliyah, 2013). Di dalam peraturan yang tertera pada UU No. 20 Tahun 2003 perihal SISDIKNAS disebutkan bahwasannya pendidikan merupakan fungsi pengembangan untuk membentuk karakter siswa supaya tercipta insan yang berakhlakul karimah, inovatif, kreatif dan mempunyai daya saing yang tinggi sehingga tercipta insan yang demokratis dan tanggung jawab (Presiden Republik Indonesia, 2003). Terkait dengan hal ini, guna menghasilkan sumber daya manusia yang baik, tidak dapat terjadi secara instan pada suatu lembaga pendidikan (Yuniarno, 2019). Kualitas yang baik dalam suatu pendidikan disebabkan oleh banyak hal di antaranya yakni manajemen pembelajaran. Manajemen pembelajaran antara lain perencanaan, pelaksanaan dan penilaian. Manajemen pembelajaran termasuk seni untuk melaksanakan pekerjaan melalui orang-orang tertentu dengan memanfaatkan berbagai sumber daya yang tersedia (Mukhlisoh & Suwarno, 2019). Manajemen pembelajaran aqidah akhlak sudah banyak dikaji oleh peneliti sebelumya, Manajemen Pembelajaran Akidah Akhlak dalam Pembentukan Akhlakul Karimah Peserta Didik oleh Abdul Halik, fokus penelitian ini untuk mengetahui peran manajemen pembelajaran Akidah Akhlak dalam pembentukan Akhlakul Karimah peserta didik, metode penelitian yang digunakan adalah metode 784 Implementasi Manajemen Pembelajaran Aqidah Akhlak Dalam Meningkatkan Prestasi Belajar Siswa kualitatif, hasil penelitiannya menunjukkan bahwa, penerapan pembelajaran Akidah Akhlak, pembentukan akhlakul karimah peserta didik meningkat sangat pesat (Halik & Saira, 2018). Perbedaan penelitian di atas dengan penelitian penulis adalah penelitian oleh Halik tentang pembentukan karakter, sedangkan penelitian penulis fokus tentang prsetasi belajar, dari perbedaan tersebut maka masih ada kesempatan penulis untuk melakukan penelitian ini. Kemudian penelitian yang berjudul Manajemen Pembelajaran Aqidah Akhlak untuk Menanamkan Kecerdasan Interpersonal Peserta Didik oleh Nur’aini dan Hamzah, penelitian ini fokus pada implementasi manajemen pembelajaran dalam mata pelajaran aqidah akhlak. Metode penelitiannya menggunakan metode fenomenologi. Temuan peneliti ini adalah implementasi manajemen pembelajaran aqidah akhlak dalam menanamkan kecerdasan interpersonal di SMA Al-Azhar dilakukan melalui tiga kegiatan yaitu pembukaan, pembentukan kompetensi dan penutup (Nur’aini, 2023). Pendahuluan Ada perbedaan penelitian oleh Nur’aini dengan penelitian penulis, penelitian Nur’aini fokus pada pembentukan kecerdasan interpersonal, sedangkan penelitian penulis fokus pada peningkatan prestasi belajar. Berdasarkan perbedaan tersebut masih ada peluang penulis untuk melakukan penelitian ini. Penelitian ini bertujuan untuk mendeskripsikan implementasi manajemen pendidikan aqidah akhlak dalam meningkatkan prestasi belajar siswa. Metode Penelitian ini menggunakan jenis peelitian kuantitatif, yaitu data yang dikumpulkan berbentuk kumpulan angka, melalui lembar observasi dan angket (Unaradjan, 2019). Populasi penelitian ini yakni siswa Kelas XI MIPA MAN 2 Takengon yang berisi siswa dari XI MIPA 1 s.d XI MIPA 6 dengan jumlah siswa 198 siswa. Adapun sampel dalam penelitian ini yakni peserta didik kelas XI MIPA 1 sebanyak 33 siswa. Pengumpulan data dilakukan dengan wawancara, dan tes. Wawancara ini dilakukan kepada guru Aqidah Akhlak dan wakil kepala sekolah bidang kurikulum untuk mengetahui Manajemen Pembelajaran Aqidah Akhlak. Kriteria Ketuntasan disesuaikan dengan KKM mata pelajaran Aqidah Akhlak di kelas XI MAN 2 Takengon yakni sebesar 75. Apabila nilai yang diterima siswa melampaui 75 (>75), maka dinyatakan tuntas dan jika nilai yang diperoleh siswa kurang dari 75 (<75) maka dinyatakan belum tuntas. Teknik analisa data menggunakan analisis kuantitatif. Uji Normalitas Data Uji normalitas yakni pengujian yang dilaksanakan guna mengetahui apakah sebaran data pada suatu kelompok data maupun variabel berdistribusi normal atau tidak. Tes Shapiro-Wilk digunakan untuk menentukan normalitas data di penyelidikan ini. Hal tersebut dilaksanakan sebab jumlah sampel penelitian < 30 responden. Uji normalitas mengasumsikan bahwasannya data berdistribusi secara normal jika tidak memiliki perbedaan yang signifikan atau standar jika dibandingkan dengan standar normal. Jika digunakan uji statistik, seperti uji Kolmogorov-Smirnov, variabel dianggap berdistribusi teratur bila nilai signifikansinya lebih besar atau sama dengan 0,05. Bila tingkat signifikansi < 0,05, maka variabel atau data tersebut tidak berdistribusi teratur. Tabel1: Uji Normalitas data Sumber:(Irwansyah, n.d.) Sumber:(Irwansyah, n.d.) Dari hasil uji normalitas data yang menerapkan uji Kolmogorov-Smirnov didapatkan nilai signifikansi untuk motivasi belajar (pretest) yakni 0,606, nilai signifikansi untuk motivasi belajar (posttest) yakni 0,535, nilai signifikansi untuk prestasi belajar (pretest) yakni 0,599 dan nilai signifikansi untuk prestasi belajar (posttest) yakni 0,104. Karena nilai signifikansi yang didapatkan pada masing-masing pengukuran lebih dari 0,05 maka mampu disimpulkan bahwasannya data penelitian berdistribusi normal. Impleentasi Manajemen Pembelajaran Aqidah Akhlak Impleentasi Manajemen Pembelajaran Aqidah Akhlak Dalam pelaksanaannya, manajemen pembelajaran aqidah akhlah dilakukan dengan beberapa tahapan. Pertama tahap perencanaan proses pembelajaran dilakukan sebelum awal semester. Sebelum awal semester / tahun ajaran baru, pengelola Madrasah Aliyah Negeri 2 Takengon bersama dengan jajaran guru mengadakan rapat yang membahas mengenai manajemen pembelajaran yang akan diterapkan kepada peserta didik Madrasah Aliyah Negeri 2 Takengon Wakil kepala sekolah bidang Kurikulum di bawah koordinasi Kepala Sekolah melakukan pembagian tugas kepada guru dan mewajibkan setiap guru untuk menyiapkan dokumen pembelajaran dan sekaligus laporan capaian pelaksanaan kegiatan pembelajaran (Paridah, 2023). Kedua, pelaksanaan pembelajaran. Setelah perangkat pembelajaran disusun dengan Tim Musyawaroh guru mata pelajaran aqidah akhlak, tahapan berikutnya adalah pelaksanaan pembelajaran. Dalam pelaksanaan pembelajaran ini tentunya guru harus mempunyai ide untuk menentukan metode pembelajaran yang akan diterapkan saat pembelajaran (Paridah, 2023). Ketiga, Setelah materi pembelajaran diberikan dan metode pembelajaran sudah diterapkan hendaknya guru waktunya mengukur kemampuan siswa dengan cara mengevaluasi peserta didiknya melalui pemberian tugas terstruktur atau tugas tidak terstruktur, sehingga bisa diketahui capaian hasil pembelajaran saat itu (Paridah, 2023). 785 Uji Validitas Ketika suatu tes memenuhi fungsi pengukuran yang dimaksudkan atau menghasilkan hasil pengukuran yang tepat dan akurat, itu dianggap memiliki validitas tinggi. Tes yang menyampaikan data yang tidak berkaitan dengan tujuan pengukuran dianggap memiliki validitas rendah. Ada dua jenis validitas: validitas faktor serta validitas item. Validitas faktor dinilai ketika item dibangun memakai banyak komponen. Satu elemen dan elemen lainnya memiliki beberapa kesamaan. Uji validitas di penelitian ini memakai uji korelasi product moment. Berdasarkan hasil uji validitas yang dilaksanakan kepada 15 butir pertanyaan penelitian didapatkan nilai r hitung di setiap butir kuesioner > nilai r tabel 0,334 berdasarkan uji signifikan 0,05 sehingga mampu disimpulkan dari 15 butir pertanyaan penelitian yang digunakan seluruhnya dinyatakan valid. Uji homogenitas Uji homogenitas (homogeneity of variance) yakni pengujian yang dilaksanakan guna mengetahui apakah data penelitian dari dua buah maupun lebih distribusi data mempunyai berbagai variansi yang sama maupun tidak. Tes homogenitas bertujuan guna membuktikan bahwasannya sampel diambil dari populasi dengan varians yang konstan. Salah satu uji homogenitas adalah uji Fisher F. Uji Fisher F dilaksanakan ketika menguji homogenitas variansi dari dua kelompok data dengan menghitung perbandingan variansi kelompok data 1 dengan variansi kelompok data 2 lalu membandingkannya dengan F tabel berdasarkan tingkat keyakinan serta derajat kebebasan kelompok data 1 dan 2. 786 Implementasi Manajemen Pembelajaran Aqidah Akhlak Dalam Meningkatkan Prestasi Belajar Siswa Tabel 2: Uji homogenitas prestasi belajar Sumber:(Irwansyah, n.d.) Sumber:(Irwansyah, n.d.) Sumber:(Irwansyah, n.d.) Berdasarkan tabel output test of homogenity of variance diketahui nilai signifikansi (Sig) variabel prestasi belajar siswa sebelum (pre-test) serta sesudah (post-test) pengukuran sebesar 20,000, maka sebagaimana dasar pengambilan keputusan pada uji homogenitas mampu diambil kesimpulan bahwasannya varians data prestasi belajar siswa yakni sama (homogen). Kesimpulan Dari hasil penelitian diatas menunjukkan bahwa penerapan manajemen pembelajaran dari segi tahapan sudah sesuai dengan teori manajemen, bahwa terdapat empat fungsi manajemen yaitu, perencanaan, pengorganisaian, pelaksanaan dan evaluasi (Maujud, 2018). Dalam perencaan pembelajaran aqidah akhlak di MAN 2 Takengon dilaksanakan pada awal semester sebelum masuk proses pembelajaran, hal ini sesuai dengan hasil penelitian suwarno bahwa rencana pembelajaran disusun sedemikian rupa untuk mencapai tujuan pembelajaran. Selanjutnya dilakukan pengorganisian, tahap ini dilakukan untuk mengkomunikasikan rencana yang telah disusun agar sesuai dengan rencana awal. Dalam pengorganisasian ini akan diketahui kendala atau hambatan yang akan dihahapi. Uji Reliabilitas Indikator seberapa dapat dipercaya atau diandalkannya suatu alat ukur disebut reliabilitas. Sebuah alat ukur dianggap dapat diandalkan jika digunakan berulang kali agar menilai gejala yang sama dan temuan pengukuran sebagian besar konsisten. Dengan kata lain, ketergantungan membuktikan seberapa konsisten suatu alat ukur mengukur suatu gejala tertentu. Sumadi Suryabrata (2004) mengklaim bahwa reliabilitas menunjukkan sejauh mana hasil pengukuran dari alat ini mampu diandalkan. Hasil pengukuran seharusnya dapat dipercaya pada artian harus konsisten dan stabil. Keseragaman urutan pengukuran atau rangkaian alat ukur disebut reliabilitas, disebut juga ketergantungan. Hal ini dapat berupa penentuan apakah suatu pengukuran dengan alat ukur yang sama (tes awal dengan tes ulang) akan mendapatkan hasil yang sama atau, untuk pengukuran yang lebih subjektif, apakah dua penilai memberikan skor yang serupa (reliabilitas antar penilai). Validitas dan reliabilitas bukanlah hal yang sama. Hal tersebut menandakan bahwasannya sementara pengukuran yang dapat dipercaya akan mengukur secara konsisten, mungkin tidak selalu mengukur apa yang dimaksudkan untuk diukur. Ketika digunakan dalam penelitian, istilah "keandalan" mengacu pada seberapa baik ukuran tes bertahan setelah beberapa contoh obat yang sama diberikan dalam kondisi yang sama. Ketika digunakan dalam penelitian, istilah "keandalan" mengacu pada seberapa baik ukuran tes bertahan setelah beberapa contoh obat yang sama diberikan pada kondisi yang sama. Penelitian diyakini mampu dipercaya ketika memberi hasil yang konsisten bagi pengukuran yang sama. Tidak mampu diandalkan bila pengukuran berulang menghasilkan hasil yang kontradiktif. Sebagai ukuran empiris reliabilitas tinggi dan rendah, nilai koefisien reliabilitas direpresentasikan sebagai angka. Nilai rxx mendekati 1 menunjukkan keandalan yang tinggi. Ambang keandalan lebih dari 0,700 secara umum diterima sebagai sudah cukup memuaskan. 787 Suwarno, Ramadan, S. A. F. (2022). Potential and Problem in Learning Tahsin Al-Qur’an to Improve Students’ Ability to Read Al-Qur’an. LITERATUS, Vol. 4(Nomor 1), 82–86. Suwarno, S. (2016). Pendekatan Kebijakan Publik dalam Politik Pendidikan Islam. Jurnal As-Salam, 1(1), 62–72. http://www.jurnal-assalam.org/index.php/JAS/article/view/45 Referensi Ahmad, M., & Nasution, D. P. (2018). Analisis kualitatif kemampuan komunikasi matematis siswa Buchari, A. (2018). Peran Guru Dalam Pengelolaan Pembelajaran. Jurnal Ilmiah Iqra’, 12(2), 106. https://doi.org/10.30984/jii.v12i2.897 Farida Isroani, The Pattern of Development the New Female Students at The Modern Islamic Boarding School Darussalam Gontor, Sustainable: Jurnal Kajian Mutu Pendidikan, Vol 1 No 6, 125-140 Latifah, A. K., Idris, M., & Prasrihamni, M. (2023). Pengembangan Media Kartu Kuartet Berbasis Gambar 2 Dimensi Untuk Keterampilan Membaca Siswa Kelas II SD Negeri 15 Gunung Megang. 06(01), 2783–2799. Nurfadhillah, S., Ningsih, D. A., Ramadhania, P. R., & Sifa, U. N. (2021). Peranan Media Pembelajaran Dalam Meningkatkan Minat Belajar Siswa Sd Negeri Kohod Iii. PENSA : Jurnal Pendidikan Dan Ilmu Sosial, 3(2), 243–255. https://ejournal.stitpn.ac.id/index.php/pensa Oktaviana, D., Prihatin, I., & Fahrizar, F. (2020). Pengembangan Media Pop-Up Book Berbasis Contextual Teaching and Learning Dalam Pencapaian Kemampuan Pemecahan Masalah Siswa Smp. AKSIOMA: Jurnal Program Studi Pendidikan Matematika, 9(1), 1. https://doi.org/10.24127/ajpm.v9i1.2543 Penulis:, T., Usep Setiawan, H. Amit Saepul Malik, Irma Megawati, Dyah Wulandari, Asri Nurazizah, Dadang Nurjaman, Tina Nurhasanah, Vina Nuranisa, D. K., & Mulyana, C. M. (2022). Media Pembelajaran (Cara Belajar Aktif: Guru Bahagia Mengajar Siswa Senang Belajar). Widina Bhakti Persada. Prastya, A. (2016). Strategi Pemilihan Media Pembelajaran Bagi Seorang Guru. Prosiding Temu Ilmiah Nasional Guru VIII Tahun 2016: Tantangan Profesionalisme Guru Di Era Digital, VIII(November), 294–302. http://repository.ut.ac.id/id/eprint/6518 Ranika Fonda, N., & Nirwana, E. S. (2018). Pengaruh Penggunaan Media Daun Untuk Meningkatkan Kemampuan Motorik Halus Anak Usia Dini 4-5 Tahun di TK Pembina Desa Simpang III Kaur Utara. Al Fitrah: Journal Of Early Childhood Islamic Education, 2(1), 229–241. Samuel Patra Ritiauw, Bunyamin Maftuh, & E. M. (2017). THE DEVELOPMENT OF DESIGN MODEL OF CONFLICT RESOLUTION EDUCATION BASED ON CULTURAL VALUES OF PELA. Cakrawala Pendidikan, 36(3), 357–368. https://journal.uny.ac.id/index.php/cp/article/view/14353 Suwarno, Ismet Nur, Rahmanita Zakaria, E. (2022). Optimisation of the WhatsApp Application in Learning Ta ḥ s ī n al- Qur ’ ā n To Improve Students ’ Reading al-Qur ’ ā n Ability. Al-Ishlah: Jurnal Pendidikan, 14(4), 5811–5818. https://doi.org/10.35445/alishlah.v14i4.2068 788 Implementasi Manajemen Pembelajaran Aqidah Akhlak Dalam Meningkatkan Prestasi Belajar Siswa Suwarno. (2023). Shari’ah Tourism Islamic Boarding School Model (Case Study of Salafiah Biharu Bahri Asali Fadlailir Rahmah Islamic Boarding School Malang). Jurnal As-Salam, 7(1), 72–84. https://jurnal-assalam.org/index.php/JAS/article/view/431 Suwarno. (2023). Shari’ah Tourism Islamic Boarding School Model (Case Study of Salafiah Biharu Bahri Asali Fadlailir Rahmah Islamic Boarding School Malang). Jurnal As-Salam, 7(1), 72–84. https://jurnal-assalam.org/index.php/JAS/article/view/431 Suwarno, S. (2016). Suwarno. (2023). Shari’ah Tourism Islamic Boarding School Model (Case Study of Salafiah Biharu Bahri Asali Fadlailir Rahmah Islamic Boarding School Malang). Jurnal As-Salam, 7(1), 72–84. https://jurnal-assalam.org/index.php/JAS/article/view/431 Referensi Pendekatan Kebijakan Publik dalam Politik Pendidikan Islam. Jurnal As-Salam, 1(1), 62–72. http://www.jurnal-assalam.org/index.php/JAS/article/view/45 789
https://openalex.org/W4295260773	https://www.qeios.com/read/FNMA8J/pdf	English	null	Review of: "Patient Positivity through Photos- What NHS Patients Really Think of Digital Healthcare"	null	2,022	cc-by	160	Qeios, CC-BY 4.0 · Review, September 12, 2022 Qeios ID: FNMA8J · https://doi.org/10.32388/FNMA8J Review of: "Patient Positivity through Photos- What NHS Patients Really Think of Digital Healthcare" Daniel Gensorowsky1 1 Universität Bielefeld Potential competing interests: The author(s) declared that no potential competing interests exist. This short article presents an interesting approach to capturing the mood and emotions of video consultation users. However, due to the lack of a comprehensive presentation and discussion of the methodology and due to the rather narrative presentation of the results, it does not provide a sufficient basis for a peer-reviewed research article. Thus, publication as a discussion paper or editorial would be more appropriate. Furthermore, the title "What NHS Patients Really Think of Digital Healthcare" is misleading given the limitations described. It can hardly be assumed that the photos depicted ensure a comprehensive unselected picture of patient attitudes. In addition, there seems to be no systematic evaluation of results. Qeios ID: FNMA8J · https://doi.org/10.32388/FNMA8J 1/1
https://openalex.org/W3089818420	https://iiste.org/Journals/index.php/JEP/article/download/54267/56073	English	null	The Effect of Organizational Culture, Teamwork, Work Motivation and Ethic on Lecturers’ Performance at Sari Mutiara Indonesia University in Medan	null	2,020	cc-by	7,634	1. INTRODUCTION Higher Education is one of the educational systems that produces graduates who can study, analyze, research, and develop knowledge and technology as well as the arts needed by the community. Higher education is also expected to be a center for the development of human civilization, so that expectations for higher education are very high. Higher education also called an organization of change agents, and becomes the hope of solving many problems that arise in people's lives. The law of Indonesia Republic Number 12 of 2012 concerning Higher Education, chapter 1 verse 2 states that Higher Education is a level of education after secondary education that includes diploma programs, undergraduate programs, master programs, doctoral programs, and professional programs, as well as specialist programs, which are organized by universities based on culture the nation of Indonesia. In line with this, the implementation of higher education in studying, analyzing, researching, and developing science and technology must be based on the nation's culture. gy The academic community in institutions is called tridarma of institution which means tertiary institution. It includes three processes, namely the learning process, the research process, and the community service process. Learning process is carried out to examine the depth of science so that the essence of something is found, which is then transformed to students so that it becomes a common property. Research is a scientific process that carried out systematically based on scientific principles. The discovery or development of science and technology can be proven and it brings theoretical and practical progress. Community service is an academic community activity in order to educate the community through the dissemination of science and technology and its application. It can solve problems in the community. The assignment of the lecturer is called the tridharma of the institution. It is an obligation that must be carried out by the tertiary institution The quantity and quality of tridharma of instituoin depends on the quality of the college as a whole in which there are subsystems of learning, research, community service, governance, facilities and infrastructure, curriculum, extracurricular activities, community relations, and so on. Sadig (2010) explains that the quality of higher education is seen from the quality of learners, the learning environment, and the content of learning, processes, and graduates. Atawijaya (2007: 509) states that the indicators of the success of institution lie in the elements of input, process, and output. www.iiste.org www.iiste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 The Effect of Organizational Culture, Teamwork, Work Motivation and Ethic on Lecturers’ Performance at Sari Mutiara Indonesia University in Medan Sondang Lucia Purba* Harun Sitompul Aman Simare Mare State University of Medan, Jl. Willem Iskandar Psr. V Medan, Indonesia Keywords: Organizational Culture, Teamwork, Work Motivation, Work Ethic, Lecturer Performance DOI: 10.7176/JEP/11-27-10 Keywords: Organizational Culture, Teamwork, Work Motivation, Work Ethic, Lecturer Performance DOI: 10.7176/JEP/11-27-10 Publication date:September 30th 2020 Publication date:September 30th 2020 Abstract The problem of this study is to investigate the effect of organizational culture, teamwork, work motivation, and work ethic on lecturer performance. It is conducted at Sari Mutiara Indonesia University in Medan as many as 128 lecturers. Data collection is carried out by using a questionnaire with five answer choices. Before testing the hypothesis, the requirements analysis are tested, namely the normality and linearity test of the data. The results show (1) organizational culture has a direct positive effect on work motivation, (2) teamwork has a direct positive effect on work motivation, (3) organizational culture has a positive direct effect on work ethic , (4) teamwork has a direct positive effect on work ethic, (5) organizational culture has a direct positive effect on lecturer performance, (6) teamwork has a direct positive effect on lecturer performance, (7) work motivation has a direct positive effect on lecturer performance, and ( 8) work ethic has a direct positive effect on lecturer performance. Based on the acceptance of research hypotheses, a theoretical model or fixed model of lecturers performance at Sari Mutiara Indonesia University in Medan illustrates the structure of causal relationships between organizational culture variables, teamwork, work motivation, work ethics and lecturer performance. To improve the performance of lecturers at Sari Mutiara Indonesia University in Medan, it can be done by improving organizational culture, teamwork, work motivation and work ethics. 1. INTRODUCTION The success of a tertiary institution is determined by the success of management in optimizing their inputs, processes and outputs. Lunenburg and Ornstein (2000: 19) say that input from educational organizations includes human resources, finance, theory and knowledge, central and state governments, local governments, the legal structure of government, and other groups. The transformation process includes school organizational structure, culture, motivation, leadership, decision making, communication, change, curriculum, teaching process, career development; outputs include student learning achievement, teacher 85 Journal of Education and Practice Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 performance, student development, worker development, student dropout, employee discharge, student absenteeism, employee absenteeism, employee management relations, school community relations, student attitudes to promote school, job satisfaction of workers. Human resources consist of educators, educator staff and lecturers. Lecturers are one of the subsystems that play an important role, because lecturers interact directly with other academics and the communities to transfer knowledge, attitude and skills to students to study, analyze, and research science and technology, make new discoveries, change agents, build civilization that are creatively adaptive. The Law of Indonesia Republic Number 12 of 2012 concerning Higher Education, chapter 1 verse 14 defines that lecturers are professional educators and scientists with the main task of transforming, developing and disseminating Science and Technology through Education, Research, and Community Service. Lecturers act as educators, instructors, student advisers, researchers, and devotees. Their activities tend to interact with students. Lecturers and students often do activities together, and in their togetherness there is a transformation of knowledge, attitudes, skills, so that the quality of lecturers greatly affects the quality of students. Lecturers tend to educate students to become qualified. The performance of lecturers at university is a major thing that needs to get the attention of all parties, especially from the heads of departments, university leaders, and other stakeholders. This can be understood by the performance of professional lecturers who will be able to support the achievement of higher quality educational processes, outputs and outcomes. However, the problem of lecturer performance is not a simple problem, but rather a very complex problem because it involves many elements that are interrelated, mutually influencing, and interdependent with each other. The quality of lecturers can be seen from their performance. 1. INTRODUCTION Qualified lecturers have high performance in tridharma of higher education, namely the field of education and teaching, the field of research and the field of community service. In addition to be an educator and instructor, lecturers also play a role as mentors in academic field in final project or thesis. They are also required to participate in self-capacity building activities through seminars, resource persons as support activities. All of these activities, each lecturer must plan their workload at the beginning of the new academic year and will be assessed at the end of the academic year. Each of lecturer workload plans must meet the teaching, research, and community service burden in each semester in academic year. Although the workload plan has been made by lecturers, in the process there are still constraints, including the limited facilities and infrastructure as well as the competence of lecturers in their knowledge, limited research funds and community service available, guidance and other supporting activities. High competition occurs to get these limited funds. In addition, the lecturer also has difficulty to carry out community service. These difficulties cannot be separated from psychological factors, personality, science and funds that will be used for the process. A preliminary study of lecturers’ performance at Sari Mutiara Indonesia University is conducted in academic year 2017/2018. It shows that three indicators of lecturer performance, namely: 1) education and teaching, 2) research and 3) community service. Obtained lecturer performance data from 9 (nine) study programs with accreditation B are: (1) very good category 14 people (16%), (2) good 52 people (59.7%), and (3) fair 21 people (24.1%). Related to the performance of lecturers at the state university and compared to the performance of lecturers at private universities, especially Sari Mutiara Indonesia University it can be said to be still low. The tendency categories are very good, good, fairly good, poor, and very poor. Performance in education and teaching is in the good and fairly good category. Performance in research is not good enough, and performance in writing articles in journals is not good. The performance in community service is in the poor and very poor categories. Although it has been urged by lecturers of the University of Sari Mutiara Indonesia to improve its performance, especially in research performance, publishing articles in journals, and community service, but there has not been much change in 2018/2019. 1. INTRODUCTION Someone succeeds due to the results of his efforts. External locus of control is a belief in the success of a person is under control outside himself, such as due to interference or help from others, luck and destiny. Both of locus controls have advantages and disadvantages. Internal locus of control builds personality which tends to be individual, hard-working, while external locus of control builds a personality that tends to be social, like to work together. In line with this, locus of control has a positive or negative effect on teamwork, but it has a positive effect on work ethic and performance. p p Work ethic has a positive effect on performance (Hardiansyah and Yanwar, 2015; 150). Furthermore, Dewi and Utomo (2015: 55) investigate that work ethic has a direct positive effect on employee performance. Based on the results of the research presented, it is concluded that work ethic has a direct positive effect on employees; and it is considered that work ethic also has a direct positive effect on the lecturers’ performance at Sari Mutiara Indonesia University. Lecturers in carrying out their work are expected to work together and help each other so that the work carried out as a duty obligation can be increased. Lecturers who have relatively similar scholarship are formed into a team called the Lecturer in the Study Group. It examines researches, develops, and devotes the knowledge being studied to the community, so it must work together. It becomes a teamwork and it is expected to be effective in solving the problem, effectively achieving its performance targets. The effectiveness of the team lies in the communication and cooperation that is built. In line with this, Lawasi & Triatmanto (2017: 47) find that teamwork influences positively and directly on employee performance. Hatta, Musnadi, and Mahdani (2017: 70) explain that teamwork also has a direct effect on performance. In addition to teamwork, Rahma, Fuad, and MIR (2011: 11) organizational culture variables are also variables that can affect performance. Based on the results of this research, it is synthesized that teamwork had a direct positive effect on employee performance. Based on the description and research results above, it show that performance is influenced by many factors, both those found based on the results of the study or based on theoretical explanations. In addition, the description above also shows the gap between the expected performances. 1. INTRODUCTION Based on preliminary studies on the lecturers performance at Sari Mutiara Indonesia University, it turns out that the results of the research have not been much, and nothing has been published in accredited or international journals that indexed by Scopus, as well as the results of community service have not shown satisfactory results. It shows that there are problems in the lecturers' performance. Many causes of lecturer performance is not as expected. The Organizational Behavior Integration Model developed by Colqiutt, LePine and Watson (2009: 8) shows that performance is influenced by a number of variables in three mechanisms, namely individuals, groups and organizations, and one individual characteristic, these effects directly or indirectly. Organizational mechanism has organizational culture variables and organizational structure. Group mechanism consists of leadership style, behavior, power, and influence variables. Individual characteristics consist of personality variables, cultural values, and abilities. Individual mechanisms have variables of job satisfaction, stress, motivation, beliefs, ethics, fairness, learning, and decision making. Theory of Mathis and Jackson (2006: 115) reveals the factors that influence individual performance consists of 3 major parts, namely: (1) effort devoted: motivation, work ethics, attendance, and task design, (2) Individual ability: talent , interests, personality, and innovation, (3) organizational support: training and development, equipment and technology, work standards, and colleagues. It is further said that organizational culture, 86 Journal of Education and Practice www.iiste.org iste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 organizational commitment, interpersonal communication, the effectiveness of performance management control system has a direct and positive effect on lecturer performance. In addition, self-efficacy, self-control (locus of control), work ethic, and teamwork influence lecturers' performance. organizational commitment, interpersonal communication, the effectiveness of performance management control system has a direct and positive effect on lecturer performance. In addition, self-efficacy, self-control (locus of control), work ethic, and teamwork influence lecturers' performance. Saraswathi, Dewi, and Piartini (2017: 2257) investigate that self-efficacy directly affects the performance of land employees. Iroegbu (2015: 170) examines based on Albert's theory of social cognition for workers, self- efficacy is related to performance. Focus of control also has a positive direct effect on lecturer performance. Sumijah (2015) says that locus of control is one of important aspect in human personality. Focus of control consists of two types, namely internal locus of control and external locus of control. Internal locus of control is a belief in the success of a person is under his own control. 1. INTRODUCTION If the problem does not receive serious attention and is immediately addressed, the consequences affect efforts in the field of education and are the main source for the decline in the quality of graduates. Therefore, in order to improve the lecturers’ performance of Sari Mutiara Indonesia University, the research on the theoretical model development of lecturer performance can be conducted. In line with the explanation above, lecturer performance is influenced by various factors, including: leadership, organizational culture, interpersonal communication, organizational commitment, work motivation, work ethic and teamwork. Therefore, in order to overcome the problems of lecturer performance, research on the effects of Organizational Culture, Team Cooperation, Work Motivation and Work Ethics on the Performance of Lecturers at Sari Mutiara Indonesia University in Medan should be carried out. 2.1. Lecturer Performance 2.1. Lecturer Performance Performance means the implementation of work achievement or work results . George and Gareth (2005: 176) state that performance is the result of assessing one's behavior, which includes how well someone has completed a task or job. On the other hand, Owen (1987: 7) defines performance as the result of an assessment of how effectively and efficiently a manager utilizes resources to achieve goals. While, Gibson (1997: 118) explains performance is the level of success in carrying out tasks and the ability to achieve goals predetermined. Performance is a management measurement tool used to assess the level of accountability of someone in carrying out their duties. Based on the description above, the understanding of the lecturers 'performance in this study is the lecturers' performance and efforts in carrying out their roles and functions. The performance indicators of the lecturers are: (1) education and teaching, among others: compiling learning plans, conducting learning, and guiding the final project, (2) research fields including: involvement in research, making art or technology, writing books / modules, and writing journals, and (3) community service, among others: conducting activities in the community, conducting academic guidance, and fostering student activity units. 2.3. Work Motivation Work motivation in an organization can be considered simple and complex problem, because basically humans are easy to be motivated by giving what they want. Motivation is used to encourage employees to complete tasks that have been charged. Creating high performance can be grown through motivational encouragement. Sutrisno (2013: 109) states that motivation is an actor who encourages someone to do a certain activity. It is often interpreted as a factor driving a person's behavior. Mangkunegara (2012: 61) states that motivation is a condition or energy that moves employees to be directed to achieve organizational goals. Hamalik (2004: 173) explains motivation can be basic or internal impulses and intensive outside the individual. Nawawi (2003) states motivation is a condition that encourages or becomes the cause of someone doing an activity that takes place consciously in order to achieve company goals. There are several objectives that can be obtained. Based on the above theory, it is concluded that work motivation is a desire that can encourage lecturers to work well to achieve organizational goals expressed through a questionnaire filled by lecturers with indicators of responsibility in carrying out assignments, having a happy feeling at work, always try to outperform others, prefers the achievements of what they do, works in the hope of getting incentives, and likes to get praise from what they do. 2.4. Work Ethic Work ethic is a person's ethics in the workplace which is based on a constellation of attitudes and beliefs about work values. Marsky (2010: 4) says that ethic explains to someone about the existence of rules where behavior can be justified or not justified. The meaning of the work ethic is more referring to the quality of the personality of the worker which is reflected through the full performance in various dimensions and lives. Slocum & Hellriegel (2009: 20) views the importance of work ethics in the organization. It is one of the competencies that make the organization effective. Therefore, employees should provide an optimal work ethic that can advance the institution well. Thus, work ethic influences employee performance. Based on some of the explanations above, it is concluded that the work ethic is the attitude shown by someone who is the totality of his personality in expressing, viewing, believing, and giving meaning to something, which drives him to act and work optimally. Indicators of work ethics, namely, work sincerely and responsibly, work completion, spirit working, love work, creativity, diligence, work performance orientation, and work are services. 2.2. Organizational Culture In accordance with the context of empowering human resources, in order to produce professional employees an In accordance with the context of empowering human resources, in order to produce professional e 87 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 Journal of Education and Practice Journal of Education and Practice ste.org organization is needed. Pabundu (2010: 1) explains that in Indonesia, organizational culture is introduced in the 1990s when it is widely discussed about cultural conflicts, how to maintain Indonesian culture and the cultivation of new values. Along with that, organizational culture is then included in the curriculum of various education, training, guidance and counseling programs, both within the tertiary institutions and government agencies as well as in various companies. organization is needed. Pabundu (2010: 1) explains that in Indonesia, organizational culture is introduced in the 1990s when it is widely discussed about cultural conflicts, how to maintain Indonesian culture and the cultivation of new values. Along with that, organizational culture is then included in the curriculum of various education, training, guidance and counseling programs, both within the tertiary institutions and government agencies as well as in various companies. Organizational culture is a system of spreading trust and values that develop in an organization and direct the behavior of its members. According to Wirawan (2007: 10) organizational culture as norms, values, assumptions, beliefs, philosophies, organizational habits, develop over a long period of time by the founders, leaders and members of the organization that are socialized and taught to new members and applied in organizational activities that affect the mindset, attitudes, and behavior of organizational members in producing products, serving consumers, and achieving organizational goals. Meanwhile according to Robbins (2001: 510), Organizational culture refers to a system of shared meaning held by members that distinguishes the organization from other organizations. g Based on the study above, it is concluded that organizational culture is a set of values, norms and basic assumptions that become the organizational guidelines adopted by lecturers in carrying out their work to achieve organizational goals revealed through a questionnaire filled by lecturers with indicators of risk taking, attention to detail, orientation to results , people orientation, team orientation, aggressiveness, and stability. 2.5. Teamwork William (2000: 6) teamwork is the ability to work together towards a common vision, the ability to direct individual achievement toward organizational goals. It is a stimulus that enables people to achieve extraordinary results. Furthermore Stephen (2000: 4) teamwork produces many ideas to come in which this will be the key to move from a control to commitment. Sharpen (1996: 19) teamwork is collaboration formed to achieve goals, each of member has part of the tasks assigned, to make the team more cohesive and how they will work together. Based on some of the explanations above, it can be concluded that teamwork is feelings and actions taken by a group of individuals through working and interacting in terms of achieving shared goals, with indicators, namely knowing goals, giving freedom, having a sense of belonging, making continuous improvements, and mutual respect. 88 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 3.2. Data Analysis Technique Analysis of the data used in this study includes descriptive analysis, test requirements analysis, and hypothesis testing. There are five variables analyzed, namely: organizational culture, teamwork, work ethic, work motivation, and lecturer performance. According to the research hypothesis and research objectives, Hypothesis testing is done using path analysis. 3.1. Place and Time of Research 3.1. Place and Time of Research This research is conducted at Sari Mutiara Indonesia University in Medan, for three months, from April to June 2019. The first month is used to administer research licenses, instrument trials, and instrument refinements. Furthermore, the last two months are used to collect data, analysis data, and writing reports on research results. Population in this study is all Sari Mutiara Indonesia University Lecturers in Medan 2019/2020, consisted of 193 lecturers, while the sample is 128 lecturers. 3.3. Research Hypothesis Test To test the hypothesis uses path analysis with the help of SPSS for Windows version 25.0. In testing hypotheses uses a significance level of α of 0.05. In connection with the use of path analysis must be described in a diagrammatic structure of causal relationships with cause and effect variables. Substructure 1 is the influence of organizational culture (X1) and teamwork (X2) on work motivation (X3). Sub-structure 2, namely the influence of organizational culture (X1) and teamwork (X2) on work ethic (X4). Sub-structure 3: the influence of organizational culture (X1) and teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5). Furthermore, the results of statistical calculations are obtained so that correlation coefficients and path coefficients can be obtained, as presented in Table 2 below. 3.4. Research Result Summary of Calculation Results for Path Coefficients * All significant correlation coefficients {tcount is greater than t table (5%) = 1.96} Based on the price of the correlation coefficient and the path coefficient obtained from the calculation results, a path diagram can be drawn which is a fixed model or theoretical model that illustrates the causalities relationships between the research variables that determine the performance of Sari Mutiara Indonesia University lecturers in Medan as shown in Figure 1 lecturers in Medan as shown in Figure 1 Figure 1 The Empiric Casual Realtionship of X1, X2, X3, dan X4 toward X5 Based on the calculation results obtained, it shows the value of Fcount = 36.071 with a significance value of 0.000, or a significance value of Fcount <0.05, this means that Ho is rejected or Ha is accepted. Thus, it can be concluded that organizational culture, teamwork, work motivation, and work ethics have a direct positive effect on the performance of Sari Mutiara Indonesia University lecturers in Medan so that individual testing can be carried out. In accordance with the path diagram of the research, variables obtained based on the theory and the results of hypothesis testing, the relative influence of exogenous variables on endogenous variables. Based on the results of calculations that refer to the decomposition of correlations, a summary of the relative influence of organizational culture (X1), teamwork (X2), work motivation (X3) and work ethic (X4) on the lecturers' performance (X5) is summarized in Table 3 below. lecturers in Medan as shown in Figure 1 Figure 1 The Empiric Casual Realtionship of X1, X2, X3, dan X4 toward X5 Figure 1 The Empiric Casual Realtionship of X1, X2, X3, dan X4 toward X5 Based on the calculation results obtained, it shows the value of Fcount = 36.071 with a significance value of 0.000, or a significance value of Fcount <0.05, this means that Ho is rejected or Ha is accepted. Thus, it can be concluded that organizational culture, teamwork, work motivation, and work ethics have a direct positive effect on the performance of Sari Mutiara Indonesia University lecturers in Medan so that individual testing can be carried out ca ed out. In accordance with the path diagram of the research, variables obtained based on the theory and the results of hypothesis testing, the relative influence of exogenous variables on endogenous variables. 3.4. Research Result Based on the results of data processing from research instruments, descriptive statistical calculation results obtained from variables of organizational culture (x1), teamwork (x2), work motivation (x3), work ethic (x4), and lecturer performance (x5), including calculating the value of central tendency and the size of its spread. This is done to know the general description of the meaning contained in the distribution of data obtained, such as the mean, median, modus, the size of the spread tendency, such as standard deviation, variance, range, minimum and maximum score, minimum and maximum ideal, mean ideal, standard deviation ideal. Calculations and summaries of the results of descriptive analysis can be seen in Table 1. p y Table 1. Summary of Results of Descriptive Statistical Analysis of Research Variables Statistic X1 X2 X3 X4 X5 N Valid 128 128 128 128 128 Mean 112,56 116,47 116,70 122,38 241,55 Median 113,00 115,50 117,00 121,00 240,00 Mode 113 115 117 121 240 Std. Deviation 7,933 14,059 12,981 11,191 24,479 Variance 62,925 197,653 168,497 125,230 599,226 Range 33 60 61 56 111 Minimum 98 84 84 100 190 Maximum 131 144 145 156 301 Minimum Ideal 29 30 30 33 68 Maximum Ideal 145 150 150 165 340 Mean Ideal 87 90 90 99 204 Stand. Deviation Ideal 19,33 20,00 20,00 22,00 45,33 h h l f i i l l l i b i d h l i ffi i Furthermore, the results of statistical calculations are obtained so that correlation coefficients and path coefficients can be obtained, as presented in Table 2 below. 89 Journal of Education and Practice iiste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 Table 2. Summary of Calculation Results for Path Coefficients Number of hypothesis Correlation Coefficient * Path coefficient Tcount significance Information 1. = 0,464 p = 0,348 4,473 0,000 Mean Paths 2. = 0,466 p = 0,351 4,504 0,000 Mean Paths 3. = 0,491 p = 0,390 5.005 0,000 Mean Paths 4. = 0,436 p = 0,307 3,949 0,000 Mean Paths 5. = 0,531 p = 0,205 2,812 0,006 Mean Paths 6. = 0,493 p = 0,170 2,378 0,019 Mean Paths 7. = 0,621 p = 0,298 3,757 0,000 Mean Paths 8. = 0,607 p = 0,267 3,362 0,001 Mean Paths * All significant correlation coefficients {tcount is greater than t table (5%) = 1 96} Table 2. 4. DISCUSSION Based on the results of testing, the first hypothesis is obtained a significant path coefficient between organizational culture and work motivation, namely: p31 = 0.348, and based on the calculation results obtained direct influence of organizational culture on work motivation of 0.121. Organizational culture has a direct positive effect on work motivation, of which 12.10% of changes in work motivation can be determined by organizational culture. The findings of this study support the theory used as a basis for proposing theoretical models of research variables, namely the integration model of organizational behavior that explains that organizational culture directly influences work motivation (Colqiutt, LePine and Watson, 2009: 8). The findings of this study, namely: organizational culture in direct significant positive effect on work motivation lecturers are in accordance with the results of research and theory referred to in this study. The results of Koesmono's research (2005) finds that organizational culture directly has a significant positive effect on work motivation. Thus, the results of this study find that organizational culture has a direct positive effect on work motivation. The second hypothesis testing results is obtained a significant path coefficient between teamwork with work motivation, namely: p32 = 0.351, and based on the calculation results is obtained direct influence of teamwork on work motivation of 0.123. Teamwork has a direct positive effect on work motivation, where 12.30% of changes in work motivation can be determined by teamwork. The findings of this study support the theory used as a basis for proposing a theoretical model of research variables, namely the integration model of organizational behavior which explains that teamwork directly influences work motivation. The findings of this study, teamwork directly has a significant positive effect on student work motivation is in accordance with the results of the study and the theory referred to in this study. y y The third hypothesis testing results is obtained a significant path coefficient between organizational culture with work ethics, namely: p41 = 0.390. Based on the calculation results, it is obtained direct influence of organizational culture on work ethic of 0.152. Organizational culture has a direct positive effect on work ethic, of which 15.20% changes in work ethic can be determined by organizational culture. The fourth hypothesis testing results is obtained a significant path coefficient between teamwork and work ethic, p42 = 0.307. 3.4. Research Result Based on the results of calculations that refer to the decomposition of correlations, a summary of the relative influence of organizational culture (X1), teamwork (X2), work motivation (X3) and work ethic (X4) on the lecturers' performance (X5) is summarized in Table 3 below. 90 Journal of Education and Practice www.iiste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 Table 3. Summary of Calculation Results of Relative Effects of Organizational Culture (X1), Teamwork (X2), Work Motivation (X3), and Work Ethic (X4) on Lecturer Performance (X5) Var Effect Non pathway Total Correlati on Directly to X5 Indirectly to X5 trough X1 X2 X3 X4 S U X1 0,205 - - 0,138 0,131 - 0,056 0,530 r15 X2 0,170 - - 0,139 0,116 - 0,068 0,493 r25 X3 0,298 - - - - 0,174 0,149 0,621 r35 X4 0,267 - - - - 0,175 0,165 0,607 r45 The total effect consisting of direct and indirect influence of organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5) of 0.331. The strength of organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) determine changes in lecturer performance (X5) of 33.90%, while the rest are in the spurious component of 0.098 and unanalyzed components of 0.111. Total direct and indirect effects, spurious, and unanalyzed due to organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5) = 0.331 + 0.098 + 0.111 = 0.540 (correction: r2 = 0.540), while the influence of other factors outside the organizational culture (x1), teamwork (x2), work motivation (x3), and work ethic (x4) that is equal to 1-0.540 = 0.460 = 46% with path coefficient, namely: ρx5e5 = √1 - 0.540 = 0.678. Journal of Education and Practice www.iiste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 Table 3. 3.4. Research Result Summary of Calculation Results of Relative Effects of Organizational Culture (X1), Teamwork (X2), Work Motivation (X3), and Work Ethic (X4) on Lecturer Performance (X5) Var Effect Non pathway Total Correlati on Directly to X5 Indirectly to X5 trough X1 X2 X3 X4 S U X1 0,205 - - 0,138 0,131 - 0,056 0,530 r15 X2 0,170 - - 0,139 0,116 - 0,068 0,493 r25 X3 0,298 - - - - 0,174 0,149 0,621 r35 X4 0,267 - - - - 0,175 0,165 0,607 r45 The total effect consisting of direct and indirect influence of organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5) of 0.331. The strength of organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) determine changes in lecturer performance (X5) of 33.90%, while the rest are in the spurious component of 0.098 and unanalyzed components of 0.111. Total direct and indirect effects, spurious, and unanalyzed due to organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5) = 0.331 + 0.098 + 0.111 = 0.540 (correction: r2 = 0.540), while the influence of other factors outside the organizational culture (x1), teamwork (x2), work motivation (x3), and work ethic (x4) that is equal to 1-0.540 = 0.460 = 46% with path coefficient, namely: ρx5e5 = √1 - 0.540 = 0.678. The total effect consisting of direct and indirect influence of organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5) of 0.331. The strength of organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) determine changes in lecturer performance (X5) of 33.90%, while the rest are in the spurious component of 0.098 and unanalyzed components of 0.111. Total direct and indirect effects, spurious, and unanalyzed due to organizational culture (X1), teamwork (X2), work motivation (X3), and work ethic (X4) on lecturer performance (X5) = 0.331 + 0.098 + 0.111 = 0.540 (correction: r2 = 0.540), while the influence of other factors outside the organizational culture (x1), teamwork (x2), work motivation (x3), and work ethic (x4) that is equal to 1-0.540 = 0.460 = 46% with path coefficient, namely: ρx5e5 = √1 - 0.540 = 0.678. 4. DISCUSSION Based on the calculation results, it is obtained that the direct influence of teamwork on work ethic by 0.094. Teamwork has a direct positive effect on work ethic, in which 9.40% changes in work ethic can be determined by teamwork. These findings support the results of Ajeng's (2016) concludes that several benefits of good work are among them (1) creating a comfortable working atmosphere, (2) creating cohesiveness in work, (3) increasing collaboration and (4) increasing productivity. The fifth hypothesis testing result is obtained a significant path coefficient between organizational culture and lecturer performance, namely: p51 = 0.205. Based on the calculation results, it is obtained direct influence of organizational culture on performance by 0.042. Organizational culture has a direct positive effect on organizational commitment, where 4.20% of changes in performance can be determined by organizational culture. Based on the relationship model of organizational culture with performance and satisfaction, Robbins and Judge (2009: 608) explain that organizational culture directly influences performance and satisfaction. The integration model of organizational behavior from Colquitt, LePine, and Wesson (2009: 9) explain that the organizational culture influences indirectly on performance through motivation, and job satisfaction. The concordance of the findings of this study supports the results of previous studies. It shows that the theory is still accurately used to answer relevant issues organization to performance. 91 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 The sixth hypothesis testing results is obtained a significant path coefficient between teamwork and performance, namely: p52 = 0.170. Based on the calculation results, it is obtained direct influence of teamwork on performance by 0.029. Teamwork has a direct positive effect on performance, of which 2.90% of performance changes can be determined by teamwork. The findings of this study shows that teamwork directly has a significant positive effect on lecturers' performance is in accordance with the results of the research and the theories referenced in this study. The seventh hypothesis testing result is obtained a significant path coefficient between work motivation and performance, namely: p53 = 0.298. Based on the calculation results, it is obtained direct influence of work motivation on performance by 0.089. Work motivation has a direct positive effect on performance, of which 8.90% changes in performance can be determined by work motivation. 4. DISCUSSION The findings of this study support the theory used as a basis for proposing a theoretical model of research variables, namely the integration model of organizational behavior which explains that motivation directly influences performance (Colqiutt, LePine and Watson, 2009: 152). The performance component model from Mathis and Jackson in Purba (2009: 10) which explains that motivation directly influences individual performance. The findings of this study support the research results of Oluseyi & Hammed (2009) who find a significant positive correlation between work motivation and performance. The findings of this study show that work motivation directly has a significant positive effect on lecturer performance. It is in accordance with the results of the study and the theory referred to in this research. The eighth hypothesis testing results is obtained a significant path coefficient between work ethic and performance, namely: p54 = 0.267. Based on the calculation results, it is obtained direct influence of work ethic on performance of 0.071. Work ethic has a direct positive effect on performance, of which 7.10% changes in performance can be determined work ethic. The findings of this study show that the work ethic has a positive and significant direct effect on the performance of lecturers at Sari Mutiara Indonesia University in Medan. In accordance with the theory with the results of the study through the fourth, fifth, sixth, seventh and eighth hypothesis testing result, it is found that the variable work motivation provides the greatest total effect on performance compared to the total effect given by organizational culture, teamwork and work ethic on performance. It can be stated that the effect of total work motivation is 8.88%, work ethic 7.13%, organizational culture 4.20% and teamwork 2.89% on performance. Based on the eight hypotheses testing result as described above, a fixed model or theoretical model is found that casuistic relationships between research variables that determine performance which is the development of several theories, primarily the Integrative Model of Organizational Behavior" from Colquitt, LePine, and Wesson. Thus, the results of research through hypothesis testing accept the eight proposed research hypotheses. It finds a new finding in the form of a fixed model or theoretical model of lecturer performance. It illustrates the structure of causal relationships between organizational culture variables, teamwork, work motivation, work ethics, and lecturer performance as in Figure 2. p g Figure 2. 5. CONCLUSION Based on the research results, it is obtained that organizational culture, teamwork, work motivation and work ethic have a direct positive effect on lecturer performance. This shows that lecturers' performance can be improved if organizational culture, teamwork, work motivation and work ethic are improved. The findings in this study are consistency with the theoretical model used. The findings are expected to be used as a comparison for relevant research in the future, especially those related to performance. REFERENCES Barsky Allan Edward. 2010. Ethics and Values in Social Works, An Integrated Approach for a Comprehensive Curriculum. Oxford: Oxford University Press, Inc. Colcuit, Jason A., Jeffery A., LePine., And Michael J. Wesson. 2009. Organizational Behavior: improving performance and commitment in the workplace. New York: McGraw-Hill. Dewi, Karina and Hardi, Utamo. 2015. 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The influence of leadership style, teamwork, and compensation, on job satisfaction and its impact on the performance of employees of PT. PLN (Persero) Aceh Territory. Journal of Masters in Management, Faculty of Economics and Business Unsyah, Vol. 1, No. September 1, 2017. g y y p Iroegbu, Manasseh N. 2015. Self Efficacy and Work Performance: A Theoretical Framework ofAlbert Bandura's Model, Review of Findings, Implications and Directions for Future Research". Journal of Psychology and Behavioral Sciences Vol. 4, No. 4. Koesmono, Friend. 2005, The Effect of Organizational Culture on Motivation and Job Satisfaction and Employee Performance in the Medium Scale Timber Processing Industry Sub Sector in East Java, Journal of Management & Entrepreneurship, Vol. 7, No. 2, September 2005. Laws of Eva Silvani and Boge Triatmanto. 2017. The Effect of Communication, Motivation, and Teamwork on Improving Employee performance. Journal of management and Entrepreneurship, Vol. 5, No. 1. Lunenburg, Fred C., and Allan C. Ornstein, 2000. Educational Administration. Concepts and Practices. Third Lunenburg, Fred C., and Allan C. Ornstein, 2000. Educational Administration. Concepts and Practic Edition. USA: Wadsworth, Thomson Learning. Mangkunegara, Anwar Prabu. 2012. HR Performance Evaluation, sixth print. Bandung: Refika Aditama. Mathis Robert L, and John H. Jackson. 4. DISCUSSION The Lectures’ Performance Models of Sari Mutiara Indonesia University in Medan Figure 2. The Lectures’ Performance Models of Sari Mutiara Indonesia University in Medan Figure 2. The Lectures’ Performance Models of Sari Mutiara Indonesia University in Medan 92 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.11, No.27, 2020 Based on the model findings as shown in Figure 2, it can be explained that increasing the strengthening of organizational culture directly causes an increase in work motivation and work ethic. Furthermore, increasing the strengthening of teamwork directly leads to increas work motivation and work ethic. Increasing work motivation and work ethic directly lead to strengthening lecturer performance. In addition, strengthening organizational culture and increasing teamwork directly lead to strengthening the performance of Sari Mutiara Indonesia University lecturers in Medan. REFERENCES 2006, Human Resources Management; Human Resource Management, Mangkunegara, Anwar Prabu. 2012. HR Performance Evaluation, sixth print. Bandung: Refika Aditama. 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https://openalex.org/W2895919447	https://research.tue.nl/files/111194087/1_s2.0_S0022509618303636_main.pdf	English	null	Towards acoustic metafoams: The enhanced performance of a poroelastic material with local resonators	Journal of the mechanics and physics of solids/Journal of the Mechanics and Physics of Solids	2,019	cc-by	16,193	Document status and date: Published: 01/03/2019 Document status and date: Published: 01/03/2019 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. p • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights C i ht d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners 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 Y t f th di t ib t th t i l it f fit ki ti it i l i • You may not further distribute the material or use it for any profit-making activity o • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Towards acoustic metafoams Citation for published version (APA): Lewińska, M. A., van Dommelen, J. A. W., Kouznetsova, V. G., & Geers, M. G. D. (2019). Towards acoustic metafoams: the enhanced performance of a poroelastic material with local resonators. Journal of the Mechanics and Physics of Solids, 124, 189-205. https://doi.org/10.1016/j.jmps.2018.10.006 Document license: CC BY Document license: CC BY DOI: 10.1016/j.jmps.2018.10.006 Document status and date: Published: 01/03/2019 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There c important differences between the submitted version and the official published version of record. Peopl interested in the research are advised to contact the author for the final version of the publication, or vis DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication DOI: 10.1016/j.jmps.2018.10.006 Download date: 24. Oct. 2024 ∗Corresponding author. E-mail address: v.g.kouznetsova@tue.nl (V.G. Kouznetsova). Towards acoustic metafoams: The enhanced performance of a poroelastic material with local resonators wi ´nska, J.A.W. van Dommelen, V.G. Kouznetsova ∗, M.G.D. Geers M.A. Lewi ´nska, J.A.W. van Dommelen, V.G. Kouznetsova ∗, M.G.D. Geers M.A. Lewi ´nska, J.A.W. van Dommelen, V.G. Kouznetsova ∗, M.G.D. Geers Department of Mechanical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, PO Box 513, The Netherlands t of Mechanical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, PO Box 513, The Netherlands a r t i c l e i n f o Article history: Received 2 May 2018 Revised 7 September 2018 Accepted 3 October 2018 Available online 12 October 2018 Keywords: Acoustic metamaterials Poroelastic materials Local resonance Viscothermal dissipation Acoustic foams Metafoams Acoustic foams are commonly used for sound attenuation purposes. Due to their porous microstructure, they eﬃciently dissipate energy through the air ﬂowing in and out of the pores at high frequencies. However, the low frequency performance is still challenging for foams, even after optimisation of their microstructural design. A new, innovative, approach is therefore needed to further improve the acoustic behaviour of poroelastic materials. The expanding ﬁeld of locally resonant acoustic metamaterials shows some promising exam- ples where resonating masses incorporated within the microstructure lead to a signiﬁcant enhancement of low frequency wave attenuation. In this paper, a combination of tradi- tional poroelastic materials with locally resonant units embedded inside the pores is pro- posed, showing the pathway towards designing acoustic metafoams : poroelastic materials with properties beyond standard foams. The conceptual microstructural design of an ide- alised unit cell presented in this work consists of a cubic pore representing a foam unit cell with an embedded micro-resonator and ﬁlled with a viscothermal ﬂuid (air). Analysis of complex dispersion diagrams and numerical transmission simulations demonstrate a clear improvement in wave attenuation achieved by such a microstructure. It is believed that this demonstrates the concept, which serves the future development of novel poroelastic materials. © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) https://doi.org/10.1016/j.jmps.2018.10.006 0022-5096/© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 Contents lists available at ScienceDirect 1. Introduction Although such solutions, based on meso-scale modiﬁcations of porous materials, can offer an im- provement of the attenuation level at low frequencies, they also require signiﬁcantly different designing and manufacturing steps. Acoustic metamaterials are another class of materials that are promising in the context of low frequency sound at- tenuation. The dedicated microstructural design of these materials results in higher attenuation level relative to what is achieved with traditional materials. A number of metamaterials achieve their extraordinary properties through a local res- onance phenomenon. The prime manufactured example of a solid material generating so called band gaps and acting as a total wave reﬂector due to resonating inclusions, which effectively prohibits the propagation of waves at certain frequencies, has been reported rather recently in Liu et al. (20 0 0) . Boutin and Becot (2015) have proposed a material entirely composed of Helmholtz resonators (of signiﬁcant size in order to attain low frequency performance), also achieving an improvement in sound attenuation. This concept was further developed in Griﬃths et al. (2017) , proposing a porogranular material design with resonators made of soft elastomer shells. Recently, the presence of shear wave band gaps and left-handed behaviour of a cellular material entrained with water have been demonstrated experimentally in Dorodnitsyn and Van Damme (2016) , where the dispersive properties of the material are achieved through resonance of the lattice walls. A special type of acoustic metamaterials are the structures incorporating membrane resonance. Such materials are typ- ically designed to improve absorption performance as is the case for a “dark” acoustic metamaterial ( Mei et al., 2012 ) or in the work of Ma et al. (2014) where hybrid resonance of a decorated membrane is exploited. Yang et al. (2015) have proposed a combination of a membrane-type and a Helmholtz resonator, showing that such degenerate resonators, when properly coupled, can also serve as an effective absorber. Recently, atypical acoustic behaviour of rigid permeable materials with thin elastic membranes embedded in a rigid microstructure has been demonstrated numerically and experimentally in Venegas and Boutin (2017) . In many studies concerning metamaterials, especially those involving ﬂuid structure interaction, material (ﬂuid) losses are either not included in the model or incorporated in a simpliﬁed way through a phenomenological parameter. 1. Introduction Protection against noise pollution is nowadays a compelling problem due to progressing industrialisation and om- nipresent sources of disturbing and harming sounds. In particular, low frequency noise attenuation still awaits for an ef- ﬁcient solution, preferably based on a material suitable for mass production. In terms of sound absorption, porous materials like acoustic foams, ﬁber glass or mineral wool are usually quite eﬃ- cient due to their large microstructural air-solid interface area, which results in high viscothermal dissipation. However, the eﬃciency of these materials for low frequencies is signiﬁcantly lower than for mid and high frequency sounds ( Yang and Sheng, 2017 ). Moreover, studies investigating the relationship between the microstructure of conventional acoustic foams and their performance ( Chevillotte and Perrot, 2017; Doutres et al., 2011; 2014; Gao et al., 2017 ) show that the shift of the M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 190 absorption peak towards lower frequencies by means of a detailed pore design or by increasing the reticulation rate is rather limited (if it can be achieved, it comes at a price of lower eﬃciency). An alternative to improvements at the microstructural level is to modify the materials at a scale larger than the pore size (i.e. meso scale). It has been observed that meso-perforations (air inclusions) introduced to a porous material may be beneﬁcial for its acoustic performance at low frequencies (see the review paper Sgard et al. (2005) on double porosity me- dia and the references therein). Embedding other types of meso-scale inclusions in foam-like materials has been explored for the same purpose. A distribution of Helmholtz resonators in the bulk of a poroelastic material has been introduced in Lagarrigue et al. (2013) and Doutres et al. (2015) , and was recently optimised in Park et al. (2017) . Note that, in order to achieve low frequency performance of such panels, the sizes of resonating cavities need to be rather large (order of cen- timetres). Moreover, inspired by the concept of phononic crystals ( Deymier, 2013 ) (i.e. a periodic arrangement of scatterers showing sound attenuation via destructive wave interference), a periodic arrangement of metal rods has been proposed for example in Weisser et al. (2016) and a double porosity material with an array of mass inclusions has been reported in Cui and Harne (2017) . 1. Introduction / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 191 M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 191 Fig. 1. (a) Unit cell without a resonator, (b) unit cell with a light resonator, (c) unit cell with a resonator with a heavy mass. Light grey represents polyurethane (PU), dark grey is the heavy mass at the tip of the resonator, light blue is the air domain. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 1. (a) Unit cell without a resonator, (b) unit cell with a light resonator, (c) unit cell with a resonator with a heavy mass. Light grey represents polyurethane (PU), dark grey is the heavy mass at the tip of the resonator, light blue is the air domain. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) −ρs ω 2 u = ∇ · σ, (1) where u is the displacement vector, ω the angular frequency, ρs the solid density and σ is the stress tensor given by σ = 4 C s : ∇ u , with 4 C s being the total symmetric fourth order elasticity tensor, symbol ∇ denotes the gradient operator. At the microstructural level, dissipative effects result from the viscothermal behaviour of the ﬂuid. Therefore a complex description of the ﬂuid domain is adopted. The governing equations for a lossy compressible ﬂuid can be written as: where u is the displacement vector, ω the angular frequency, ρs the solid density and σ is the stress tensor given by σ = 4 C s : ∇ u , with 4 C s being the total symmetric fourth order elasticity tensor, symbol ∇ denotes the gradient operator. At the microstructural level, dissipative effects result from the viscothermal behaviour of the ﬂuid. Therefore a complex description of the ﬂuid domain is adopted. The governing equations for a lossy compressible ﬂuid can be written as: i ωρ f 0 v = −∇p + ∇ · μ f (∇ v + (∇ v ) T ) −2 3 μ f (∇ · v ) I , i ω ρ f 0 c f p θ = i ω p −∇ · −κ f ∇θ , i ωρ f 0 v = −∇p + ∇ · μ f (∇ v + (∇ v ) T ) −2 3 μ f (∇ · v ) I , i ωρ f 0 v = −∇p + ∇ · μ f (∇ v + (∇ v ) T ) −2 3 μ f (∇ · v ) I , (2) i ω ρ f 0 c f p θ = i ω p −∇ · −κ f ∇θ , (3) (2) i ω ρ f 0 c f p θ = i ω p −∇ · −κ f ∇θ , (3) i ω p p 0 = i ω θ θ0 −∇ · v , (4) which represent the balance of momentum, the energy balance and mass conservation (considering the ideal gas law), respectively. 2.1. Geometry of the unit cell The geometry and dimensions of the unit cell have been chosen on the basis of polymer foams ( Gao et al., 2017 ), aiming at high absorption for low frequencies. A cubic idealised representation of the pore has struts with a square cross-section along all edges and membranes in the faces ( Fig. 1 ). A small cell size of a = 100 μm is adopted, with the thicknesses of the struts t s = 25 μm and of the membranes t m = 1 μm . These values are close to the parameters measured experimentally in Gao et al. (2016) . The membranes of four out of six faces are partially open, with a small opening ratio of 1% (where the opening ratio represents the area fraction of the hole in the face). A local resonator is introduced within the microstructure of the idealised cubic cell in the form of a cantilever decorated at its tip with a heavy (relatively to the unit cell weight) mass of m hea v y = 10 −10 kg . In order to reduce the role of the geometry in this study and to focus on the contribution of the local resonance effect to the material performance, the dimensions of the resonating mass are minimised to 0.01mm × 0.01mm × 0.01mm. In this way, the dissipation is not being enhanced due to an increase of the solid-ﬂuid interface. Moreover, two reference cases are considered, the case of a unit cell without a resonator and a unit cell with a light resonator (i.e. the cantilever only, omitting any external mass at its tip). The three geometries are depicted in Fig. 1 . 1. Introduction For some materials, incorporating the inﬂuence of realistic damping occurring through thermal and viscous dissipation is not just a reﬁnement of the model but may be crucial for the correct predictions of their response. For instance, in the study by Molerón et al. (2016) , focusing on a metamaterial consisting of rigid slabs embedded in air, the inclu- sion of ﬂuid losses changed the predicted acoustic behaviour from perfect transmission to perfect reﬂection. Furthermore, Henríquez et al. (2017) have reported that viscothermal dissipation in the air, even for geometries much larger than the boundary layer thicknesses, may completely suppress the double negative behaviour of the analysed rigid periodic struc- ture. In this paper, the acoustic performance of a poroelastic material enriched with resonators embedded in the pores is inves- tigated. Unlike previous studies ( Chevillotte and Perrot, 2017; Doutres et al., 2011; 2014; Gao et al., 2017 ) aiming at optimis- ing the pore geometry and morphology for low frequency performance, here, a major change in terms of micro-dynamical phenomena is introduced. The micro-resonator is represented by a cantilever beam with a heavy mass attached at its tip, which represents a particle embedded in the pore during the manufacturing process. Bloch analysis performed for the pro- posed unit cell design predicts a signiﬁcant attenuation in the low frequency range for both fast and slow compressional waves propagating through the system. This behaviour is also conﬁrmed by a transmission analysis of a ﬁnite size set-up by direct simulations. Moreover, it is shown that the shear viscosity of the ﬂuid is crucial for revealing the resonance-related attenuation mechanisms. In order to numerically demonstrate the concept of embedding local resonators within the pores of poroelastic material, a simple cubic unit cell is used. Supporting the recent progress in innovative approaches to manu- facturing poroelastic/cellular materials ( Kaur et al., 2017 ), this work contributes towards the development of a new type of foams: acoustic metafoams. The paper is organised as follows. In Section 2 , the unit cell and the modelling approach are described. In Section 3 , the simulation results are shown, including the analysis of the dispersion diagrams and transmission studies of the response of a single unit cell as well as a ﬁnite size conﬁguration. In Section 4 the main results are further discussed, after which conclusions are presented. M.A. Lewi ´nska et al. 2.2. Material description The microstructure of the unit cell ( Fig. 1 ) consists of ﬂuid and solid phases which are described in the frequency domain. Conventional descriptions for each component are adopted from Gao et al. (2015) and Yamamoto et al. (2011) . p p p ( ) ( ) The solid part is modelled as in general an isotropic elastic material. Within the solid domain, the equation of motion holds: −ρs ω 2 u = ∇ · σ, balance and continuity equations are: balance and continuity equations are: (5) i ωρ f 0 v + ∇p = 0 , i ωρ f 0 v + ∇p = 0 , i ωp + c 2 0 ρ f 0 ∇ · v = 0 , i ωp + c 2 0 ρ f 0 ∇ · v = 0 , (6) where c 0 denotes the wave speed in the ﬂuid. By combining Eqs. (5) and (6) , the acoustic Helmholtz equation is obtained: ω where c 0 denotes the wave speed in the ﬂuid. By combining Eqs. (5) and (6) , the acoustic Helmholtz equation is obtained: where c 0 denotes the wave speed in the ﬂuid. By combining Eqs. (5) and (6) , the acoustic H ∇ · (∇p) + k 2 0 p = 0 , with the wavenumber k 0 = ω c 0 . ∇ · (∇p) + k 2 0 p = 0 , with the wavenumber k 0 = ω c 0 . (7) (7) This domain will be referred in the text as inviscid isothermal. The coupling between the domains is prescribed in the following way. At the interface between the viscothermal ﬂuid and the solid, continuity of velocities and tractions is assumed, along with an isothermal condition (justiﬁed by the large difference between thermal conductivity of the solid and ﬂuid phases). Continuity of tractions and normal acceleration is adopted at the interfaces between the solid and the inviscid isothermal ﬂuid as well as between the viscothermal and inviscid isothermal ﬂuid. At the latter interface, also adiabatic conditions for the temperature are assumed. The material parameters adopted for the ﬂuid (air) and solid (polyurethane, PU) domains are presented in Tables 1 and 2 , respectively. The material parameters adopted for the ﬂuid (air) and solid (polyurethane, PU) domains ar 2 , respectively. 2.3. Bloch analysis Free wave propagation in an inﬁnite periodic medium is typically analysed based on dispersion diagrams. By considering the complex band structures, the assessment of the wave dispersion in the presence of local resonators is possible, as well as the description of the wave attenuation due to (thermo) viscous dissipation inside the unit cell. The wave attenuation is characterised by the imaginary part of the wavenumber (attenuation factor), where higher values indicate stronger at- tenuation in space. In this study, the complex dispersion diagrams are obtained with the standard k (ω) approach ( Hussein et al., 2014; Krushynska et al., 2016; Wang et al., 2015 ) based on the Bloch-Floquet theorem, which states that the wave solution inside the Brillouin Zone ( Kittel, 1971 ) is a superposition of a periodic function and a plane wave ( Deymier, 2013 ). The Bloch-Floquet theorem is applied to all considered ﬁeld variables and substituted in the governing Eqs. (1) , (2) , (4) and (7) of the domains. Although thermal effects are included in the transmission analysis, due to their marginal contribution demonstrated in Section 3.3 , they are neglected in this part of the study. COMSOL Multhiphysics is used to solve the cou- pled eigenvalue problem, exploiting a user deﬁned weak form for a unit cell with periodic boundary conditions. The details of the strong and weak formulations used for the Bloch analysis are given in the Appendix. The study is restricted to the X direction of the wave propagation ( Deymier, 2013 ) since it coincides with the conditions realised in the corresponding transmission analysis. −ρs ω 2 u = ∇ · σ, In these equations, v denotes the velocity vector, p the pressure, θ the temperature, ρ f 0 the equilibrium density, μ f the viscosity, c f p the heat capacity at constant pressure, κf the thermal conductivity, i the imaginary unit, p 0 and θ 0 the ambient pressure and temperature, respectively. which represent the balance of momentum, the energy balance and mass conservation (considering the ideal gas law), respectively. In these equations, v denotes the velocity vector, p the pressure, θ the temperature, ρ f 0 the equilibrium density, μ f the viscosity, c f p the heat capacity at constant pressure, κf the thermal conductivity, i the imaginary unit, p 0 and θ 0 the ambient pressure and temperature, respectively. In order to reduce the computational costs, external ﬂuid domains (outside the unit cells), used in transmission simu- lations (see Section 2.4 ), are modelled in a simpliﬁed way, neglecting viscothermal damping. Then, the local momentum 192 M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 Table 1 Material properties of air ( ρ f 0 denotes ﬂuid equilibrium density, κf ther- mal conductivity, c f p heat capacity at constant pressure, μf dynamic vis- cosity, θ 0 and p 0 equilibrium temperature and pressure, respectively) Deckers et al. (2015) . ρ f 0 κf c f p μf θ 0 p 0 (kg/m 3 ) (W/(m · K)) (J/(kg · K)) (Pa · s) (K) (Pa) 1.2 0.0257 1005 1.84 ·10 −5 293 1.01 · 10 5 Table 2 Material properties of the solid phase polyurethane (PU) ( ρs denotes solid density, E Young’s modulus, ν Poisson’s ratio) Gao et al. (2017) . ρs E ν (kg/m 3 ) (MPa) ( −) 10 0 0 1 0.4 Table 2 balance and continuity equations are: Table 1 Table 1 Material properties of air ( ρ f 0 denotes ﬂuid equilibrium density, κf ther- mal conductivity, c f p heat capacity at constant pressure, μf dynamic vis- cosity, θ 0 and p 0 equilibrium temperature and pressure, respectively) Deckers et al. (2015) . 2.4. Transmission analysis In order to distinguish the mechanisms underlying the absorption, the fractions of viscous and thermal dissipated powers are respectively obtained by ( Cambonie et al., 2018; Pierce, 1981 ): P v = V σ : ∇ v d V, P t = V κ f T 0 T d V, P v = V σ : ∇ v d V, P v = V σ : ∇ v d V, (10) P t = V κ f T 0 T d V, (11) (10) P t = V κ f T 0 T d V, (11) and will be normalised by incident power. In the above expressions, V denotes the volume of the viscothermal ﬂuid and the symbol is the Laplace operator. and will be normalised by incident power. In the above expressions, V denotes the volume of the viscothermal ﬂuid and the symbol is the Laplace operator. 2.4. Transmission analysis A numerical three-dimensional transmission set-up ( Cui and Harne, 2017 ) is used in this paper to assess the performance of the material. The ﬁnite element method simulations have also been conducted with COMSOL Multiphysics. The set-up is inspired by an impedance tube test, typically utilised for measuring the acoustic properties of poroelastic materials. A three point method ( Henríquez et al., 2017; Ho et al., 2005 ) is used to measure transmission, absorption and reﬂection. The tested sample is placed in between two air domains, with a pressure plane-wave excitation assigned to the left boundary M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 193 Fig. 2. Transmission set-up used in the numerical simulations. Light grey represents polyurethane (PU), dark grey is the heavy mass at the tip of the resonator, light blue is the air domain. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 2. Transmission set-up used in the numerical simulations. Light grey represents polyurethane (PU), dark grey is the heavy mass at the tip of the resonator, light blue is the air domain. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) and a perfectly matched layer (PML) used at the right boundary to reduce spurious reﬂections. Lateral boundaries (in y and z directions) are periodic in this analysis. The mid-plane cross-section of the numerical model with the position of the measurement points (mic) is shown in Fig. 2 . Following Henríquez et al. (2017) , reﬂection r ( ω) and transmission t ( ω) coeﬃcients are obtained by: and a perfectly matched layer (PML) used at the right boundary to reduce spurious reﬂections. Lateral boundaries (in y and z directions) are periodic in this analysis. The mid-plane cross-section of the numerical model with the position of the measurement points (mic) is shown in Fig. 2 . Following Henríquez et al. 3. Results In this section, the numerical results are presented. First, the behaviour of an inﬁnite periodic arrangement of unit cells is assessed based on the Bloch analysis. Next, the acoustic performance of a ﬁnite material sample (considering single and multiple cells) is studied using the transmission set-up. 2.4. Transmission analysis (2017) , reﬂection r ( ω) and transmission t ( ω) coeﬃcients are obtained by: r(ω) = p 2 exp (−i kx 1 ) −p 1 exp (−i kx 2 ) p 1 exp (i kx 2 ) −p 2 exp (i kx 1 ) , (8) t(ω) = p 3 ( exp (−i kx 2 ) + r(ω) exp (i kx 2 )) p 2 exp (−i kx 3 ) exp (−i kL ) , (9) r(ω) = p 2 exp (−i kx 1 ) −p 1 exp (−i kx 2 ) p 1 exp (i kx 2 ) −p 2 exp (i kx 1 ) , r(ω) = p 2 exp (−i kx 1 ) −p 1 exp (−i kx 2 ) p 1 exp (i kx 2 ) −p 2 exp (i kx 1 ) , t(ω) = p 3 ( exp (−i kx 2 ) + r(ω) exp (i kx 2 )) p 2 exp (−i kx 3 ) exp (−i kL ) , (8) t(ω) = p 3 ( exp (−i kx 2 ) + r(ω) exp (i kx 2 )) p 2 exp (−i kx 3 ) exp (−i kL ) , (9) where p 1 , p 2 and p 3 are complex pressure values calculated at three measurement points x i (at the positions of the micro- phones 1, 2 and 3, respectively), L is the length of the sample, k denotes the wavenumber. 2 2 where p 1 , p 2 and p 3 are complex pressure values calculated at three measurement points x i (at the positions of the micro- phones 1, 2 and 3, respectively), L is the length of the sample, k denotes the wavenumber. Power transmittance and reﬂectance are then expressed as: R (ω) = \| r(ω ) \| 2 and T (ω ) = \| t(ω) \| 2 , respectively, and sum up to 1 in the absence of losses, due to the conservation of energy. In the presence of losses, the dissipated energy is given by an absorption term: A (ω) = 1 −R (ω) −T (ω) . 3.1. Dispersion diagrams 3.1. Dispersion diagrams Fig. 3 shows the dispersion diagrams obtained for the three considered 3D unit cell geometries, of Fig. 1 , using an inviscid isothermal ﬂuid. The wave polarisations are identiﬁed by the ratio between the amplitudes of the displacement in the solid along the x axis and the total displacement in the solid, both integrated over the solid domain. The compressional and shear wave polarisations are then distinguished by colours varying from red (1) to blue (0), respectively. As can be seen in Fig. 3 , two shear and two compressional waves propagate in the material consisting of 3D unit cells with the solid and ﬂuid phases, where two shear waves (S1, S2) relate to the two perpendicular displacement directions, and two compressional (longitudinal) waves (L1, L2) correspond to the in-phase (fast, ﬂuid-born) and out-of-phase (slow, solid-born) motion of the ﬂuid and solid, respectively ( Allard and Atalla, 2009; Bruneau and Potel, 2013 ). Dispersion curves for the inviscid cases without the resonator and with the light resonator presented in Fig. 3 a overlap in the considered frequency range and do not exhibit any dispersive or dissipative effects (imaginary parts of the wavenumbers all equal zero). Yet, signiﬁcant dispersion can be observed for the case with the heavy resonator in Fig. 3 b. A band gap is formed in the frequency range 445–560 Hz for two wave polarisation: the slow (solid-born) compressional wave (L2) and one of the shear waves (S2), as evident from the absence of the dispersion curves in the real plane and high imaginary values of the wavenumber. It should be emphasised that due to the presence of the two other wave types (L1, S1) inside the M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 194 Fig. 3. 2D projections of the complex dispersion diagrams for the unit cells with inviscid ﬂuid domains: (a) the unit cells without the resonator (ﬁlled circles) and with the light resonator (open circles); (b) the unit cell with the heavy resonator. Colours represent wave polarisations from shear (blue) to longitudinal (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 3. 3.1. Dispersion diagrams 2D projections of the complex dispersion diagrams for the unit cells with inviscid ﬂuid domains: (a) the unit cells without the resonator (ﬁlled circles) and with the light resonator (open circles); (b) the unit cell with the heavy resonator. Colours represent wave polarisations from shear (blue) to longitudinal (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 4. Complex dispersion diagrams for the unit cell with the heavy resonator and a viscous ﬂuid domain (air viscosity): (a) 3D band structure; (b) 2D projections. Colours represent wave polarisations from shear (blue) to longitudinal (red). Black dots depict the case with inviscid ﬂuid. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 4. Complex dispersion diagrams for the unit cell with the heavy resonator and a viscous ﬂuid domain (air viscosity): (a) 3D band structure; (b) 2D projections. Colours represent wave polarisations from shear (blue) to longitudinal (red). Black dots depict the case with inviscid ﬂuid. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) band gap region, a complete band gap is not formed. Note, that due to the small dimensions of the unit cell, the dispersive effects related to the cavity resonance for the wave type L1 occur at much higher frequencies, exceeding the considered range (not shown here). Fig. 4 presents the 3D band structure and its 2D projections obtained for the geometry with the heavy resonator when a viscous ﬂuid is considered (with the viscosity of air). As a reference, the lossless case discussed previously is also shown in the graph (in black). The viscosity of the ﬂuid has a signiﬁcant inﬂuence on the dispersion curves. In particular, both compressional waves (L1, L2) are attenuated in the considered frequency range, with attenuation peaks located around 440 Hz. The 3D view of the band structure ( Fig. 4 a) reveals how both dispersion curves associated with these waves bend towards the complex wavenumber domain, exhibiting a spin in the band gap region, characteristic for dissipative systems ( Krushynska et al., 2016; Lewi ´nska et al., 2017 ). 3.1. Dispersion diagrams The shear wave S2 (forming a band gap in the lossless case) now shows a slight broadening of the attenuation regime in the viscous case. No inﬂuence of the air viscosity can be observed for the other shear wave polarisation (S1). In order to assess the effect of the resonance on the attenuation performance of the unit cell with the heavy resonator entrained with viscous ﬂuid, the corresponding dispersion curves obtained are compared with those calculated for the unit cell with a light resonator (also accounting for the ﬂuid viscosity). Fig. 5 shows that for low frequencies (below 450 Hz), M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 195 Fig. 5. Complex dispersion diagrams for the unit cell with the heavy resonator (ﬁlled circles) and light resonators (open circles), both ﬁlled with a viscous ﬂuid (air): (a) 3D band structure; (b) 2D projections. Colours represent wave polarisations from shear (blue) to longitudinal (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 5. Complex dispersion diagrams for the unit cell with the heavy resonator (ﬁlled circles) and light resonators (open circles), both ﬁlled with a viscous ﬂuid (air): (a) 3D band structure; (b) 2D projections. Colours represent wave polarisations from shear (blue) to longitudinal (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 6. Velocity ﬁelds in the longitudinal x direction at the mid-plane cross section of the unit cells at the points indicated in Fig. 5 b: (a) AL (with light resonator), (b) A (with heavy resonator), (c) BL (with light resonator), (d) B (with heavy resonator). Fig. 6. Velocity ﬁelds in the longitudinal x direction at the mid-plane cross section of the unit cells at the points indicated in Fig. 5 b: (a) AL (with light resonator), (b) A (with heavy resonator), (c) BL (with light resonator), (d) B (with heavy resonator). the fast (ﬂuid-born) compressional wave (L1) reveals a higher level of attenuation in the case with the heavy resonator. On the other hand, for frequencies above 450 Hz the attenuation factor is higher for the light resonator. 3.1. Dispersion diagrams An attenuation peak located around frequency 440 Hz, which is observed for the slow (solid-born) wave (L2) with the heavy resonator, is not present in the case of the light resonator, for which the attenuation is slightly lower at the higher frequencies as well. the fast (ﬂuid-born) compressional wave (L1) reveals a higher level of attenuation in the case with the heavy resonator. On the other hand, for frequencies above 450 Hz the attenuation factor is higher for the light resonator. An attenuation peak located around frequency 440 Hz, which is observed for the slow (solid-born) wave (L2) with the heavy resonator, is not present in the case of the light resonator, for which the attenuation is slightly lower at the higher frequencies as well. p g g y g q The attenuation of the compressional waves shown in the dispersion diagrams can be associated with the dynamic be- haviour of the unit cell. Therefore, in Fig. 6 the velocity ﬁelds in the longitudinal direction at the mid-plane cross section for different points (marked in Fig. 5 b by A, AL, B, BL), are presented for both unit cells. The mode shape for the fast wave, for which the ﬂuid motion is in-phase with the solid, shows high velocity gradients occurring around the membrane opening, if the light resonator is considered ( Fig. 6 a). For the heavy resonator unit cell, the resonating cantilever signiﬁcantly enhances the velocity gradient ﬁeld, in particular, through its out-of-phase oscillations ( Fig. 6 b). Analogous effects can be observed for the slow wave ( Fig. 6 c,d). The presence of the heavy resonator contributes to the higher attenuation level by, enhancing both dissipative effects and increasing reﬂection, as will be demonstrated in the following sections. In Figs. 7 and 8 , the band structures obtained for the unit cell with heavy resonators and viscous ﬂuid with different viscosities are presented. For clarity, only the compressional waves are shown and the distinction between fast (ﬂuid-born) and slow (solid-born) waves is introduced using colours, where red and green colours denote ﬂuid-born and solid-born waves, respectively. Intermediate colours reﬂect the ratio between the ﬂuid and solid velocities in the longitudinal direction integrated over the front face of the unit cell and averaged over the solid and ﬂuid parts of this surface, allowing to identify the in-phase and nearly out-of-phase motion between ﬂuid and solid. 3.1. Dispersion diagrams The 3D dispersion diagram ( Fig. 7 a) also shows dispersion curves obtained for a ﬂuid viscosity lower than that of air i.e. μ = 0 . 1 μair . In this case, both ﬂuid-born and solid-born waves exhibit less attenuation preserving the general shape of the dispersion curves (with attenuation peaks around 440 Hz for both wave polarisations). The velocity ﬁelds for the points M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 196 196 M.A. Lewi nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189 205 Fig. 7. Complex dispersion diagrams for the heavy resonator unit cell and different ﬂuid viscosities: μ = μair marked with ﬁlled circles, μ = 0 . 1 μair marked with open circles: (a) 3D band structure; (b) 2D projections. Colours represent compressional wave types from solid-born (green) to ﬂuid-born (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 7. Complex dispersion diagrams for the heavy resonator unit cell and different ﬂuid viscosities: μ = μair marked with ﬁlled circles, μ = 0 . 1 μair marked with open circles: (a) 3D band structure; (b) 2D projections. Colours represent compressional wave types from solid-born (green) to ﬂuid-born (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 8. Complex dispersion diagrams for the heavy resonator unit cell and different ﬂuid viscosities: μ = μair marked with ﬁlled circles, μ = 10 μair marked with open circles: (a) 3D band structure; (b) 2D projections. Colours represent compressional wave types from solid-born (green) to ﬂuid-born (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 8. Complex dispersion diagrams for the heavy resonator unit cell and different ﬂuid viscosities: μ = μair marked with ﬁlled circles, μ = 10 μair marked with open circles: (a) 3D band structure; (b) 2D projections. Colours represent compressional wave types from solid-born (green) to ﬂuid-born (red). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) marked in Fig. 7 b as A01 ( Fig. 3.1. Dispersion diagrams 9 a) and C01 ( Fig. 10 a) reveal a reduced coupling between the solid and ﬂuid domains, with a lower viscosity: higher velocity gradients are present in the unit cell in comparison with the reference air viscosity case (cf. Figs. 6 b and 10 b). Fig. 9 a) and C01 ( Fig. 10 a) reveal a reduced coupling between the solid and ﬂuid domains, with a ocity gradients are present in the unit cell in comparison with the reference air viscosity case (cf. g ) Fig. 8 shows that the inﬂuence of an increased ﬂuid viscosity ( μ = 10 μair ) on the dispersion diagrams depends on the wave type. For the fast wave smoothing of the attenuation factor is observed, with a decrease of the attenuation peak at 440 Hz and a minor increase of the imaginary parts of the wavenumbers in the frequency range 450–650 Hz. For the slow wave, the attenuation increases in almost the entire frequency range. Velocity ﬁelds for the points marked in Fig. 8 are shown in Fig. 9 and 10 . At the point A10 signiﬁcant motion of the entire solid skeleton can be observed together with reduced velocity gradients, in comparison with the point A01 ( Fig. 9 a) and A ( Fig. 6 b). On the other hand, the velocity ﬁeld for the point C10 ( Fig. 10 c) is characterised by signiﬁcant velocity concentrations around the membrane opening region. Note, that the attenuation of the slow wave is stronger than the attenuation of the fast wave for all considered viscosities due to the relative viscous ﬂow of the pore-ﬂuid relative to the solid frame (see Fig. 10 ) ( Allard and Atalla, 2009; Borocin, 2003 ). Fig. 8 shows that the inﬂuence of an increased ﬂuid viscosity ( μ = 10 μair ) on the dispersion diagrams depends on the wave type. For the fast wave smoothing of the attenuation factor is observed, with a decrease of the attenuation peak at 440 Hz and a minor increase of the imaginary parts of the wavenumbers in the frequency range 450–650 Hz. For the slow wave, the attenuation increases in almost the entire frequency range. Velocity ﬁelds for the points marked in Fig. 8 are shown in Fig. 9 and 10 . 3.2. Single cell performance The dispersion analysis allows to assess the wave propagation and attenuation in the inﬁnite material domain. In order to analyse the behaviour of ﬁnite structures and distinguish between the mechanisms underlying the wave attenuation, a transmission calculation is conducted using the numerical set-up detailed in Section 2.4 . In this section, the performance of a single unit cell as a material sample is investigated. Fig. 11 shows the acoustic properties of a single unit cell with viscothermal losses for the unit cells with and without resonators. The heavy resonator cell reveals, a transmission dip at 440 Hz, which is not visible for the case with a light or no resonator ( Fig. 11 a). Based on the reﬂection ( Fig. 11 b) and absorption ( Fig. 11 c) curves, it can be stated that at this frequency part of the energy is dissipated within the ﬂuid and a similar part is reﬂected. In Fig. 12 , the velocity ﬁelds at the mid-plane cross-section of the three unit cells are depicted. The unit cells without the resonator ( Fig. 12 a) and with the light resonator ( Fig. 12 b) behave quite similar. A minor increase of the velocities is present around the membrane opening, and additionally around the resonator tip for the corresponding unit cell. The presence of the heavy resonator signiﬁcantly changes the response of the unit cell ( Fig. 12 c). At a frequency of 440 Hz, the elastic cantilever resonates and high velocity gradients can be observed within the ﬂuid. Note, that this velocity ﬁeld qualitatively resembles the one obtained through the dispersion analysis ( Fig. 6 b), suggesting that the dominant role for the transmission analysis in the considered set-up is played by the fast (ﬂuid-born) compressional wave. As a consequence, a high level of viscous dissipation is obtained, as well as an increase in the acoustic impedance, resulting in a reﬂection peak, see Fig. 11 b. In Fig. 13 , the acoustic properties obtained for the unit cell with a heavy resonator and viscothermal ﬂuid are compared with those obtained for the inviscid isothermal ﬂuid (described by Eq. (21) ). A clear difference between both unit cells emerges. The transmission dip observed with the viscothermal ﬂuid is not present in the analysis without viscothermal losses. Only an isolated reﬂection peak is found at 550 Hz ( Fig. 3.1. Dispersion diagrams Velocity ﬁeld in the longitudinal x direction at the mid-plane cross section of the heavy resonator unit cells, and different ﬂuid viscosities at the band structure points marked in Figs. 7 and 8 : (a) for μ = 0 . 1 μair indicated with A01, (b) for μ = 10 μair indicated with A10. Fig. 10. Velocity ﬁeld in the longitudinal x direction at the mid-plane cross section of the heavy resonator unit cells, and different ﬂuid viscosities at the band structure points marked in Figs. 7 and 8 : (a) for μ = 0 . 1 μair indicated with C01, (a) for μ = μair indicated with C, (d) for μ = 10 μair indicated with C10. Fig. 10. Velocity ﬁeld in the longitudinal x direction at the mid-plane cross section of the heavy resonator unit cells, and different ﬂuid viscosities at the band structure points marked in Figs. 7 and 8 : (a) for μ = 0 . 1 μair indicated with C01, (a) for μ = μair indicated with C, (d) for μ = 10 μair indicated with C10. 3.1. Dispersion diagrams At the point A10 signiﬁcant motion of the entire solid skeleton can be observed together with reduced velocity gradients, in comparison with the point A01 ( Fig. 9 a) and A ( Fig. 6 b). On the other hand, the velocity ﬁeld for the point C10 ( Fig. 10 c) is characterised by signiﬁcant velocity concentrations around the membrane opening region. Note, that the attenuation of the slow wave is stronger than the attenuation of the fast wave for all considered viscosities due to the relative viscous ﬂow of the pore-ﬂuid relative to the solid frame (see Fig. 10 ) ( Allard and Atalla, 2009; Borocin, 2003 ). Fig. 8 shows that the inﬂuence of an increased ﬂuid viscosity ( μ = 10 μair ) on the dispersion diagrams depends on the wave type. For the fast wave smoothing of the attenuation factor is observed, with a decrease of the attenuation peak at 440 Hz and a minor increase of the imaginary parts of the wavenumbers in the frequency range 450–650 Hz. For the slow wave, the attenuation increases in almost the entire frequency range. Velocity ﬁelds for the points marked in Fig. 8 are shown in Fig. 9 and 10 . At the point A10 signiﬁcant motion of the entire solid skeleton can be observed together with reduced velocity gradients, in comparison with the point A01 ( Fig. 9 a) and A ( Fig. 6 b). On the other hand, the velocity ﬁeld for the point C10 ( Fig. 10 c) is characterised by signiﬁcant velocity concentrations around the membrane opening region. Note, that the attenuation of the slow wave is stronger than the attenuation of the fast wave for all considered viscosities due to the relative viscous ﬂow of the pore-ﬂuid relative to the solid frame (see Fig. 10 ) ( Allard and Atalla, 2009; Borocin, 2003 ). 197 M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 Fig. 9. Velocity ﬁeld in the longitudinal x direction at the mid-plane cross section of the heavy resonator unit cells, and different ﬂuid viscosities at the band structure points marked in Figs. 7 and 8 : (a) for μ = 0 . 1 μair indicated with A01, (b) for μ = 10 μair indicated with A10. Fig. 9. 3.2. Single cell performance 13 b), which can be associated with the global eigenfrequency of the ﬁnite system. Note, that the reﬂection dips are present in both cases at the frequency 560 Hz (see logarithmic scale insert graph in Fig. 13 c), which is the frequency closing the band gap in Fig. 3 c. Naturally, no absorption is observed if the losses are not considered ( Fig. 13 c). Based on the above results it can be therefore concluded that viscothermal losses play a key role for the performance of the unit cell. To further scrutinise this effect, in Fig. 14 , the results of a variation of the ﬂuid viscosity around the regular M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 198 Fig. 11. Transmission, reﬂection and absorption of a single unit cell with a viscothermal ﬂuid: without resonator (dashed line); with the light resonator (red line); with heavy resonator (green line). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 11. Transmission, reﬂection and absorption of a single unit cell with a viscothermal ﬂuid: without resonator (dashed line); with the light resonator (red line); with heavy resonator (green line). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 12. Velocity ﬁeld in the longitudinal x direction at the mid-plane cross section of the unit cells in the transmission simulation at 440 Hz for (a) the unit cell without a resonator, (b) the unit cell with the light resonator and (c) the unit cell with the heavy resonator. Fig. 12. Velocity ﬁeld in the longitudinal x direction at the mid-plane cross section of the unit cells in the transmission simulation at 440 Hz for (a) the unit cell without a resonator, (b) the unit cell with the light resonator and (c) the unit cell with the heavy resonator. Fig. 13. Transmission, reﬂection and absorption of a single unit cell with a heavy resonator and a viscothermal ﬂuid (black line) and an inviscid ﬂuid (grey line). Fig. 13. Transmission, reﬂection and absorption of a single unit cell with a heavy resonator and a viscothermal ﬂuid (black line) and an inviscid ﬂuid (grey line). M.A. Lewi ´nska et al. 3.2. Single cell performance / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 199 Fig. 14. Transmission, reﬂection and absorption of a single unit cell with heavy resonator and viscothermal ﬂuid with different viscosity values. Fig. 14. Transmission, reﬂection and absorption of a single unit cell with heavy resonator and viscothermal ﬂuid with different viscosity values. Fig. 15. Amplitudes of the velocity ﬁeld in the longitudinal x direction at the mid-plane cross section at the frequency 440 Hz for different ﬂuid viscosities (a) μ = 0 . 1 μair , (b) μ = μair and (c) μ = 10 μair . Fig. 15. Amplitudes of the velocity ﬁeld in the longitudinal x direction at the mid-plane cross section at the frequency 440 Hz for different ﬂuid viscosities (a) μ = 0 . 1 μair , (b) μ = μair and (c) μ = 10 μair . air viscosity are presented for the unit cell with a heavy resonator. Both an increase or a decrease of the viscosity results in smoothening of the transmission spectra and a reduction of the transmission dip. The change of viscosity initially inﬂu- ences the reﬂection spectrum, where for both μ = 0 . 1 μair and μ = 10 μair the peak values are already signiﬁcantly reduced compared to the value with air viscosity ( Fig. 14 b). The absorption level is only affected strongly if the viscosity is modiﬁed considerably ( μ = 0 . 01 μair and μ = 100 μair ). If the viscosity reduction is relatively small (e.g. μ = 0 . 1 μair ) absorption is not inﬂuenced much. This amount of viscous dissipation is still achieved due to the local resonance effect resulting in high local velocity gradients. air viscosity are presented for the unit cell with a heavy resonator. Both an increase or a decrease of the viscosity results in smoothening of the transmission spectra and a reduction of the transmission dip. The change of viscosity initially inﬂu- ences the reﬂection spectrum, where for both μ = 0 . 1 μair and μ = 10 μair the peak values are already signiﬁcantly reduced compared to the value with air viscosity ( Fig. 14 b). The absorption level is only affected strongly if the viscosity is modiﬁed considerably ( μ = 0 . 01 μair and μ = 100 μair ). If the viscosity reduction is relatively small (e.g. 3.2. Single cell performance Fig. 17. Transmission, reﬂection and absorption for a ten unit cell set-up with a viscothermal ﬂuid and either the heavy or light resonator. 3.2. Single cell performance μ = 0 . 1 μair ) absorption is not inﬂuenced much. This amount of viscous dissipation is still achieved due to the local resonance effect resulting in high local velocity gradients. y g In Fig. 15 , the amplitudes of the longitudinal velocity ﬁelds are depicted for three different ﬂuid viscosities, showing that the highest resonator amplitudes can be observed if the viscosity of air is adopted, which corresponds with the largest transmission dip. This suggests that for this particular system, with the unit cell dimensions and material parameters based on the optimisation study of Gao et al. (2017) , the viscosity of air is the best among the values considered. This is also consistent with the predictions obtained in the dispersion analysis where the highest attenuation peak for the fast (ﬂuid- born) compressional wave has been achieved with the viscosity of air. Both reducing and enhancing the viscosity leads to the mitigation of the resonator dynamics, with a stronger effect for higher losses μ = 10 μair . The interplay between the viscosity level and the resonance magnitude also explains the lower absorption level for the higher ﬂuid viscosity, see Fig. 14 c. The small opening ratio of the membrane in this study has been used in order to induce the viscothermal dissipation at low frequencies. In Fig. 16 , transmission, reﬂection and absorption for different opening ratios are shown, from fully open to closed unit cells. It is clear that for the high attenuation to occur, a suﬃciently small membrane opening is required, since the increase of the membrane opening size reduces both reﬂection and absorption levels. On the other hand, the presence of small membrane openings results in broadening of the absorption zone also beyond the regime of local resonance. M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 200 Fig. 16. Transmission, reﬂection and absorption of a single unit cell with the heavy resonator and viscothermal ﬂuid for different membrane opening ratios. Fig. 16. Transmission, reﬂection and absorption of a single unit cell with the heavy resonator and viscothermal ﬂuid for different membrane opening ratios. and absorption of a single unit cell with the heavy resonator and viscothermal ﬂuid for different membrane opening ratios. Fig. 17. Transmission, reﬂection and absorption for a ten unit cell set-up with a viscothermal ﬂuid and either the heavy or light resonator. 3.3. Multiple cell performance Dissipation in the ten unit cell set-up. Fig. 19. Transmission, reﬂection and absorption for different set-up sizes with the heavy resonators unit cells and a viscothermal ﬂuid. Fig. 19. Transmission, reﬂection and absorption for different set-up sizes with the heavy resonators unit cells and a viscothermal ﬂuid. In Fig. 19 , the acoustic performance of set-ups of different sizes is shown. The increase in the number of unit cells results in an increase of the wave attenuation in the frequency range around 440 Hz. An increase of the reﬂection can be observed which is accompanied by a decrease and a shift of a broadened absorption peak. 3.3. Multiple cell performance In this section, results obtained for the transmission set-up with multiple unit cells as a material sample are presented. In Fig. 17 , transmission, reﬂection and absorption are shown for a row of ten unit cells with heavy resonators and a vis- cothermal ﬂuid with the viscosity of air. The performance of the unit cells with light resonators is also depicted. In analogy to the single unit cell study, a transmission dip around 440 Hz can be observed, which is not present in the case without added mass. Moreover, in this case, the attenuation range spans a broader frequency range. Note, that the behaviour of the transmission spectra for both light and heavy resonators can be directly related to the attenuation factor for the fast compressional wave (L1) shown in Fig. 5 . According to Fig. 17 b, the main mechanism underlying the reduction in transmis- sion is reﬂection, since a high reﬂection peak exceeding 0.8 emerges around 440 Hz. The reﬂection peak is followed by a reﬂection dip, which explains the increase of transmission observed around 560 Hz. Such a behaviour is typical for locally resonant materials, where at the end of the band gap region there is again a high transmissibility (e.g. Lewi ´nska et al. (2017) ; Liu et al. (20 0 0) ). The absorption performance of the multiple cell set-up is rather moderate ( Fig. 17 c). Moreover, the ab- sorption peak is shifted to higher frequencies in comparison with the single cell behaviour, which is a result of the strong reﬂection at the resonance frequency. q y Among the different dissipative mechanisms occurring in the unit cells, the major role is played by viscous losses, as can be seen in Fig. 18 , where the lines denoting total absorption and viscous dissipation practically overlap. Indeed, as stated in Gao et al. (2017) , for small unit cell sizes, thermal dissipation is highly reduced. This justiﬁes why this contribution was neglected in the Bloch analysis. 201 M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 Fig. 18. Dissipation in the ten unit cell set-up. Fig. 19. Transmission, reﬂection and absorption for different set-up sizes with the heavy resonators unit cells and a viscothermal ﬂuid. Fig. 18. Dissipation in the ten unit cell set-up. Fig. 18. Dissipation in the ten unit cell set-up. Fig. 18. 4. Discussion The results of this analysis show that the proposed microstructural conﬁguration, in spite of its simplicity clearly in- dicates a potential way for improving the acoustic attenuation of foams by combining the mechanisms of viscothermal dissipation with local resonance. This combination constitutes a pathway towards the design of acoustic metafoams . In this study, based on transmission, reﬂection and absorption spectra, as well as complex dispersion diagrams, the behaviour of foam-like unit cells with and without resonators has been assessed, showing that the presence of resonating masses locally improves the noise isolation properties of the material. The enhancement of the sound attenuation is based on the increase of both reﬂection and absorption properties due to the speciﬁc design of this coupled solid-ﬂuid system. Moreover, by increasing the number of cells in the material, the reﬂection mechanism becomes the dominating one. To obtain the desired transmission dip, it is necessary to properly incorporate the losses in the ﬂuid, i.e. a realistic viscothermal description of air needs to be adopted. A complex description of the ﬂuid domain is fully justiﬁed since the characteristic lengths of the pores are comparable with the thicknesses of the viscous and thermal boundary layers. Coupling between the ﬂuid and solid domains plays an important role in inducing the resonance and amplifying viscothermal dissipa- tion. Although the idea of designing a dynamic absorber using tuned resonators (also called Lanchester damper) introduced M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 202 by Den Hartog ( Den Hartog, 1985 ), is well known in the literature (and could be explored further in the metamaterial com- munity), the concept proposed here goes clearly beyond that and relies on the more complex interaction between solid and ﬂuid including viscous effects resulting from the non-slip interface condition. Particularly, due to the presence of the viscous stresses, the solid and ﬂuid domains are strongly coupled and the local resonance has a visible effect on the performance of the material. As demonstrated through the dispersion analysis, the combination of the local resonance of the solid and ﬂuid viscosity allows for the attenuation of the fast (ﬂuid-born) compressional wave, which is key for the development of the next generation acoustic porous materials. 4. Discussion This study has also identiﬁed the main parameters governing the attenuation performance of acoustic metafoams, namely the characteristic size of the pores, the membrane opening ratio and the viscosity of the ﬂuid. The characteristic pore size determines the frequencies at which absorption can be expected ( Gao et al., 2017 ). The opening ratio inﬂuences the at- tenuation performance (see Fig. 16 ). In particular, small membrane openings broaden the absorption peak, but they should be suﬃciently small for obtaining high attenuation levels. Finally, the ﬂuid viscosity is essential for enhancing attenuation by ensuring a strong coupling between solid and ﬂuid phases. Depending on other material and geometrical parameters, an optimal level of viscosity exists for which dissipation in the ﬂuid is enhanced by suﬃciently high amplitudes of the local res- onator (in analogy to viscoelastic solid metamaterials, as shown in Lewi ´nska et al. (2017) ). In other words, a viscosity value exists for which the attenuation factor for the fast compressional wave is (locally) the highest. Therefore, an optimal design of the geometry could be pursued, by controlling the characteristic dimensions of the unit cell and the viscosity of the ﬂuid such that the effect of the attenuation is maximised. Intrinsic parameters of the resonators (mass, stiffness) can also be var- ied in order to tune the operating frequency range, as done for purely solid metamaterials e.g. in Krushynska et al. (2014) . Moreover, material damping (viscosity) of the solid should also be taken into account, since it directly inﬂuences the eﬃ- ciency of the local resonator as demonstrated e.g. in Krushynska et al. (2016) . In terms of applications, the microstructural design proposed in this paper can be used to improve the performance of porous materials by the distribution of resonating particles inside their pores. This work might open new paths for designing porous materials, especially considering recent advancements in the control of foam manufacturing processes ( Lesov et al., 2014 ) and the developments in 3D printing techniques for cellular materials ( Kaur et al., 2017 ). Limited by the computational resources for direct simulations of multiphase lossy material with ﬁne geometrical features, the performance of up to ﬁfteen identical unit cells in a row has been analysed, whereas the typical thickness of foam panels is of the order of several centimetres using high-performance computing tools e.g. van Tuijl et al. 5. Conclusion This paper presented an acoustic metafoam concept, based on a poroelastic unit cell with an embedded resonating mass. The acoustic attenuation at low frequencies has been improved by combining the effects of viscothermal dissipation in the ﬂuid (air) with local resonance of the solid. In contrast to many previous studies, the resonators are introduced within the pore and in the form of a resonating particle at the tip of an elastic micro-cantilever. Analysis of complex dispersion diagrams and numerical transmission spectra showed that the proposed unit cell enriched with a resonator performs signiﬁcantly better at low frequencies (below 450 Hz) than its light or non-resonating equivalent. The resulting transmission dip is profound even for a single unit cell. However, due to the resonant origin of the attenuation the affected frequency range remains limited. It has been shown that the enhanced attenuation only emerges if viscothermal losses in the ﬂuid are included. This underlines the role of the ﬂuid-solid coupling due to which not only the local resonance is induced but also the viscous dissipation is increased. The complex ﬂuid description is another feature distinguishing this work among other metamate- rials involving acoustic-structure design ( Kook and Jensen, 2017 ), where a phenomenological description of losses is usually suﬃcient. This work contributes to a novel design towards acoustic metafoams and to the development of new porous materials, with improved performance at low frequencies. 4. Discussion (2018) , these larger thickness could be reached. On the other hand, based on the studies of band structures, the behaviour of an inﬁnite periodic material has been assessed, leading to consistent conclusions. For the proposed unit cell geometry, the observed transmission dip is followed by a transmission peak, which is typical for locally resonant acoustic metamaterials ( Lewi ´nska et al., 2017; Liu et al., 20 0 0 ) and coherent with the attenuation spectra obtained based on Bloch analysis. This fact implies that the proposed idealised structure is speciﬁcally effective for wave attenuation at a selected low frequency range. A random microstructure with non-uniform resonators, however, may have the potential of providing even broader frequency attenuation ranges. This would require further analyses which will be the subject of future investigations. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement no 339392 . Acknowledgements The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement no 339392 . M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 203 M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 203 203 Appendix In the appendix the details concerning the complex band structure calculation for a two phase unit cell consisting of a solid frame entrained with either a complex viscous or an inviscid ﬂuid are presented. For this purpose, the weak forms of governing equations with the application of Bloch theorem are presented. Acoustic ﬁeld with viscous effects The governing equations (see Eqs. (2) , (3) , and (4) assuming that thermal dissipation is negligible allowing to discard the temperature ﬁeld) are: i ωρ f 0 v = −∇p + ∇ · μ f (∇ v + (∇ v ) T ) −2 3 μ f (∇ · v ) I , (12) ρ f 0 ∇ · v = −i ω p c 2 0 . (13) Next, Bloch wave solutions are assumed in the form: (14) p = ˜ p exp (i k · x ) , (15) v = ˜ v exp (i k · x ) , ∇p = ∇ ˜ p + i ˜ p k exp (i k · x ) , (16) ∇ v = ∇ ˜ v + i k ˜ v exp (i k · x ) , ∇ v = ∇ ˜ v + i k ˜ v exp (i k · x ) , (17) ∇ · v = ∇ · ˜ v + i k · ˜ v exp (i k · x ) , (18) where ˜ p and ˜ v are the Bloch pressure and velocity functions with spacial periodicity (identical to the periodicity of the structure), x is the position vector and k is the wavevector. In these equations, the dyadic product is denoted as: a b = a n b m e n e m . The ﬁnal weak form describing the acoustic ﬁeld with viscous effects is obtained after the substitution of the Bloch wave solutions in Eqs. ∇ u = (∇ ˜ u + i k ˜ u ) exp ( i k · x ) , ∇ u = (∇ ˜ u + i k ˜ u ) exp ( i k · x ) , (25) where ˜ u is the Bloch displacement function with spatial periodicity (identical to the periodicity of the structure). The weak form obtained for Eq. (23) after the substitution of the Bloch wave solution is written as: e e u s t e oc d sp ace e t u ct o t spat a pe od c ty ( de t ca to t e pe od c ty o t e st uctu e). The weak form obtained for Eq. (23) after the substitution of the Bloch wave solution is written as: − V s ρω 2 ˜ u · φs d V −i V s φs k : 4 C s : (∇ ˜ u + i k ˜ u ) d V + V s (∇φs ) T : ( 4 C s : (∇ ˜ u + i k ˜ u ) d V − ∂V s n s · 4 C s : (∇ ˜ u + i k ˜ u ) · φs d = 0 , (26) (26) φs is a test function, V s denotes the solid domain, n s is the unit outward normal to the boundary where φs is a test function, V s denotes the solid domain, n s is the unit outward normal to the b V s . where φs is a test function, V s denotes the solid domain, n s is the unit outward normal to the boundary ∂V s of the domain V s . Coupling conditions at the interface between ﬂuid and solid are adopted as described in Section 2.2 through the bound- ary terms in the weak forms of the governing equations (using weak contributions assigned to surfaces). A set of fully coupled equations is solved similarly as done for instance in Matuszyk et al. (2012) . p q y y ( ) In order to obtain an eigenvalue problem, the k -vector is written as k = k α, where k is the amplitude of the wave vector along the unit propagation direction α. Since only the X direction of wave propagation is considered here, wavenumbers k = k x e x are calculated for given frequencies. Appendix (12) and (13) , followed by the multiplication with their respective test functions φf , φf and integration over the ﬂuid domain V f : −i V f ωρ f 0 ˜ v · φ f d V −i V f ˜ p k · φ f d V + V f ˜ p ∇ · φ f d V − V f (∇ φ f ) T : μ f ∇ ˜ v + i k ˜ v + ∇ ˜ v + i k ˜ v T d V +i V f k · μ f ∇ ˜ v + i k ˜ v + ∇ ˜ v + i k ˜ v T · φ f d V + V f (∇φ f ) T : 2 3 μ f ∇ · ˜ v + i k · ˜ v I d V −i V f k · 2 3 μ f ∇ · ˜ v + i k · ˜ v I · φ f d V − ∂V f n f · ˜ p φ f d − ∂V f n f · 2 3 μ f ∇ · ˜ v + i k · ˜ v I · φ f d + ∂V f n f · μ f ( ∇ ˜ v + i k ˜ v + ∇ ˜ v + i k ˜ v T ) · φ f d = 0 , (19) −i V f ωρ f 0 ˜ v · φ f d V −i V f ˜ p k · φ f d V + V f ˜ p ∇ · φ f d V − V f (∇ φ f ) T : μ f ∇ ˜ v + i k ˜ v + ∇ ˜ v + i k ˜ v T d V +i V f k · μ f ∇ ˜ v + i k ˜ v + ∇ ˜ v + i k ˜ v T · φ f d V + V f (∇φ f ) T : 2 3 μ f ∇ · ˜ v + i k · ˜ v I d V −i V f k · 2 3 μ f ∇ · ˜ v + i k · ˜ v I · φ f d V − ∂V f n f · ˜ p φ f d − ∂V f n f · 2 3 μ f ∇ · ˜ v + i k · ˜ v I · φ f d + ∂V f n f · μ f ( ∇ ˜ v + i k ˜ v + ∇ ˜ v + i k ˜ v T ) · φ f d = (19) ρ f 0 V f ∇ · v φ f d V −ρ f 0 V f ∇φ f · v d V + i ρ f 0 V f v · k φ f d V + i ω c 2 0 V f ˜ p φ f d V = 0 , ρ f 0 V f ∇ · v φ f d V −ρ f 0 V f ∇φ f · v d V + i ρ f 0 V f v · k φ f d V + i ω c 2 0 V f ˜ p φ f d V = 0 , (20) ρ f 0 V f ∇ · v φ f d V −ρ f 0 V f ∇φ f · v d V + i ρ f 0 V f v · k φ f d V + i ω c 2 0 V f ˜ p φ f d V = 0 , (20) where n f is the unit outward normal to the boundary ∂V f of the domain V f . Boutin, C. , Becot, F. , 2015. Theory and experiments on poro acoustics with inner resonators. Wave M Bruneau, M. , Potel, C. , 2013. Materials and acoustics handbook. NY John Wiley & Sons, New York . Allard, J.F. , Atalla, N. , 2009. Propagation of sound in porous media: Modelling sound absorbing materials, 2nd. John Wiley & Borocin, F. , 2003. Reﬂection and transmission coeﬃcients in ﬂuid-saturated poroelastic sediments. The University of Edinbur Boutin, C. , Becot, F. , 2015. Theory and experiments on poro-acoustics with inner resonators. Wave Motion 54, 76–99 . Allard, J.F. , Atalla, N. , 2009. Propagation of sound in porous media: Modelling sound absorbing materials, 2nd. John Wiley & Borocin F 2003 Reﬂection and transmission coeﬃcients in ﬂuid saturated poroelastic sediments The University of Edinbur Allard, J.F. , Atalla, N. , 2009. Propagation of sound in porous media: Modelling sound absorbing materials, 2nd. John Wiley & Sons, United Kingdom . Borocin, F. , 2003. Reﬂection and transmission coeﬃcients in ﬂuid-saturated poroelastic sediments. The University of Edinburgh, Edinburgh . Boutin, C. , Becot, F. , 2015. Theory and experiments on poro-acoustics with inner resonators. Wave Motion 54, 76–99 . Appendix where φa is a test function, V a denotes the ﬂuid domain, n a is the unit outward normal to the boundary ∂V a of the domain V a . Elastic solid Elastic solid Elastic solid The equation of motion for elastic solid is given (see Eq. (1) ) by: −ρs ω 2 u = ∇ · ( 4 C s : ∇ u ) . (23) The Bloch wave solutions assumed in this case are: u = ˜ u exp ( i k · x ) , (24) ∇ u = (∇ ˜ u + i k ˜ u ) exp ( i k · x ) , (25) where ˜ u is the Bloch displacement function with spatial periodicity (identical to the periodicity of the structure). The weak form obtained for Eq. (23) after the substitution of the Bloch wave solution is written as: The equation of motion for elastic solid is given (see Eq. (1) ) by: The equation of motion for elastic solid is given (see Eq. (1) ) by: (23) −ρs ω 2 u = ∇ · ( 4 C s : ∇ u ) . −ρs ω 2 u = ∇ · ( 4 C s : ∇ u ) . The Bloch wave solutions assumed in this case are: (24) u = ˜ u exp ( i k · x ) , u = ˜ u exp ( i k · x ) , (24) ∇ u = (∇ ˜ u + i k ˜ u ) exp ( i k · x ) , (25) Appendix M.A. Lewi ´nska et al. / Journal of the Mechanics and Physics of Solids 124 (2019) 189–205 204 Acoustic inviscid isothermal ﬂuid equation is given (see Eq. (7) ) by: The acoustic Helmholtz equation is given (see Eq. (7) ) by: with k 0 = ω c0 . (21) ∇ · (∇p) + k 2 0 p = 0 , with k 0 = ω c 0 . ∇ · (∇p) + k 2 0 p = 0 , with k 0 = ω c 0 . ∇ · (∇p) + k 2 0 p = 0 , with k 0 = ω c 0 . (21) The weak form obtained for Eq. (21) after the substitution of Bloch wave solution is written as: The weak form obtained for Eq. 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https://openalex.org/W2981301687	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0224141&type=printable	English	null	Understanding self-reported importance of religion/spirituality in a North American sample of individuals at risk for familial depression: A principal component analysis	PloS one	2,019	cc-by	8,906	RESEARCH ARTICLE Connie SvobID1,2, Lidia Y. X. WongID2, Marc J. Gameroff1,2, Priya J. Wickramaratne1,2, Myrna M. Weissman1,2, Ju¨rgen KayserID1,2* 1 Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, New York, United States of America, 2 New York State Psychiatric Institute, New York, New York, United States of America * Jurgen.Kayser@nyspi.columbia.edu Abstract Several studies have shown protective effects between health outcomes and subjective reports of religious/spiritual (R/S) importance, as measured by a single self-report item. In a 3-generation study of individuals at high or low familial risk for depression, R/S importance was found to be protective against depression, as indicated by clinical and neurobiological outcomes. The psychological components underlying these protective effects, however, remain little understood. Hence, to clarify the meaning of answering the R/S importance item, we employed a comprehensive set of validated scales assessing religious beliefs and experiences and exploratory factor analysis to uncover latent R/S constructs that strongly and independently correlated with the single-item measure of R/S importance. A Varimax- rotated principal component analysis (PCA) resulted in a 23-factor solution (Eigenvalue > 1; 71.5% explained variance) with 8 factors that, respectively, accounted for at least 3% of the total variance. The first factor (15.8%) was directly related to the R/S importance item (r = .819), as well as personal relationship with the Divine, forgiveness by God, religious activities, and religious coping, while precluding gratitude, altruism, and social support, among other survey subscales. The corresponding factor scores were greater in older indi- viduals and those at low familial risk. Moreover, Spearman rank-order correlations between the R/S importance item and other subscales revealed relative consistency across genera- tions and risk groups. Taken together, the single R/S importance item constituted a robust measure of what may be generally conceived of as “religious importance,” ranking highest among a diverse latent factor structure of R/S. As this suggests adequate single-item con- struct validity, it may be adequate for use in health studies lacking the resources for more extensive measures. Nonetheless, given that this single item accounted for only a small fraction of the total survey variance, results based on the item should be interpreted and applied with caution. OPEN ACCESS Citation: Svob C, Wong LYX, Gameroff MJ, Wickramaratne PJ, Weissman MM, Kayser J (2019) Understanding self-reported importance of religion/spirituality in a North American sample of individuals at risk for familial depression: A principal component analysis. PLoS ONE 14(10): e0224141. https://doi.org/10.1371/journal. pone.0224141 * Jurgen.Kayser@nyspi.columbia.edu Understanding self-reported importance of religion/spirituality in a North American sample of individuals at risk for familial depression: A principal component analysis Connie SvobID1,2, Lidia Y. X. WongID2, Marc J. Gameroff1,2, Priya J. Wickramaratne1,2, Myrna M. Weissman1,2, Ju¨rgen KayserID1,2* Introduction information on data access requests, interested readers may contact IRBMail@nyspi.columbia.edu. There has been growing interest in the role of religiosity/spirituality (R/S) in health research [1], with findings generally focusing on three broad R/S domains based on single-item mea- sures, namely, R/S importance, service attendance, and religious affiliation. R/S importance (i.e., How important to you is religion or spirituality?) is often associated with protective effects —presumably independently of religious service attendance and religious affiliation, which have also proven beneficial [2–7]–and this has been particularly true in our studies [8–16]. As such, we were motivated to investigate what underlies the largely subjective and complex con- struct of R/S importance. Funding: This study was supported in part by the John Templeton Foundation (https://www. templeton.org/) grant #54679 (MMW), #61330 (MMW), and the National Institute of Mental Health (https://www.nimh.nih.gov/index.shtml) grant 2- R01-MH36197 (MMW). In the past three years, Dr. Weissman received funding from the National Institute of Mental Health (NIMH), the Sackler Foundation, the John Templeton Foundation, and receives royalties from the Oxford University Press, Perseus Press, the American Psychiatric Association Press, and MultiHealth Systems. Dr. Kayser received funding from NIMH and the John Templeton Foundation. The remaining authors have no disclosures to report. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. As reviewed by Koenig [1], approximately 80% of research on R/S and health focuses on mental health. Previous research by our team has investigated extensively the ways R/S importance, service attendance, and denomination impact families at-risk for major depres- sive disorder. In most instances, we found that R/S importance was the only item associated with many of the health-related outcomes. For example, we found high self-report ratings of R/S importance to be protective against recurrence of depression [11] and childhood suicidal behaviors [12]. We have also observed protective effects of R/S importance in bio- markers of high-risk as compared to low-risk families, including decreased default mode network connectivity [13], thicker cortices [8–9], and greater posterior EEG alpha [15–16]. Moreover, these effects were transmitted across generations for both depression [10] and suicidal behavior [14], and extended to correlates of genetic markers [17]. Taken together, R/S importance has evidenced a role in the resilience of individuals at high risk for depres- sion [18–19], yet the specific mechanisms that underlie its protective effects remain little understood. Editor: Rosemary Frey, University of Auckland, NEW ZEALAND Received: April 1, 2019 Accepted: October 6, 2019 Published: October 18, 2019 Copyright: © 2019 Svob 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: Data were obtained as part of an ongoing, multi-generational study of families at risk for depression that started in 1982 (before data sharing existed); therefore, consent was not obtained. According to the Institutional Review Board at Columbia University and New York State Psychiatric Institute, public data sharing, even anonymously, is restricted by participants’ informed consent. For further 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Introduction Competing interests: The authors have declared that no competing interests exist. There is general consensus among social scientists that a single item is insufficient to describe the depth and complexity of personal religiosity and spirituality [20–30]. To this end, various efforts have been made to (a) differentiate explicitly between religion and spirituality [1,28–29], and (b) to design multidimensional instruments that capture different aspects of religious/spiritual experience [20,28,31]. Importantly, how participants understand or respond to the single R/S Importance item, whose terms are not defined and are, moreover, conflated in the single item, remains unknown. Efforts to expand measures of this single item have primarily been driven through a top- down process, that is, they have been based on theoretical constructs that hold significance to psychologists and religious scholars. In contrast, McClintock, Lau, and Miller [28] developed a questionnaire to identify common dimensions of spirituality across three diverse cultures (China, India, USA). Their objective was to examine the spiritual constructs that may not be captured by more traditional scales administered to primarily Judeo-Christian populations. Using exploratory factor analysis and cross-validating exploratory structural equation modeling identified five factors: (a) Altruistic Engagement (i.e., altruism), (b) Love, (c) Con- templative Practice (e.g., meditation, yoga), (d) Unifying Interconnectedness (viewing the environment and all living things from a spiritually unifying perspective), and (e) Religious and Spiritual Reflection and Commitment. Using confirmatory factor analysis on a selected subset of their original 34 R/S instruments, McClintock et al. [27] were able to replicate this factor structure in the present sample of individuals at high and low risk for major depressive disorder. Importantly, the R/S Reflection and Commitment factor correlated strongly (r = .80) with the single-item of R/S importance, and lower religious/spiritual commitment was associ- ated with previous MDD diagnosis, particularly among high risk individuals. Because the recent McClintock et al. [27] study was motivated by the findings of McClintock et al. [28], it only included about two-thirds of the available R/S variables for this sample (i.e., to maximize 2 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis variable overlap across these two McClintock et al. studies), and clarifying the meaning of the single-item of R/S importance was not its primary focus. Participants Data were derived from a 3-generation, 35-year longitudinal study of families at high- and low-risk for major depressive disorder (MDD) that also included reports of R/S importance [18–19]. Over the 35 years, data have been collected at Year 0 (baseline) and in subsequent waves at Year 2, 10, 20, 25, 30, and 35. High and low risk was defined by the original proband (1st generation; G1) having a diagnosis of major depressive disorder (MDD), or no psychiatric diagnosis, as related to their 2nd generation (G2) and 3rd generation (G3) offspring. G1 partic- ipants were all European Caucasian and predominantly Catholic. In the present study, partici- pants (N = 282) were drawn from both risk groups and across all generations, with ages ranging from 18.5 years to 87.4 years. All participants provided written informed consent and all interviews were approved by the Institutional Review Boards at Columbia University and New York State Psychiatric Institute. Data for the present study were collected 35 years into the study (Yr35) and included an extensive survey of R/S measures as detailed below, comprising Likert-scale items that were administered using paper and pencil (n = 88) or online (n = 194) through a secure, HIPAA-compliant internet application (Qualtrics.com). Introduction Therefore, the purpose of the present study was to conduct an in-depth investigation of the single item of R/S importance through a bottom-up, data-driven process, which would reveal the psychological components underlying the R/S importance item. The obvious advantage of a data-driven approach is its bottom-up nature to inform our understanding of the psychologi- cal construct of R/S importance, which should, in turn, provide important clarifications for previous and future health-related findings. Hence, the goal of the present study was to better understand what underlies the protective effects observed in prior reports that have found associations with high ratings of R/S importance. To this end, we employed a comprehensive survey to examine the relationship between the single item measure of R/S importance and other validated constructs within the psychology of religion. By doing so we sought to help bridge previous theoretical and empirical findings across the biomedical and social sciences and aid in their interpretation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis Table 1. Religious/Spiritual constructs comprising survey. R/S Construct LABEL Number of Items Description Reference Altruism ALTRUISM 6 Giving of oneself for the good of another. [33] Belief Salience BEL_SALIENCE 5 Degree to which religious beliefs influence one’s personal life and God is considered an intimate part of it. [34] Compassion COMPASSION 5 Empathy for others. [31] Contemplative Practice CONT_PRACT 4 Mind-body practices, such as yoga and meditation. [28] Eco-Awareness ECO_AWARE 6 Degree to which communion is sensed with creation and all living things. [35] Forgiveness FORGIVE 3 A measure of forgiveness extended to self, others, and God. [31] Gratitude GRATITUDE 4 Recognition and appreciation of an inherent good. [31] Intrinsic Religiosity INTRINS_REL 3 Degree to which a person is motivated by religious precepts for their own sake, rather than the perceived benefits they receive from being a part of a religious group. [36] Ontological Love ONT_LOVE 4 Attitudes toward love. [37] Psychological Love PSYC_LOVE 4 Experience of being loved. [37] Religious Community Social Support REL_SUPPORT 2 Degree to which a religious community itself lends support to a person, aside from the support found in other parts of one’s life. [20] Religious Coping REL_COPING 6 Degree to which a person uses religion to help them cope with life stressors by either framing situations in a positive light or seeking refuge and support in God. [31] Religious Engagement REL_ENGAGE 4 Degree to which a person engages in religious activities, such as prayer, reading sacred texts, attending services, participating in religious groups like Bible Studies. [33] Self-Transcendence SELF_TRANS 26 Degree to which religion or spirituality elevate a person’s awareness beyond themselves. [38] Social Love SOC_LOVE 4 Relational love. [37] Social Support SOC_SUPPORT 3 Support derived from friends and family. [31] Spirituality in Nature SP_NATURE 7 Sensing a greater spiritual power through nature. [28] Universality UNIVERSALITY 9 Awareness of interconnection between oneself, others, and all of life. [39] Volunteering VOLUNTEER 1 Freely giving of one’s time to those in need, whether religious or non-religious. [31] https://doi.org/10.1371/journal.pone.0224141.t001 Table 1. Religious/Spiritual constructs comprising survey. spiritual dimension of the human person not dependent on affiliation with formal institutional religion). It drew heavily upon Hill and Pargament’s [32] suggestions for areas of growth con- cerning religious conceptualization and measurement in health research, as well as McClin- tock et al.’s [28] global spirituality measures to encompass a diverse array of religious and spiritual constructs. Notably, the present survey included several R/S instruments (i.e., com- passion, forgiveness, gratitude, religious community support, religious coping, self-transcen- dence, social support, volunteering) that were not considered by McClintock et al. [27]. See Table 1 for complete listing (in alphabetical order). Religiosity/Spirituality variables Data collection in this ongoing longitudinal study has typically been separated by approxi- mately 5 to 10 year increments since Year 10 (i.e., at Years 0, 2, 10, 20, 25, 30, and 35). Starting at Year 10 and continuing to the present (Year 35), two single items of religious/spiritual items of importance and service attendance have been measured. R/S importance (R/S IMPORTANCE) was measured by responses to the question How important to you is religion or spirituality? on a 4-point Likert scale (i.e., highly, moderately, slightly, or not at all important). Religious service attendance (REL_ATTENDANCE) was determined by responses to the question How often, if at all, do you attend church, synagogue, or other religious or spiritual ser- vices? on a 5-point Likert scale (Once a week or more, About once a month, About once or twice a year, Less than once a year, or Never). In addition to measures of R/S importance and attendance at Year 35, we also obtained data for an extensive array of religious and spiritual constructs taken from previously vali- dated, published scales. This survey sought to include a reasonably comprehensive set of religious (facets of organized religion) and spiritual constructs (encompassing the broader 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Statistical analyses Given the research objective, a multi-pronged statistical approach was adopted. First, Spear- man rank order correlations were run between R/S importance and the survey items at Yr35 across high and low risk groups, as well generations 2 and 3. Generation 1 (G1) was precluded in the refined analyses as the G1 probands defined risk status for all following generations. Sur- vey items were then ordered by their degree of association with the R/S importance item at Yr35. Second, all survey items (excluding items related to follow-up questions) were submitted to exploratory factor analysis using SPSS 24.0 (SPSS Inc., 2011). Beforehand, missing values were imputed in the statistical platform R on a variable-by-variable basis using the package MICE (Multivariate Imputation via Chained Equations) [40]. After excluding follow-up items on Religious Coping (REL_COPING_1 to REL_COPING_6) and Religious Support (REL_SUPPORT_1 and REL_SUPPORT_2), 101 variables were used for imputation of 0.54% 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis of data assumed to be missing at random. Five imputed datasets were created using predictive mean matching (50 iterations) and then combined to an average dataset with no missing val- ues. The final imputed data set comprising 101 variables was submitted to principal compo- nent analyses (PCA) using a correlation association matrix, followed by an orthogonal rotation (Varimax) [41] to maximize item variance and simplify interpretability of the latent factors. As there was no a priori assumption about the factor structure, that is, to what degree factors are correlated, this process was repeated using an oblique rotation (Promax) [42]. Fac- tor extraction was limited to an Eigenvalue > 1 criterion, resulting in 23 extracted factors. Pearson’s correlations were then run between the factor scores of the first eight high-variance components deemed meaningful (consistent with a Scree test criterion and, respectively, accounting for at least 3% of the total variance after Varimax rotation) and the eight excluded items comprising Religious Coping and Religious Support (i.e., for the subset of participants who provided responses to these items). As these correlations are essentially equivalent to the factor loadings of the individual items if they had been part of the original PCA, this provided a convenient means to gauge the relationship of these additional variables to the R/S impor- tance item. Statistical analyses This combined data-driven approach allowed for a comprehensive evaluation of the interrelationship between the different religious scales and instruments with a special emphasis on their association with the R/S importance item. To test the robustness of the PCA solution with regard to the High Risk design, analogous PCA solutions were obtained for critical subgroups, that is, for generations 2 (n = 140) and 3 (n = 99), and for high (n = 150) and low (n = 89) risk subgroups. Tucker’s factor congruence coefficient [43] was used to establish “equality” ( .95) or “fair similarity” ( .85) of factor loadings. To accomplish this, Tucker’s congruence coefficient was computed for pairwise com- parisons of all factors of the original PCA solution (N = 282) with all factors of each of the four additional subgroup PCA solutions. Factors were deemed robust if they had a single corre- sponding factor matching equality in all subgroup PCA comparisons, or almost robust if unique factor correspondence reached at least fair similarity. For robust and almost robust fac- tors, the corresponding factor scores were submitted to an analysis of covariance (i.e., one for each factor), using generation (2, 3) and risk (high, low) as between-subject variables, and gen- der and age as covariates. The analyses allowed evaluating whether stable latent factors stem- ming from the survey variables differed among offspring of the original probands as a function of familial risk status and generation. Tucker’s factor congruence coefficient was also used to compare the PCA solutions obtained when using Varimax versus Promax rotations. PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Results Table 2 summarizes the sample characteristics, including demographics, clinical and religious characteristics at the 35-year follow-up. Significant differences in frequency distributions of these variables between G2 and G3, which were the focus of this report, are reported in the last column of Table 2. The majority of the sample were female (60.6%), Catholic (49.1%), and from families at high risk for depression (63.1%). The oldest generation (G1) rated R/S Impor- tance as being highly important more often than did the following generations (58.5% [G1] compared to 27.1% [G2] and 21.2% [G3]). Similarly, older generations attended weekly reli- gious services more frequently than subsequent younger generations (46.3% [G1] as compared to 13.6% [G2] and 9.1% [G3]); this effect was also observed when directly comparing G2 and G3. Finally, the oldest generation (G1) identified primarily as being “religious and spiritual” (61%), whereas G2 and G3 were fairly evenly divided between identifying as “religious and spiritual” and “spiritual, but not religious.” As for a lifetime history of clinical diagnoses, G2 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 5 / 15 Religious/spiritual importance: A principal component analysis Table 2. Demographic, clinical, and religiosity characteristics of families at high and low risk for depression at most recent wave (Yr35). Characteristics Total N = 282 Generation 1 n = 41 Generation 2 n = 141 Generation 3 n = 99 G2 vs. s one participant from Generation 4 G3 ϕ Age [Mean (SD)] 46.61 (17.91) 76.26 (0.49) 51.72 (7.48) 27.34 (5.88) χ2 df p N (%) N (%) N (%) N (%) Gender Male 111 (39.4) 16 (39.0) 52 (36.9) 42 (42.4) .75 1 .39 Female 171 (60.6) 25 (61.0) 89 (62.4) 57 (57.6) Risk for Depression (MDD) Low Risk 104 (36.9) 15 (36.6) 50 (35.5) 39 (39.4) .39 1 .54 High Risk 178 (63.1) 26 (63.4) 91 (64.5) 60 (60.6) Clinical Diagnoses (Lifetime) MDD 117 (41.5) 15 (36.6) 78 (55.7) 24 (24.2) 23.48 1 < .001 Anxiety Disorder 116 (41.1) 12 (29.3) 68 (48.6) 36 (36.4) 3.52 1 .06 Alcohol/Drug Disorder 106 (37.6) 13 (31.7) 68 (48.6) 25 (25.3) 13.27 1 < .001 Disruptive Disorder 42 (14.9) 1 (2.4) 27 (19.3) 14 (14.1) 1.08 1 .30 Suicide Attempts 4 (1.4) 1 (2.4) 3 (2.1) 0 (0) 2.15 1 .14 Religiosity/Spirituality (R/S) Denomination Catholic 138 (49.1) 25 (61.0) 68 (48.2) 45 (45.5) 1.54 2 .46 Protestant 48 (17.1) 8 (19.5) 26 (18.4) 14 (14.1) Other 95 (33.8) 8 (19.5) 47 (33.3) 40 (40.4) R/S Importance High 83 (29.4) 25 (58.5) 38 (27.1) 21 (21.2) 2.70 3 .44 Moderate 84 (29.8) 10 (24.4) 46 (32.9) 28 (28.3) Slight 80 (28.4) 7 (17.1) 39 (27.9) 34 (34.3) Not at All 34 (12.1) 0 (0) 17 (12.1) 16 (16.2) Religious Service Attendance Once a Week 47 (16.7) 19 (46.3) 19 (13.6) 9 (9.1) 9.55 4 .05 Once a Month 38 (13.5) 4 (9.8) 23 (16.6) 11 (11.1) 1–2 Times a Year 76 (27.0) 9 (22.0) 34 (24.3) 33 (33.3) < Once a Year 58 (20.6) 5 (12.2) 37 (26.4) 16 (16.2) Never 62 (22.0) 4 (9.8) 27 (19.3) 30 (30.3) Religious Identity Religious and Spiritual 113 (40.1) 25 (61.0) 49 (34.8) 39 (39.4) 1.28 3 .74 Spiritual, Not Religious 95 (33.7) 9 (22.0) 53 (37.6) 33 (33.3) Religious, Not Spiritual 22 (7.8) 4 (9.8) 9 (6.4) 9 (9.1) Neither Spiritual or Religious 49 (17.4) 2 (4.9) 28 (19.9) 18 (18.2) Includes one participant from Generation 4 ical, and religiosity characteristics of families at high and low risk for depression at most recent wave (Yr35). had significantly higher rates of depression and alcohol/drug disorders than G3, presumably because they were older and had more time to develop the disorders. S1 Table (see Supplement) displays the Spearman rank order correlations of the R/S Importance item at the most recent wave (Yr35) with all survey items. The purpose of a non- parametric ranking was to provide an intuitive understanding which survey items and R/S scales associate most strongly with R/S Importance. Several scale constructs correlated highly with R/S Importance, including (a) belief salience, (b) religious engagement, (c) religious Several scale constructs correlated highly with R/S Importance, including (a) belief salience, (b) religious engagement, (c) religious PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 6 / 15 Religious/spiritual importance: A principal component analysis coping, and (d) self-transcendence. By and large, all religious/spiritual constructs retained the same order across generations and risk groups. Importantly, several items failed to correlate with R/S Importance, including items related to contemplative practice, eco-awareness, spirituality in nature, social support, love, and gratitude. Again, this lack of correlation was observed in the overall sample, and held its consistency across generations and risk groups. As a more stringent test of association between R/S Importance and the other R/S con- structs measured in the survey, we conducted a Varimax-rotated PCA to reveal the latent fac- tor structure underlying all survey items, that is, to determine the R/S items that load on an independent, orthogonal dimension alongside the single-item of R/S Importance. The PCA resulted in a 23-factor solution (Eigenvalue > 1; 71.5% explained variance) with each of the first 8 extracted factors accounting for at least 3% of the total variance (see Table 3). The factor containing the single item of R/S importance (to which we will refer to as the R/S Importance Factor) explained 15.8% of the overall variance after rotation (factor 1) and had high internal consistency (Cronbach’s alpha = .84). Similar to the highest Spearman rank order correlations with R/S importance, the R/S Importance Factor included items related to Belief Salience, Reli- gious Engagement, Religious Coping, Self-Transcendence, and Forgiveness by God. Apart from the R/S Importance Factor (factor 1), 7 other out of the 8 high-variance factors (> 3%) were readily interpretable and included, by order of extraction: (2) Spirituality in Nature, (3) Self-transcendence, (4) Altruism, (5) Love, (6) Gratitude, (7) Social Support, and (8) Mind Wandering. Five of these 8 high-variance factors were also found to be consistent across the four identi- fied subgroups: high vs. low risk, and generation 2 vs. 3 (see Supplemental S2 Table). The comparison of the factor loadings stemming from the subgroup PCA solutions via Tucker’s congruence coefficient (ϕ) indicated that factors 1 (R/S Importance Factor) and 2 (Spirituality in Nature Factor) were robust (equality correspondence of factor loadings in each subgroup), and factors 4 (Altruism Factor), 6 (Gratitude Factor) and 7 (Social Support Factor) were almost robust (at least fair similarity correspondence in each subgroup). The ensuing repeated measures ANOVAs for the corresponding factor scores revealed sig- nificant effects only for the R/S Importance Factor: a generation main effect, F[1,233] = 6.43, p = 0.01, stemming from greater R/S importance for generation 2 than 3, and a risk main effect, F[1,233] = 7.18, p = 0.008, with low risk individuals having greater R/S importance than high risk individuals; however, there was no significant generation x risk interaction (F[1,233] < 1.0). No significant main or interactions effects were observed for the other four factors deemed robust or almost robust (i.e., Spirituality in Nature Factor, Altruism Factor, Gratitude Factor, and Social Support Factor). This supports the notion that the R/S Importance Factor is a stable and reliable factor, one which allows for meaningful interpretability of results, and which differs between G2 and G3 and between low and high risk individuals. Finally, the Promax-rotated PCA solution, which allowed for correlated factors, was remarkably similar to the Varimax-PCA solution (see Supplement S3 Table). All 23 extracted factors showed a 1:1 correspondence of at least fair similarity (0.88 ϕ 0.98) between solu- tions. Accordingly, the 8 high-variance factors identified by the Varimax rotation loaded highly and by-and-large on the same variables, thereby resulting in virtually the same factor structure. Demographic, clinical, and religiosity characteristics of families at high and low risk for depression at most recent wave Includes one participant from Generation 4 had significantly higher rates of depression and alcohol/drug disorders than G3, presumably because they were older and had more time to develop the disorders. S1 Table (see Supplement) displays the Spearman rank order correlations of the R/S Importance item at the most recent wave (Yr35) with all survey items. The purpose of a non- parametric ranking was to provide an intuitive understanding which survey items and R/S scales associate most strongly with R/S Importance. Several scale constructs correlated highly with R/S Importance, including (a) belief salience, (b) religious engagement, (c) religious Includes one participant from Generation 4 The clinical diagnoses are not mutually exclusive; multiple diagnoses are possible. ϕ Pearson’s Chi-Square test statistics are reported to compare the sampling distributions for Generations 2 and 3 only. https://doi.org/10.1371/journal.pone.0224141.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 6 / 15 had significantly higher rates of depression and alcohol/drug disorders than G3, presumably because they were older and had more time to develop the disorders. S1 Table (see Supplement) displays the Spearman rank order correlations of the R/S Importance item at the most recent wave (Yr35) with all survey items. The purpose of a non- parametric ranking was to provide an intuitive understanding which survey items and R/S scales associate most strongly with R/S Importance. Several scale constructs correlated highly with R/S Importance, including (a) belief salience, (b) religious engagement, (c) religious Includes one participant from Generation 4 The clinical diagnoses are not mutually exclusive; multiple diagnoses are possible. ϕ Pearson’s Chi-Square test statistics are reported to compare the sampling distributions for Generations 2 and 3 only. https://doi.org/10.1371/journal.pone.0224141.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 6 / 15 had significantly higher rates of depression and alcohol/drug disorders than G3, presumably because they were older and had more time to develop the disorders. S1 Table (see Supplement) displays the Spearman rank order correlations of the R/S Importance item at the most recent wave (Yr35) with all survey items. The purpose of a non- parametric ranking was to provide an intuitive understanding which survey items and R/S scales associate most strongly with R/S Importance. Discussion To better understand the mechanisms implicated in the demonstrated protective effects on health when affirmatively answering the question how important to you is religion or spirituality [2–16], the present report assessed latent psychological components comprising a 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis Table 3. Varimax-rotated principal component loadings for questionnaire items at most recent wave (Yr35). Factor Items (n = 282 unless otherwise specified) 1 2 3 4 5 6 7 8 R/S Importance Spirituality in Nature Self-transcendence Altruism Love Gratitude Social Support Mind Wandering BEL_SALIENCE_2 .869 BEL_SALIENCE_3 .852 BEL_SALIENCE_1 .848 INTRINS_REL_2 .825 R/S IMPORTANCE .819 INTRINS_REL_3 .812 INTRINS_REL_1 .799 REL_ENGAGE_1 -.765 SELF_TRANS_16 .745 BEL_SALIENCE_5 .691 REL_ATTENDANCE .655 REL_ENGAGE_2 -.654 REL_ENGAGE_3 -.648 FORGIVE_3 .646 SELF_TRANS_17 .640 SELF_TRANS_4_Rev .583 SELF_TRANS_14 .544 .410 SELF_TRANS_7 .544 .400 SP_NATURE_3 .820 SP_NATURE_4 .811 ECO_AWARE_1 .798 SP_NATURE_1 .766 SP_NATURE_6 .760 SP_NATURE_5 .717 SP_NATURE_2 .334 .670 SP_NATURE_7 .385 .609 ECO_AWARE_6 .564 .464 ECO_AWARE_2 .551 SELF_TRANS_3 .538 .530 SELF_TRANS_5 .743 SELF_TRANS_8 .686 SELF_TRANS_10 .626 SELF_TRANS_18 .609 SELF_TRANS_6 .592 SELF_TRANS_2 .334 .558 SELF_TRANS_25 .333 .550 SELF_TRANS_13 .522 SELF_TRANS_1 .514 .383 SELF_TRANS_11 .396 SELF_TRANS_15 .387 .351 ALTRUISM_1 .785 ALTRUISM _2 .765 ALTRUISM_4 .756 ALTRUISM_3 .664 ALTRUISM_6 .608 (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 8 / 15 N.B. Religious Engagement (REL_ENGAGE) items were scored on a scale ranging from Highest to Lowest, rather than Lowest to Highest, as were the remaining scales. Variable/item not included in PCA due to missing data. Values listed are Pearson’s correlations, which are in this case equivalent to factor loadings. Correlations in bold are loadings > .5. Loadings < .3 are not listed. https://doi.org/10.1371/journal.pone.0224141.t003 are loadings > .5. Loadings < .3 are not listed. Correlations in bold are loadings > .5. Loadings < .3 are not listed. N.B. Religious Engagement (REL_ENGAGE) items were scored on a scale ranging from Highest to Lowest, rather than Low Variable/item not included in PCA due to missing data. Values listed are Pearson’s correlations, which are in this case equ For example, measures of love, altruism, gratitude, contemplative/medi- tative practice, communing with nature, and social support were weakly correlated with R/S importance and, accordingly, loaded on separate PCA factors. This suggests that these aspects of religion, spirituality, and positive psychology, while important psychological constructs in and of themselves, may contribute minimally to the protective effects of R/S importance observed in previous studies. Critically, the PCA solution obtained for the full sample was stable when compared to PCA solutions obtained for important subgroups of this cohort of families at risk for depression. Several factors were either robust (R/S Importance Factor, Spirituality in Nature Factor) or almost robust (Altruism Factor, Gratitude Factor, Social Support Factor), strongly suggesting that these represent genuine latent R/S constructs or constituents. However, only the R/S Importance Factor revealed differences between the cohort subgroups that are consistent with previously reported findings—that is, greater ratings of R/S importance in generation 2 than 3 [14] and in low than in high risk participants [11]. The factor structure observed here appeared to closely match the common dimensions of spirituality reported by McClintock et al. [28], which were religious commitment, contempla- tive practice, interconnectedness, love, and altruism. Their five-dimensional structure was uncovered in a large sample (N = 5512) of cross-cultural spirituality involving participants from China, India, and the United States across various religious backgrounds (Buddhism, Christianity, Hinduism, Islam, Judaism, non-religious, or other). Initially, the authors employed a data-driven approach on two subsamples (i.e., exploratory factor analyses with oblique rotation), which was then followed-up by confirmatory factor analyses on two other subsamples (i.e., validating the original factor structure via exploratory structural equation modeling), which suggested that these five dimensions of spirituality were universal (i.e., pres- ent across cultures, religions and societies). In a recent study relying virtually on the same par- ticipants included in the present report, McClintock et al. [27] were able to further support the validity of their five-factor structure when again relying on confirmatory factor analyses, com- paring the present high and low risk sample (N = 281) to a Caucasian Christian American sub- sample (N = 602) from their prior study. The present study differed from McClintock et al. [27] in two critical aspects: 1) it included all of the data collected in the survey (i.e., it was not restricted to the subset of measures that had been included in the earlier McClintock et al. Religious/spiritual importance: A principal component analysis Table 3. (Continued) Factor Items (n = 282 unless otherwise specified) 1 2 3 4 5 6 7 8 R/S Importance Spirituality in Nature Self-transcendence Altruism Love Gratitude Social Support Mind Wandering ALTRUISM_5 .517 .381 PSYC_LOVE_2 .795 ONT_LOVE_1 .736 ONT_LOVE_4 .662 PSYC_LOVE_1 .637 ONT_LOVE_2 .338 .334 .612 PSYC_LOVE_4 .602 ONT_LOVE_3 .438 GRATITUDE_2 .834 GRATITUDE_1 .786 GRATITUDE_3 .781 GRATITUDE_4 .729 SOC_SUPPORT_3 .835 SOC_SUPPORT_1 .776 SOC_SUPPORT_2 .769 PSYC_LOVE_3 .519 SELF_TRANS_24 .771 SELF_TRANS_19 .744 SELF_TRANS_12 .726 SELF_TRANS_9 .367 .600 UNIVERSALITY_7 .473 UNIVERSALITY_6 .478 UNIVERSALITY_5 .363 COMPASSION_2 .305 ECO_AWARE_4 .353 REL_ENGAGE_4 -.594 SELF_TRANS_23_Rev .396 BEL_SALIENCE_4 .548 SELF_TRANS_21 .339 .351 SELF_TRANS_20 .331 REL_COPING_1 (n = 139) .408 REL_COPING_2 (n = 139) .430 REL_COPING_3 (n = 139) .437 REL_COPING_4 (n = 139) .592 REL_COPING_5 (n = 139) .606 REL_COPING_6 (n = 138) .579 REL_SUPPORT_1 (n = 126) .318 REL_SUPPORT_2 (n = 126) .285 Correlations in bold are loadings > .5. Loadings < .3 are not listed. g > g < N.B. Religious Engagement (REL_ENGAGE) items were scored on a scale ranging from Highest to Lowest, rather than Lowest to Highest, as were the remaining scales. Variable/item not included in PCA due to missing data. Values listed are Pearson’s correlations, which are in this case equivalent to factor loadings. 9 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis comprehensive survey of religious beliefs and experiences: R/S importance emerged as the pri- mary factor. Several religious and spiritual constructs, as defined by existing instruments, cor- related highly with the single-item measure of R/S importance across generation and familial risk status, namely, salient R/S beliefs that were internalized and personally experienced, as well as externalized religious practices (prayer, service attendance). Additionally, a personal relationship with the Divine played a central role in perceived R/S importance, such as having a personal relationship with God, feeling forgiven by God, and leaning on God through times of stress. These findings suggest that these particular characteristics of religiosity/spirituality are implicated most strongly in its protective effects, although future work will need to address this hypothesis directly. Notably, not all previously defined sub-constructs of R/S correlated highly or consistently with R/S importance (i.e., both the single-item measure and the corresponding PCA compo- nent identified here). PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 [28] study); and 2) it employed a fully data-driven approach with all survey items, as opposed to being confined by an a priori factor solution. While a detailed discussion of the nuanced differ- ences between the multivariate data-reduction techniques employed in these two reports, which may have contributed to any differences in the factor structure, is beyond the scope of the present paper, it is nevertheless reassuring that several factors revealed a close correspon- dence, particularly the present R/S Importance Factor and McClintock et al.’s R/S Reflection PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 10 / 15 Religious/spiritual importance: A principal component analysis and Commitment factor, with both factors sharing high loadings on the same survey items (e.g., intrinsic religiosity, belief salience, religious engagement). The present R/S Importance Factor, however, also included religious coping, certain aspects of self-transcendence, and for- giveness by God, all survey items that were not included by McClintock et al. [27–28]. Our other factors were seemingly consistent with most of the independent factors observed by McClintock et al. [28], with the exception of Contemplative Practice, the reasons of which are not immediately clear. Interestingly, McClintock et al. [28] found that in the USA and India, individuals scoring in the top quartile of the R/S commitment factor (the one that correlated highly with R/S impor- tance) were about 50% less likely to experience major depressive disorder, suicidal thoughts, and generalized anxiety disorder. Moreover, in our High Risk sample, McClintock et al. [27] found that previous diagnoses of major depression were associated with lower R/S commit- ment scores in high risk individuals, lower contemplation scores in low risk individuals, and lower R/S importance scores across both risk groups. These findings support our proposal that the protective effects of R/S importance are limited to only certain aspects of R/S (primarily, a personal relationship with the Divine and engagement with religious practices) and preclude others (i.e., those that load on other factors—gratitude, love, social support, altruism and spiri- tuality in nature). The consistencies and discrepancies in factor structure between the present and the two McClintock et al. reports underscore the importance of the R/S variable selection as the most crucial decision when employing factor analytic techniques. By contrast, the extracted factor structure was not dependent on the choice of component rotation (i.e., orthogonal vs. PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 oblique); rather, the present factor structure was evidently stable and primarily determined by the given set of input variables. It is also worth noting that the single item of R/S Importance loaded on the same factor as Religious Service Attendance (another widely used single item measure used in mental health research). These two items were moderately correlated at rho = .52 (p < .01). Although it is true that people who reported religion or spirituality as being highly important to them were also more likely to attend religious services with greater frequency, this was not always the case and suggests that R/S importance encompasses something more than attendance at religious services. At the same time, religious attendance evidently contributes to what renders R/S as being personally important. Critically, only R/S importance was protective against depression in our previous studies, but not religious attendance [8–14]. The present findings hold impor- tant implications for health psychologists, psychiatrists, and clinicians in the interpretation and application of R/S importance in relation to health outcomes. The R/S importance item includes both internal (e.g., intrinsic religiosity, belief salience) and external aspects of religion and spirituality (engaging in religious activities)–and not only internal components as is often suggested [2]. Importantly, this single item appears to relate specifically to the heart of the indi- vidual through a personal belief in and relationship with God, one that is externalized through religious practices. On the other hand, the single item measure evidently does not capture other aspects of spirituality (e.g., communion with nature, gratitude, altruism). Of note, the possibility that religiosity exceeds spirituality in its protective effects against depression was also observed in several longitudinal studies [44–46]. Limitations of the present study include the cross-sectional design of the extensive array of R/S measures with clinical diagnoses. It should also be noted that the sample upon which the study is based was predominantly Caucasian and Catholic; nonetheless, given the universality of the common R/S dimensions reported by McClintock et al. [28] and repli- cated in this same sample [27], this may not be a crucial limitation. Still, further research is needed to determine whether our results generalize to other faith traditions and religious groups. Conclusion Although using a multi-dimensional questionnaire to measure religiosity/spirituality may be desirable, time and energy-expenditure constrains feasibility of comprehensive instru- ments for various populations, community samples, and mental disorders alike. Our find- ings, however, strongly suggest that the single R/S importance item has adequate construct validity and may therefore be sufficient for many applied and basic research purposes. Our findings accordingly corroborate the item’s obvious face validity: it captures much of what may be intuitively conceived as personal R/S importance when individuals are asked—to provide an integrative summary estimate of the R/S construct—which is the intent in the first place. Accordingly, it also captures both internal and external aspects of religion and spirituality and maintains considerable consistency across generations and risk groups for familial depression. Nonetheless, it is important for researchers to keep in mind the specific aspects of R/S importance included and precluded by the single item, as detailed by this report, and to apply these considerations when interpreting future findings. As such, the single-item measure alone fails to capture important aspects of religiosity and spirituality, including spirituality experienced through nature, meditative practices, gratitude, love, and compassion. Moreover, theoretical distinctions between religiosity and spirituality remain undifferentiated by this single item, and whether specific activities and forms of religious or spiritual engagement provide protection against depression (and other mental health dis- orders) is left to future research using a broader conceptualization and measurement of the R/S construct. As discussed above, our findings are also naturally limited by the constructs and PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 11 / 15 Religious/spiritual importance: A principal component analysis subscales employed in our survey despite the effort to employ a comprehensive selection of R/S scales. We also note the limitation of the single-item measure of R/S importance itself. More com- plex measures may distinguish between religiosity and spirituality, whereas religiosity involves formal or informal religious practices (public or private), spirituality involves the individual’s relationship to a transcendent force (God or higher power [47]; cf. [48]). Spirituality is said to represent an integrative force in the individual’s life [49], providing meaning, core values, and principles for organizing one’s life, which may or may not be a part of religion. To the extent that spirituality and religion constitute unique constructs, they are necessarily conflated by a single-item measure. As another possible limitation, the sample size of N = 282 may be too small for a PCA with 101 variables because it violates a popular rule calling for a cases-to-variables ratio of no less than 5:1, although this rule—without further qualification—lacks both empirical support and a theoretical rationale [50–51]. For the current data, 7 out of the 8 factors described in Table 3 included four or more variables with loadings above .6 (the R/S importance factor had 15), thereby warranting their interpretation independent of sample size [50]. More importantly, the systematic comparisons of PCA solutions using Tucker’s congruence coefficient identified several factors as being robust, including the R/S importance factor, despite the fact that each of the subgroup PCAs violated the item ratio rule to an even greater degree. Acknowledgments This project was made possible through the support of a grant from the John Templeton Foun- dation. The opinions expressed in this publication are those of the authors and do not neces- sarily reflect the views of the John Templeton Foundation. Author Contributions Conceptualization: Connie Svob, Priya J. Wickramaratne, Ju¨rgen Kayser. Data curation: Marc J. Gameroff. Formal analysis: Connie Svob, Lidia Y. X. Wong, Marc J. Gameroff, Ju¨rgen Kayser. Funding acquisition: Myrna M. Weissman. Investigation: Connie Svob, Ju¨rgen Kayser. Methodology: Connie Svob, Marc J. Gameroff, Priya J. Wickramaratne, Ju¨rgen Kayser. Project administration: Connie Svob, Lidia Y. X. Wong, Marc J. Gameroff, Ju¨rgen Kayser. Supervision: Myrna M. Weissman, Ju¨rgen Kayser. Writing – original draft: Connie Svob, Lidia Y. X. Wong, Ju¨rgen Kayser. Conceptualization: Connie Svob, Priya J. Wickramaratne, Ju¨rgen Kayser. Formal analysis: Connie Svob, Lidia Y. X. Wong, Marc J. Gameroff, Ju¨rgen Kayser. Methodology: Connie Svob, Marc J. Gameroff, Priya J. Wickramaratne, Ju¨rgen Kayser. Project administration: Connie Svob, Lidia Y. X. Wong, Marc J. Gameroff, Ju¨rgen Kayser. Supervision: Myrna M. Weissman, Ju¨rgen Kayser. Writing – original draft: Connie Svob, Lidia Y. X. Wong, Ju¨rgen Kayser. Writing – review & editing: Connie Svob, Lidia Y. X. Wong, Priya J. Wickramaratne, Myrna M. Weissman, Ju¨rgen Kayser. Supporting information S1 Table. Nonparametric Spearman rank-order correlations between religious/spiritual importance (at Yr35) and each quantitative variable included in the religiosity/spirituality (R/S) survey for full sample and subgroup categories reflecting risk status and generation. (PDF) 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0224141 October 18, 2019 Religious/spiritual importance: A principal component analysis References 1. Koenig HG. Religion, spirituality, and health: The research and clinical implications. ISRN, Article ID 278730. 2012: 1–33. https://doi.org/10.5402/2012/278730 PMID: 23762764 2. Hoffman S, Marsiglia F. The impact of religiosity on suicidal ideation among youth in central Mexico. J Relig Health. 2014; 53(1): 255–266. https://doi.org/10.1007/s10943-012-9654-1 PMID: 23054483 3. Kendler KS, Gardner CO, Prescott CA. Religion, psychopathology, and substance use and abuse; a multimeasure, genetic-epidemiologic study. 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https://openalex.org/W4381337162	http://usnsj.id/index.php/JME/article/download/1338/pdf	English	null	Student’s Junior High School Mathematical Connection Ability Based On The Big Five Personality	Journal of Mathematics Education	2,021	cc-by	7,187	AUTHORS INFO © 2021 JME All rights reserved © 2021 JME All rights reserved g p y Wardina,A.S., Nurjanah, & Sudihartinih, E. (2021). Student’ Junior High School Mathematical Connection Ability Based on The Big Five Personality. Journal of Mathematics Education, 6(1), 1-15. http://doi.org/10.31327/jme.v6i1.1338 Journal of Mathematics Education ∼JME∼ Website: http://usnsj.com/index.php/JME Email: editor.jme@usnsj.com Student’s Junior High School Mathematical Connection Ability Based on The Big Five Personality Suggestion for the citation and bibliography Citation in text: Wardina,A.S., Nurjanah, & Sudihartinih, E. (2021) Bibliography: Wardina,A.S., Nurjanah, & Sudihartinih, E. (2021). Student’ Junior High School Mathematical Connection Ability Based on The Big Five Personality. Journal of Mathematics Education, 6(1), 1-15. http://doi.org/10.31327/jme.v6i1.1338 Aulia Suci Wardina Universitas Pendidikan Indonesia Auliasuciwardina19@student.upi.edu +6285524452455 Nurjanah Universitas Pendidikan Indonesia Nurjanah@upi.edu +628122401402 Eyus Sudihartinih Eyuss84@upi.edu +6285624110105 o-ISSN: 2528-2026 p-ISSN: 2528-2468 Vol. 6, No. 1, June 2021 URL: http://doi.org/10.31327/jme.v6i1.1338 © 2021 JME All rights reserved Abstract Each individual has a diverse personality which is categorized by the Big Five Personality traits and it’s important for a successful education. A successful education especially in mathematics major aimed at the good of student’s mathematical abilities AUTHORS INFO ARTICLE INFO Creative Commons Attribution 4.0 International License Journal of Mathematics Education ∼JME∼ Website: http://usnsj.com/index.php/JME Email: editor.jme@usnsj.com Student’s Junior High School Mathematical Connection Ability Based on The Big Five Personality Suggestion for the citation and bibliography Citation in text: Wardina,A.S., Nurjanah, & Sudihartinih, E. (2021) Bibliography: Wardina,A.S., Nurjanah, & Sudihartinih, E. (2021). Student’ Junior High School Mathematical Connection Ability Based on The Big Five Personality. Journal of Mathematics Education, 6(1), 1-15. http://doi.org/10.31327/jme.v6i1.1338 Aulia Suci Wardina Universitas Pendidikan Indonesia Auliasuciwardina19@student.upi.edu +6285524452455 Nurjanah Universitas Pendidikan Indonesia Nurjanah@upi.edu +628122401402 Eyus Sudihartinih Eyuss84@upi.edu +6285624110105 o-ISSN: 2528-2026 p-ISSN: 2528-2468 Vol. 6, No. 1, June 2021 URL: http://doi.org/10.31327/jme.v6i1.1338 © 2021 JME All rights reserved AUTHORS INFO ARTICLE INFO Creative Commons Attribution 4.0 International License A. Introduction If students can properly connect ideas in mathematics, mathematical ideas with other disciplines, or mathematical ideas to everyday problems, then they will have good mathematical skills. The mathematical connection is an indicator of a mathematical understanding because it relates to the ability to make connections between ideas, concepts, procedures, representations, and meaning in mathematics (Berry, J., & Nyman, 2003). The ability of mathematical connections plays a role in mathematical understanding which is the internal relationship between representations of mathematical ideas, procedures, and facts so that a stronger mathematical connection allows for increased mathematical understanding (Hiebert J, 1992). Students who able to connect mathematical ideas can complete tasks related to mathematics and make connections between new ideas and relevant things (Waynberg, as cited in Zengin, 2019). Based on previous research, it’s known that the connection as a critical thinking ability in Indonesian students is still low, one of which is due to the low quality of mathematics learning in schools (Listiani, 2016). Other research shows that the percentage of student’s ability to connect between mathematical concepts is 58.33% (good enough), the ability of students to connect and apply mathematical concepts to other disciplines is 50% (less), the ability of students to apply mathematical concepts to solve the problem in daily life is 66.67% (good enough) (Wardina & Sudihartinih, 2019). This shows that the mathematical connection ability of students needs further research. The difference in the processing of the given questions can affect the academic achievement of students at school. Student academic achievement can’t be separated from the type of learning. Different types of learning are influenced by differences in their personalities (Vygotsky, Lave & Wanger, Wanger, as cited in Bhakta, Wood, & Lawson, 2010). The grouping of personality dimensions can be done through the Big Five. The Big Five traits (extraversion, agreeableness, conscientiousness, neuroticism, and openness) have been associated with a variety of behaviors including job performance, academic performance, leadership, and well being (John & Srivastava, Heckman, Hakim, Jackson, Shaw, Scott & Rich, Fairweather, Singh, as cited in Nye, Orel, & Kochergina, 2013). Conscientiousness can predict academic success, performance on exams, and cumulative grade point index, openness and agreeableness are also related to academic performance, and Neuroticism is related to disrupted academic (Komarraju, Karau, & Schmeck, 2009). Abstract Each individual has a diverse personality which is categorized by the Big Five Personality traits and it’s important for a successful education. A successful education especially in mathematics major aimed at the good of student’s mathematical abilities, one of which is good mathematical connection ability. This research aims to determine the mathematical connection ability of junior high school students based on the Big Five personality in Bekasi. This research used qualitative research case studies with data triangulation. Participants of this research were junior high school students in grade ninth. The instrument used in the form is a test description as many as four questions that refer to the indicators of mathematical connection ability and has been consulted with experts and the Big Five personality traits questionnaire that has been adopted. The results of this research describe: 1) mathematical connection ability of the student in Extraversion dimension on indicator 1 was 60%, indicator 2 was 0%, and indicator 3 was 0%, 2) the mathematical connection ability of students in Agreeableness dimension on indicator 1 was 60%, indicator 2 was 25%, and indicator 3 was 25%, 3) mathematical connection ability of students in Conscientiousness dimension on indicator 1 was 60%, indicator 2 was 35%, and indicator 3 was 25%, 4) mathematical connection ability of a student in Neuroticism dimension on indicator 1 was 20%, indicator 2 was 0%, and indicator 3 was 20%, 5) mathematical connection ability of students in Openness dimension on indicator 1 was 64.21%, indicator 2 was 27.37%, and indicator 3 was 34.21%. Keywords: connections, mathematical connection, mathematical connection ability, big five, personality Keywords: connections, mathematical connection, mathematical connection ability, big five, personality JME/6.1; 1-15; June 2021 JME/6.1; 1-15; June 2021 2 A. Introduction Based on the description that has been described, the purpose of this research was to determine student’s junior high school mathematical connection ability based on the Big Five personality. This research can be a source of information that would be studied more deeply by other studies regarding mathematical connection ability or other mathematical connection based on the personality traits so it can be a useful knowledge in mathematics subject. 1. Mathematical Connection Ability The mathematical connection is one of the characteristics of mathematics where mathematical ideas are interconnected with certain relationships and these connections can be identified a priori and independently from students (Businskas, 2008). The mathematical connection is part of the relationship between knowledge and other knowledge which consists of important concepts to understand and develop relationships between mathematical ideas, concepts, and procedures (Kenedi, Helsa, Ariani, Zainil, & Hendri, 2019). p p ( ) Mathematical connection skills include: 1) recognizing and using connections among mathematical ideas; 2) understand how mathematical ideas relate to one another and build on one another to produce a coherent whole; 3) recognize and apply mathematics in contexts outside of mathematics, if students can connect, they will have a deep mathematical understanding and be able to last longer (NCTM, 2000). There are three indicators for the ability of mathematical connections: 1) to understand the relationship between one mathematical concept and another; 2) understanding the relationship between mathematics and other disciplines of science; 3) understand the relationship between mathematics and everyday life (NCTM, 2000). y y ( 2. Big Five Personality Personality is an expression of the hierarchical integration of the dispositions and innate habits and habitual reaction tendencies of each individual socially (Walker, 2008). Personality is defined as a characteristic pattern of a person’s behavior in a broad sense including thoughts, JME/6.1; 1-15; June 2021 JME/6.1; 1-15; June 2021 3 feelings, and motivations (Uher, 2017). Personality is one of the non-cognitive factors that play an important role in the success of education or work (Rieger et al., 2017). The Big Five model has been defined as an important and productive taxonomy for classifying personality structures (Bakker, Barrick & Mount, Roberts, Caspi & Goldberg, Soldz & Vaillant, as cited in Meyer et al., 2019). The Big Five personality types are classified into extraversion, agreeableness, neuroticism, conscientiousness, and openness which is based on the idea that the real difference that can be seen from someone is behavior (John & Srivasta, 1999). The extraversion dimension (denoted by E) relates to individual engagement with the outside world and enthusiasm and other positive emotions, the agreeableness dimension (denoted by A) relates to individual cooperative values and social harmony, politeness, trust, and honesty, individuals with the Agreeableness dimension have a view that optimistic about other individuals, conscientiousness (denoted by C) is related to the value of individual planning, perseverance, and results-oriented (achievement), neuroticism (denoted by N) relates to individual negative feelings and excessive emotional stress, openness (denoted by O) is associated with curiosity, self-awareness, and individualism (Rossberger, as cited in Ali, 2019). Conscientiousness has good behavior in learning at every level, is ambitious, persistent, diligent, has learning rules, goals, systematic learning styles, and appropriate learning methods (Meyer et al., 2019). The neuroticism dimension is related to the poor self-concept and low self intelligence (Furnham, Chamorro-Premuzic, & McDougall, 2003). Openness is a personality type that describes individuals with high curiosity and motivation from within (Komarraju et al., 2009). Individuals with high scores of openness have higher critical thinking skills (McCredie & Kurtz, 2020). 1. Research Design This research is a qualitative case study which describes student’s junior high school mathematical connection ability based on the Big Five personality on the topic of the polyhedron that using data triangulation (interviews, theory, and documentation). Participants of this research were 28 female students in class IX for answering the questionnaire of big five personality traits with a duration of 15 minutes and 26 female students for doing mathematical connection ability test with a duration of 30 minutes at one of the private junior high schools in Bekasi Regency which took place on August 6 and 28, 2020. Participants who were then selected in this research were 25 female students. 2. Instruments The instruments used in this research were a questionnaire of big five personality traits that have been adopted from the previous research and 4 questions of polyhedron topic that has been consulted to experts and arranged by the indicator of mathematical connection ability: 1) students can recognize and connect between mathematical ideas, 2) students can connect and apply mathematical concepts to other disciplines, 3) students can connect and apply mathematical concepts to everyday life. These indicators are listed sequentially in question number 1, 2, 3, and 4 with the following problems: 1. It is known that the length of the base of the PBD triangle on the cube is √ . Calculate the volume of the ABCD.EFGH cube! JME/6.1; 1-15; June 2021 4 2. It is known that the following three objects are given the same force. Specify and provide the reasons: 2. It is known that the following three objects are given the same force. Specify and provide the reasons: the reasons: a. The object that exerts the least pressure b. The object which has the smallest volume 3. My father bought a prism-shaped aquarium with a hexagon-shaped base and a height of 50 cm. Father filled the aquarium with water until the water level reached 20 cm. Father again filled the aquarium with water as much as until the water rose from its height before. Calculate the base area of the aquarium! a. The object that exerts the least pressure p st volume b. The object which has the smallest volume 3. My father bought a prism-shaped aquarium with a hexagon-shaped base and a height of 50 cm. Father filled the aquarium with water until the water level reached 20 cm. Father again filled the aquarium with water as much as until the water rose from its height before. Calculate the base area of the aquarium! 3. My father bought a prism-shaped aquarium with a hexagon-shaped base and a height of 50 cm. Father filled the aquarium with water until the water level reached 20 cm. Father again filled the aquarium with water as much as until the water rose from its height before. Calculate the base area of the aquarium! 3. My father bought a prism-shaped aquarium with a hexagon-shaped base and a height of 50 cm. Father filled the aquarium with water until the water level reached 20 cm. 2. Instruments Mathematical Connection Ability Assessment Rubric (Wardina & Sudihartinih, 2019) Indicators Student responses to questions Score Students can recognize and connect mathematical ideas Didn’t answer at all 0 The answer doesn’t match the question 1 Able to answer the question, not yet able to connect, and the final result is wrong 2 Able to answer the question, not yet able to connect, but the final result is correct 3 Able to answer the question and connect, but the final result is wrong 4 Able to answer a question and connect, but the final result is correct 5 Students can connect and apply mathematical concepts to other disciplines Didn’t answer at all 0 The answer doesn’t match the question 1 Able to answer the question, not yet able to connect, and the final result is wrong 2 Able to answer the question, not yet able to connect, but the final result is correct 3 Able to answer a question and connect, but the final result is wrong 4 Able to answer a question and connect, but the final result is correct 5 Students can connect and apply mathematical concepts to everyday life Didn’t answer at all 0 The answers don’t match the question 1 Able to answer the question, not yet able to connect, and the final result is wrong 2 Able to answer the question, not yet able to connect, but the final result is correct 3 Able to answer a question and connect, but the final result is wrong 4 Able to answer a question and connect, but the final result is correct 5 JME/6.1; 1-15; June 2021 JME/6.1; 1-15; June 2021 5 Calculation of the total score proportion of each problem-solving indicator ( in Siregar & Surya, 2017 used: with the qualifications below: Calculation of the total score proportion of each problem-solving indicator ( in Siregar & Surya, 2017 used: with the qualifications below: Table 2. Qualifications of Mathematical Connection Ability on Each Indicator (Siregar & Surya, 2017) Percentage Qualifications Very Good Good Good Enough Less Very Less Table 2. Qualifications of Mathematical Connection Ability on Each Indicator (Siregar & Surya, 2017) 3. The technique Data Analysis 3. The technique Data Analysis The data analysis technique used in this research is 1) data reduction, at this stage the existing data are grouped based on the results of a personality trait questionnaire which is then scored on the mathematical connection problem, selecting samples that represent each of the Big Five personality dimensions (extraversion, conscientiousness, neuroticism, agreeableness, and openness), and summarizes the data that has been obtained, 2) data presentation by describing student’s mathematical connection ability based on the Big Five personality grouping on the topic of a polyhedron, 3) withdrawal of all activities and results obtained from research that done. 2. Instruments Father again filled the aquarium with water as much as until the water rose from its height before. Calculate the base area of the aquarium! 4. Bookstore “X” plans to sell gift boxes in the shape of cuboids with a size of The bookstore buys wrapping paper with a size of . If bookstore “X” plans to sell 150 gift boxes, then how much wrapping paper is needed to wrap the boxes? 4. Bookstore “X” plans to sell gift boxes in the shape of cuboids with a size of The bookstore buys wrapping paper with a size of . If bookstore “X” plans to sell 150 gift boxes, then how much wrapping paper is needed to wrap the boxes? Table 1. Mathematical Connection Ability Assessment Rubric (Wardina & Sudihartinih, 2019) Table 1. D. Findings and Discussion 1. Findings Based on the results of answering the Big Five personality questionnaire, there were 1 student (4%) has been categorized as extraversion, 2 students (8%) have been categorized to agreeableness, 2 students (8%) have been categorized to conscientiousness, 1 student (4%) has been categorized to neuroticism, and 19 students (76%) have been categorized to openness. The results of the student’s mathematical connection ability based on the Big Five personality were shown in the following table. Table 3. Student’s Mathematical Connection Ability Based on The Big Five personality The Big Five Personality Dimension The Numbe r of Student s Indicator 1 Indicator 2 Indicator 3 Percenta ge Qualificati on Percenta ge Qualificati on Percenta ge Qualificati on Extraversion 1 60 Good Enough 0 Very Less 0 Very Less Agreeableness 2 60 Good Enough 25 Very Less 25 Very Less Conscientiousn ess 2 60 Good Enough 35 Very Less 25 Very Less Neuroticism 1 20 Very Less 0 Very Less 20 Very Less Openness 19 64,21 Good Enough 27,37 Very Less 34,21 Very Less Table 3. Student’s Mathematical Connection Ability Based on The Big Five personality The student worksheets that will be analyzed are students who have different category of The Big Five personality and their answer to the mathematical connection test that stands out the most. The author conducts interviews to complete the data and performs analysis on the answer sheets of selected students based on The Big Five personality, which is as follows. JME/6.1; 1-15; June 2021 6 a. Worksheet and interviews of ZN who has categorized to extraversion Figure 1. The Answer of ZN On Question Number 1 Figure 1. The Answer of ZN On Question Number 1 From Figure 1, it is known that ZN with extraversion tends to be easier to socialize, active, people-oriented, optimistic, fun, and full of affection. ZN has been able to connect between mathematical concepts, namely in identifying the length of the side of the cube, but she is not shown yet how to get the value 4 as the length of the side of the cube. ZN is still incomplete in writings × s × s as the volume and unit volume used. ZN did not answer question number 2, 3, and 4. JME/6.1; 1-15; June 2021 JME/6.1; 1-15; June 2021 JME/6.1; 1-15; June 2021 was able to answer correctly, but it is not yet known how they connect between a mathematical concept in the form of a polyhedron (using the diagonal length of the plane known in the problem to find the side length of the cube). KS didn’t answer sheet number 2 which indicates that KS has not been able to connect mathematical concepts related to the volume and area of the base from polyhedron with the concept of pressure on liquids in science material (Physics). was able to answer correctly, but it is not yet known how they connect between a mathematical concept in the form of a polyhedron (using the diagonal length of the plane known in the problem to find the side length of the cube). KS didn’t answer sheet number 2 which indicates that KS has not been able to connect mathematical concepts related to the volume and area of the base from polyhedron with the concept of pressure on liquids in science material (Physics). Figure 3. The Answer of KS On Question Number 3 Figure 3. The Answer of KS On Question Number 3 Based on Figure 3, it is known that KS is still wrong in understanding the concept of volume in a prism by writing the formula for the volume of the prism which is then multiplied by half. KS did not continue his work to find the results of the problems given, so it could be concluded that KS was still unable to connect concepts in mathematics to solve sample problems in everyday life. Figure 4. The Answer of KS On Question Number 4 Figure 4. The Answer of KS On Question Number 4 The final solution of question number 4 that has been written by KS is 4, in figure 4 shows that the KS has been able to find the surface area of the gift box in the form of cuboid s, but she is still unable to connect the surface area of the gift box and the area of the wrapping paper that will be used to cover it. She is also not able to understand the related mathematical concepts that will be used to solve example problems in everyday life. P : “What did you feel when you do the test?” KS : “It’s so difficult. D. Findings and Discussion 1. Findings This indicates that ZN has not been able to understand the problem well, to connect mathematical concepts related to the area of the base and the volume of several spatial shapes with the concept of science (physics) related to pressure on solids, to work on problems and finding the final solution to the problems given, and to connect mathematical concepts related to polyhedron with some examples of mathematical problems in everyday life. p y y P : “When you answered the questions, what did you think?” ZN : “The questions were difficult. I’m confused.” P : “What were the steps to answer question 1? for what?” ZN : “It was a volume of a cube.” P : “How can you assume that the cube edge is 4 cm?” ZN : “I don’t know, sist.” P : “What did you think to answer question 2? How?” ZN : “At that time, I thought to use the formula that had been taught before, but I was confused so I didn’t answer it.” P : “When you answered question 3, what did you think to answer it? What must you do first? How to find it?” ZN : “A little bit. I gave up.” P : “When you answered question 4, what did you think?” ZN : “I don’t know, sist. Hehehe.” p y y : “When you answered the questions, what did you think?” b. Worksheet and interviews of KS who has categorized to agreeableness g g Figure 2. The Answer of KS On Question Number 1 Figure 2. The Answer of KS On Question Number 1 KS who has categorized as agreeableness tend to be kind, gentle, full of trust, forgiving, has altruistic, responsive, and empathetic traits, in figure 2 of the KS worksheet it is known that KS 7 JME/6.1; 1-15; June 2021 I’m confused and didn’t understand.” P : “For number 1, how could you answer?” KS : “I know the formula, sister.” P : “What is the formula about?” KS : “The formula of a cube, sister.” P : “Why you answer 64 directly?” KS : “I’ll write 4 as a side before, but the time was over.” P : “How could you answer number 2?” KS : “It didn’t answer yet, because I’m confused.” P : “But, did you find the way?” KS : “I must calculate it but it did not.” P : “How could you answer number 3?” KS : “I forgot the volume of a prism, so it can’t be solved.” P : “For number 4, what must we find?” KS : “First, we have to find the area of surface, size of wrapping paper. 8 JME/6.1; 1-15; J Then the surface area is divided by the number of gift boxes. But the result is not finished yet, sis” P : “Okay, thank you. When studying the previous topic, was it effective in a pandemic like this?” KS : “No, it was not. Because of that, I'm confused and not optimal yet.” 8 JME/6.1; 1-15; June 2021 Then the surface area is divided by the number of gift boxes. But the result is not finished yet, sis” P : “Okay, thank you. When studying the previous topic, was it effective in a pandemic like this?” KS : “No, it was not. Because of that, I'm confused and not optimal yet.” JME/6.1; 1-15; June 2021 8 c. Worksheet and interviews of FA who has categorized to conscientiousness c. Worksheet and interviews of FA who has categorized to conscientiousness Figure 5. The Answer of FA On Question Number 1 Figure 5. The Answer of FA On Question Number 1 FA who has categorized as conscientiousness tend to be organized, reliable, hardworking, independent, precise, conscientious, ambitious, and diligent. From Figure 5, it is known that FA has been able to answer the question correctly and determine the length of the edge on the cube, but has not shown in the worksheet the steps in obtaining the length of the edge using the diagonal length of the plane that is known in the problem. Figure 6. The Answer of FA On Question Number 2 Figure 6. JME/6.1; 1-15; June 2021 The Answer of FA On Question Number 2 From the student worksheet in Figure 6, it is known that the FA’s answering question number 2a has not been able to relate the concept of the pressure formula to the area of the base which will later be used to compare the volume between the polyhedron. FA perceived all pressure to be the same because it is known that the applied force is equal. FA has been able to answer question number 2b correctly with the reasons written on the figure of the space where she has been looking for the volume of the spatial shape of the known edge but there are still errors in the algebraic concept of finding the volume of cuboids and pyramids. FA also got JME/6.1; 1-15; June 2021 9 around by calculating the edge in the shape with a number, there is 2 to find the volume of each shape around by calculating the edge in the shape with a number, there is 2 to find the volume of each shape around by calculating the edge in the shape with a number, there is 2 to find the volume of each shape. Figure 7. The Answer of FA On Question Number 3 Figure 7. The Answer of FA On Question Number 3 Based on Figure 7, it is known that FA has been able to write the concept of prism volume correctly and can derive the formula to find the area of the prism base but FA still does not understand the volume and height of the prism that will be used to solve the given problem. Figure 8. The Answer of FA On Question Number 4 Figure 8. The Answer of FA On Question Number 4 From Figure 8, it is known that FA already knows the steps they will use to find the final solution to the problems given, but in the process, there are still mistakes in the operation so that she is not able to solve the problems given. From Figure 8, it is known that FA already knows the steps they will use to find the final solution to the problems given, but in the process, there are still mistakes in the operation so that she is not able to solve the problems given. P : “How you answer question number 1?” FA : “First, it’s confusing sis. JME/6.1; 1-15; June 2021 From √ then 4 as edge or sides. To find the volume, substitute the edge to the volume formula which it is obtained 64 .” P : “How could you answer question number 2?” FA : “Point a) there’s no smallest pressure because the force that was given is equal, b) pyramid because I calculate one by one and then pyramid has the smallest volume.” pyramid has the smallest volume. P : “How could you answer number 3?” FA : “I’m confused, didn’t understand. I know the formula but when I substitute the number it’s confusing.” P : “How could you answer number 4?” FA : “Not accurate, not multiplied by 2, so the result is 300 wrapping paper. I thought 300 cm, 600 wrapping paper, 300 top-down, 300 right, and left. 600 only get 1 gift box so if for example 150 boxes of blocks are multiplied by 2. " d. Worksheet and interviews of NI who has categorized to neuroticism 10 JME/6.1; 1-15; June 2021 Figure 9. The Answer of NI On Question Number 1 Figure 9. The Answer of NI On Question Number 1 NI was categorized as neuroticism where the personality shows emotional instability, self- control, and low motivation. This personality type is associated with poor self-concept and low self intelligence. The NI’s answer to question number 1 in figure 9 shows that she already understands the question and knows the volume formula to be used but when deriving the formula, NI only writes that the volume formula for the cube is and has not been able to determine the length of the edge cube from the length of diagonal that has been known from the question. q On the question number 2 and 3, NI can’t answer the question given at all, thus indicating that NI has not been able to connect the concept of pressure on solids with the concept of volume and area of the base in polyhedron and has not been able to connect and apply mathematical concepts that have been studied previously to solve the question as an example of daily life problem. Figure 10. The Answer of NI On Question Number 4 Figure 10. JME/6.1; 1-15; June 2021 The Answer of NI On Question Number 4 In figure 10, it is known that NI is only able to write down the surface area of the base on cuboid and has not been able to relate it to other concepts to solve the problem given. NI has not been able to apply mathematical concepts to solve the problem of daily life. P : “What did you feel when you do the test?” NI : “I thought that it was so difficult, I can’t solve it and wanna give up.” P : “When you answer question number 1, how to do it?” NI : “We have to find the volume of a cube with all of its sides is equal.” P : “How to answer question number 2?” NI : “I did not know. I am confused. Unthinkable because I am thinking about another matter.” P : “How to answer question number 3?” NI : “I’ll divide it before, but I'm not confident with my answer so I answer another one.” P : “How to answer question number 4?” NI : “I used the formula but I didn’t know further so it was just a piece." P : “What did you feel when you do the test?” NI : “I thought that it was so difficult, I can’t solve it and wanna give up.” P : “When you answer question number 1, how to do it?” NI : “We have to find the volume of a cube with all of its sides is equal.” P : “How to answer question number 2?” NI : “I did not know. I am confused. Unthinkable because I am thinking about another matter.” P : “How to answer question number 3?” NI : “I’ll divide it before, but I'm not confident with my answer so I answer another one.” P : “How to answer question number 4?” NI : “I used the formula but I didn’t know further so it was just a piece." 11 11 JME/6.1; 1-15; June 2021 Figure 11. The Answer of BHM On Question Number 1 Figure 11. The Answer of BHM On Question Number 1 BHM was categorized as openness that describes individuals with a high sense of knowledge and gets motivation from themselves. An individual with high openness scores has higher critical thinking skills. JME/6.1; 1-15; June 2021 The result of question number 1 that has seen in figure 11 shows that BHM was able to answer the question correctly and was able to determine the edge cube to find the volume, but it has not been written in the worksheet how to work on getting 4 as edge cube by utilizing the diagonal length field known in the question. Figure 12. The Answer of BHM On Question Number 2 Figure 12. The Answer of BHM On Question Number 2 From figure 12 it is known that BHM has been able to answer the question. On the worksheet, the pressure formula is written but it is not explained further about reasons and other work steps correctly. Figure 13. The Answer of BHM On Question Number 3 Figure 13. The Answer of BHM On Question Number 3 Figure 13 shows that BHM has written the correct answer to the question given. She has been able to find the area of the base of a prism by being able to determine the volume and height that will be used to answer the problem. 12 JME/6.1; 1-15; June 2021 Figure 14. The Answer of BHM On Question Number 4 Figure 14. The Answer of BHM On Question Number 4 Based on figure 14, it is known that BHM has been able to find the surface area of the gift box in the form of cuboid, but she is still not precise in the numerical calculation operation. BHM has not been able to connect the concept of wrapping paper area to the surface area of the gift box which will be used to find a solution to the example of the problem given. P : “How was the test? How did you do it?” BHM : “I’m confused in the beginning like I am blank.” “I see the question, try to understand it. JME/6.1; 1-15; June 2021 My mathematics teacher said that the formula in mathematics is not only one, using the logic instead.” P : “When you answer question number 1, what did you do first?” BHM : “I already know the answer.” P : “Why you think that the answer was it?” BHM : “Because mathematics is absolute, sis.” P : “Why you know that the side is 4 cm?” BHM : “From the Pythagoras theorem to find cube sides and then to find the volume.” P : “And then, what is 64?” BHM : “It was a volume of cube, sis, but it’s not written yet.” P : “How to answer question number 2?” BHM : “I just know the . The pressure is inversely proportional to the base, if the pressure is large it means that it is getting smaller. I see there is an addition on the cuboid so it means the pressure is getting smaller because of the large base of the cuboid. For b) the volume is less than the pyramid because the pyramid is a piece of a cuboid.” P : “Cuboid or cube?” BHM : “Cube.” “If the cube is small, then the pyramid is smaller than the cube.” P : “How you answer question number 3?” BHM : “First, it was so tricky. When I read and try to understand I know that the water was 1.440 , height 5 cm and then it substitutes to the formula and we get the base area is 288 .” P : “How to answer question number 4?” BHM : “I calculate the surface area, it was 150 and then the size of wrapping paper is 6000 so 6000/150 we get 40 wrapping papers. 150 boxes can be wrapped in 40 wrapping paper.” P : “Please, calculate it once more.” BHM : “Uh, it was wrong, sis.” “There were 600 for gift boxes, 6000 is an area of 1 paper. 1 paper for 10 boxes so 15 wrapping papers for 150 boxes. P : “How was the test? How did you do it?” BHM : “I’m confused in the beginning like I am blank.” “I see the question, try to understand it. 2. Discussion The author assesses 25 student’s worksheet and classifies them based on the Big Five personality: 1) ZN is a student who has categorized to extraversion, 2) KS is a student who has categorized to agreeableness, 3) FA is a student who has categorized to conscientiousness, 4) NI is a student who has categorized to neuroticism, 5) BHM is a student who has categorized to openness, to determine the student’s mathematical connection ability based on their Big Five personality types to solve problems about polyhedron topic. Student’s mathematical connection ability based on the Big Five personality 1) student who has categorized to extraversion on indicator 1 was good enough with the percentage of 60%, both on indicator 2 and 3 were very less with the percentage of 0%, 2) student who has categorized to agreeableness on indicator 1 was good enough with the percentage of 60%, both on indicator 2 and 3 were very less with the percentage of 25%, 3) student who has categorized to conscientiousness on indicator 1 was good enough with the percentage of 60%, on indicator 2 was very less with the percentage of 35%, and on indicator 3 was very less with the percentage of 25%, 4) student who has categorized to neuroticism on indicator 1, 2, and 3 were very less with the successive percentages of 20%, 0%, 20%, and 5) student who has categorized to openness on indicator 1 was good enough with the percentage of 64.21%, both on indicator 2 and 3 were very less with the successive percentages of 27.37% and 34.21%. The results showed that agreeableness, conscientiousness, and openness had a fairly dominant percentage of each indicator, and neuroticism had the lowest percentage. This is in line that conscientiousness, openness, and agreeableness related to academic performance and neuroticism related to disrupted academic performance (Komarraju et al., 2009). Openness has the highest percentage of mathematical connection ability and neuroticism has the lowest percentage of mathematical connection ability. This finding is reinforced by previous research that individuals with high openness scores have higher critical thinking skills (McCredie & Kurtz, 2020), and individuals who were categorized as neuroticism are associated with poor self-concept and low self-intelligence (Furnham et al., 2003). JME/6.1; 1-15; June 2021 My mathematics teacher said that the formula in mathematics is not only one, using the logic instead.” “When you answer question number 1, what did you do first?” “There were 600 for gift boxes, 6000 is an area of 1 paper. 1 paper for 10 boxes so 15 wrapping papers for 150 boxes. “There were 600 for gift boxes, 6000 is an area of 1 paper. 1 paper for 10 boxes so 15 wrapping papers for 150 boxes. 13 13 JME/6.1; 1-15; June 2021 Figure 15. The Students are doing the test of mathematical connection in class room Figure 15. The Students are doing the test of mathematical connection in class room Figure 16. The Students are doing the test of Big Five Personality traits in class room Figure 16. The Students are doing the test of Big Five Personality traits in class room F. Acknowledgment We are thanking all students and civitas of the institution who help us to complete this research and all of the supporters who supported this research. A paper is presenting the thesis partly and will be published in repository UPI. E. Conclusion From findings and discussion, it is known that the percentage of student’s mathematical connection ability is different for each personality type. The personalities which have a lower percentage than the others are extraversion with the percentage of mathematical connection JME/6.1; 1-15; June 2021 JME/6.1; 1-15; June 2021 14 ability on indicators 1, 2, and 3 were 60%, 0%, and 0% and neuroticism was 20%, 0%, 20%. The personalities with a higher percentage seen from each indicator with the percentage on indicators 1, 2, 3 are conscientiousness with 60%, 35%, and 25%, agreeableness with 60%, 25%, and 25%, and openness with 64.21%, 27.37%, and 34.21%. 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https://openalex.org/W4360615877	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/FAAC42897AE53C6A912C9BE9C7D3C3EA/S1752196323000093a.pdf/div-class-title-historical-records-reissuing-as-curatorial-practice-in-harry-smith-s-span-class-italic-anthology-of-american-folk-music-span-div.pdf	English	null	Historical Records: Reissuing as Curatorial Practice in Harry Smith's <i>Anthology of American Folk Music</i>	Journal of the Society for American Music	2,023	cc-by	17,624	© The Author(s), 2023. Published by Cambridge University Press on behalf of the Society for American Music. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unre- stricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 1“K-Tel: The Secret History!” The Independent, January 9, 2005, https://www.independent.co.uk/arts-entertainment/music/ features/k-tel-the-secret-history-14334.html. 2Stephen Thomas Erlewine, “Why the Death of Compilation Albums and Reissues Is Worth Mourning,” Pitchfork, May 2, 2016, https://pitchfork.com/features/article/9887-why-the-death-of-greatest-hits-albums-and-reissues-is-worth-mourning/. Abstract Reissues of recordings are commonplace, yet rarely examined by scholars. This article considers how reissuing is a creative act akin to museum curation. Through this lens, reissuers emerge as crucial interpretive figures, making decisions that affect how music and music history are understood by listeners. I take as a case study the Anthology of American Folk Music (1952), one of the most canonic and widely praised reissues. Created by record collector and avant-garde artist Harry Smith and distributed by Folkways, the Anthology comprises eighty-four commercially released recordings made between 1926 and 1932, accompanied by an idiosyncratic set of liner notes. At the time, folk music was beset by various mythologies, notably an idealization of racial purity and an antithetical stance toward commercialism and modernity. When reissuing the material, Smith carefully selected, sequenced, and annotated the recordings in ways that unsettled these mythologies. In 1994, the Smithsonian reissued the Anthology on compact disc. Drawing on archival documents from the Smithsonian, I demonstrate how their decisions reframed the Anthology in various ways to fit their goals and concerns. Through marketing, reproduction, cover design, and the soliciting and editing of additional notes, the Smithsonian elevated Smith as a racially progressive auteur while navigating commercial concerns and historical preservation. In particular, the editing of notes avoided controversial statements during an era of politically contentious museum exhibitions. Nevertheless in doing so, the very myths that Smith unsettled in his reissuing were replaced by an idealized race-blindness, anti-commercialism, and historicism. Reissues have been a cornerstone of the music industry for decades. K-Tel Records’ 25 Country Hits, a cheaply made and heavily marketed compilation album released in 1966, first demonstrated that reis- sues could be profitable.1 Interest in reissues expanded considerably in the 1980s. This boom was partly due to the rise of compact discs, replacing vinyl LPs, and the ensuing reissuing of albums on CD. However reissues also led to new compilations and sets, led especially by Rhino Records, an inde- pendent record company that made its name by reissuing and repackaging older records as new com- pilations. Rhino won critical praise for their attention to fidelity, finding high-quality masters to reproduce, and for their visually attractive packaging.2 p y p g g Unlike repressings of vinyl LPs, reissues often involve new creative work: Remastering and changing the format, assembling compilations, updating liner notes, or including new bonus tracks. Journal of the Society for American Music (2023), 17, 178–203 doi:10.1017/S1752196323000093 RESEARCH ARTICLE RESEARCH ARTICLE Historical Records: Reissuing as Curatorial Practice in Harry Smith’s Anthology of American Folk Music Dan Blim Denison University, Granville, Ohio, USA Email: blimd@denison.edu https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Abstract Soon after the compact disc format emerged, reissues began to include deluxe retrospectives of artists, including Bob Dylan’s Biograph in 1985 and the hugely successful Eric Clapton Crossroads four-disc set in 1988. These reissues offered connoisseurs rare, demo, and unreleased tracks as well as lavish packaging. Today, reissues maintain an active role in the music industry, from compilations like the multiplati- num Now That’s What I Call Music series to the multitude of box sets and anniversary reissues of https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Journal of the Society for American Music 179 canonic albums. Critics now regularly issue best-of-the-year lists celebrating the most elaborate box sets and reissues; 2019’s reissues, for example, included a fiftieth Anniversary Deluxe edition of The Beatles’ Abbey Road, featuring twenty-three outtakes and demos, The Bakersfield Sound, 1940–1974, a collection of over 200 tracks with a 225-page booklet, and Kate Bush: The Other Sides, consisting entirely of unreleased recordings from the artist. Although these reissues frequently gain attention in the popular press, scholars have largely ignored reissues as a subject, even as popular music scholarship has widened its perspective to consider the impact of figures beyond the original artist. In his review of Albin J. Zak’s The Poetics of Rock: Cutting Tracks, Making Records, Robert Walser writes approvingly that Zak “shifts our attention from the music’s most marketable figures—performers and songwriters—to the people who actually create the ‘musical works wrought in sound,’” namely producers, engineers, mixers, and technological inventors.3 Zak’s book effectively shows the complexity of the collaborations behind recordings and argues that lesser-known figures have been just as influential in music history. Scholars have also doc- umented how artists and fans use songs to create new meanings, whether through transformative cover versions, as George Plasketes, Alyssa Woods, and others have discussed,4 or through listener-generated mash-ups, which Aram Sinnreich and Kembrew McLeod argue can have a subversive, political poten- tial.5 Reissues differ from the above techniques in that they do not generate new sounds, but simply reproduce existing recordings. Nevertheless, like producers and engineers, reissuers assist in the man- ufacturing of a new commercial recording, and their artistic vision can be clearly felt in the final prod- uct. Like mash-ups and covers, reissues can juxtapose sounds and styles in meaningful ways. In doing so, they can be equally transformative of the way we listen to and interpret songs. 3Robert Walser, “Review of The Poetics of Rock: Cutting Tracks, Making Records by Albin J. Zak,” Journal of the American Musicological Society 60, no. 1 (Spring 2007): 253. 4 5Aram Sinnreich, Mashed Up: Music, Technology, and the Rise of Configurable Culture (Amherst: University of Massachusetts Press, 2010); Kembrew McLeod, “Confessions of an Intellectual (Property): Danger Mouse, Mickey Mouse, Sonny Bono, and My Long and Winding Path as a Copyright Activist-Academic,” Popular Music and Society 28, no. 1 (February 2005): 79–93. Musicological Society 60, no. 1 (Spring 2007): 253. 4Alyssa Woods, Violence and the Negotiation of Musical Meaning in Rock, Pop, and Rap cover songs (Master’s thesis, University of Ottawa, 2004); George Plasketes, “Like A Version: Cover Songs and the Tribute Trend in Popular Music,” Studies in Popular Culture 15, no. 1 (1992): 1–18; George Plasketes, ed. Play It Again: Cover Songs in Popular Music (Burlington, VT: Ashgate, 2010). https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 7David Fricke, “Anthology of American Folk Music edited by Harry Smith,” Rolling Stone, September 18, 1997, 101–2. Accessed via ProQuest. 6Mark Clague, “Portraits in Beams and Barlines: Critical Editions and the Art of Notation,” American Music 23, no. 1 (April 2005): 40. 3Robert Walser, “Review of The Poetics of Rock: Cutting Tracks, Making Records by Albin J. Zak,” Journal of the American Musicological Society 60, no. 1 (Spring 2007): 253. 4Alyssa Woods, Violence and the Negotiation of Musical Meaning in Rock, Pop, and Rap cover songs (Master’s thesis, University of Ottawa, 2004); George Plasketes, “Like A Version: Cover Songs and the Tribute Trend in Popular Music,” Studies in Popular Culture 15, no. 1 (1992): 1–18; George Plasketes, ed. Play It Again: Cover Songs in Popular Music (Burlington, VT: Ashgate, 2010). 5Aram Sinnreich, Mashed Up: Music, Technology, and the Rise of Configurable Culture (Amherst: University of Massachusetts Press, 2010); Kembrew McLeod, “Confessions of an Intellectual (Property): Danger Mouse, Mickey Mouse, Sonny Bono, and My Long and Winding Path as a Copyright Activist-Academic,” Popular Music and Society 28, no. 1 (February 2005): 79–93. 6Mark Clague, “Portraits in Beams and Barlines: Critical Editions and the Art of Notation,” American Music 23, no. 1 (April 2005): 40. 7David Fricke, “Anthology of American Folk Music edited by Harry Smith,” Rolling Stone, September 18, 1997, 101–2. Accessed via ProQuest. 5Aram Sinnreich, Mashed Up: Music, Technology, and the Rise of Configurable Culture (Amherst: University of Massachusetts Press, 2010); Kembrew McLeod, “Confessions of an Intellectual (Property): Danger Mouse, Mickey Mouse, Sonny Bono, and My Long and Winding Path as a Copyright Activist-Academic,” Popular Music and Society 28, no. 1 (February 2005): 79–93. 6Mark Clague, “Portraits in Beams and Barlines: Critical Editions and the Art of Notation,” American Music 23, no. 1 (April 2005): 40 Long and Winding Path as a Copyright Activist-Academic,” Popular Music and Society 28, no. 1 (February 2005): 79–93. 6Mark Clague, “Portraits in Beams and Barlines: Critical Editions and the Art of Notation,” American Music 23, no. 1 (April 2005): 40. 7D id F i k “A th l f A i F lk M i dit d b H S ith ” R lli St S pt b 18 1997 101 2 Abstract To better understand the activity of reissuers, this essay draws from museum studies scholarship to consider reissuers’ work as akin to that of a museum curator. Like museums, reissues frequently center on historical recordings as important artifacts—a sort of aural exhibition. Moreover, many deluxe box sets emphasize the physical material—notes, photographs, and elaborate designs—as much as the musical content. In his study of critical editions of American music, Mark Clague makes a similar move, comparing critical editions to portraits, suggesting that “musical notation communicates on sev- eral levels.”6 Clague analyzes the iconographic details of notation, cover art, and supplemental images, as well as the editorial decisions concerning what images, text, and music to include and how to notate and edit them. Reissues offer a similar wealth of material to analyze, from editorial (or curatorial) choices and selections to visual iconography. My essay examines in depth one important reissue, Harry Smith’s The Anthology of American Folk Music, a collection of eighty-four commercial recordings made between 1926 and 1932, compiled by Smith from his extensive record collection into a three double-LP volume set, and released by Folkways Records in 1954. Now considered a canonic work of Americana, no less than “a bedrock of our national musical identity” according to Rolling Stone’s David Fricke, it has garnered critical praise, robust scholarly attention, and even an Honorary Grammy Award for Smith.7 Much of this attention came in the 1990s, when another key development in the Anthology’s legacy occurred: A widely praised reissue of the album on compact disc by the Smithsonian in 1997. In the years since the reissue, 180 Dan Blim scholars have examined the album as a refraction of the political environment of the 1950s.8 Little attention has been given, however, to its status as a reissue—a fact taken for granted by many listeners. Examining the Anthology from this angle not only allows us to reconsider previous readings of the Anthology anew, but also illuminates the seldom-considered interpretive work done through the act of reissuing. In her book Destination Culture: Tourism, Museums, and Heritage, Barbara Kirshenblatt-Gimblett argues that exhibitions “are also exhibits of those who make them.”9 The Anthology was similarly the product of distinct choices that reflect the values of its reissuers. Abstract First I consider how Harry Smith’s agency as reissuer sought to define folk music in specific ways, accomplished through the collection, selection, and sequencing of the tracks, as well as his annotation through liner notes. These choices diverged from the methods and ideologies of other collectors. I then turn to the Smithsonian’s agency as reissuer of the Anthology on compact disc. Their work included remastering tracks, reproducing liner notes, and compiling accompanimental material for a lavish box set. Much of the production of this reissue is documented in the Center for Folklife and Cultural Heritage, allowing an unusually detailed glimpse into the act of reissuing. That documentation affords a glimpse into the choices made at various steps, reflecting the Smithsonian staff’s mission and values as curators of American history. Considering both Smith and the Smithsonian in tandem as agents repackaging older material demon- strates how museological practices of preservation, collection, and curation continually reshaped the meaning of this music as an aesthetic and political object. 8See Robert Cantwell, When We Were Good: The Folk Revival (Cambridge, MA: Harvard University Press, 1996); Paola Igliori, ed. American Magus, Harry Smith: A Modern Alchemist (New York: Inanout Press, 1996); David A. Jannsen and Edward J. Whitelock, Apocalypse Jukebox: The End of the World in American Popular Music (New York: Soft Skull Press, 2009); Greil Marcus, Invisible Republic: Bob Dylan’s Basement Tapes (New York: H. Holt and Co., 1997); Kevin M. Moist, “Collecting, Collage, and Alchemy: The Harry Smith Anthology of American Folk Music as Art and Cultural Intervention,” American Studies 48, no. 4 (Winter 2007): 111–27; and John Street, Music and Politics (Malden, MA: Polity Press, 2012). 9Barbara Kirshenblatt-Gimblett, Destination Culture: Tourism, Museums, and Heritage (Berkeley: University of California Press, 1998), 2. 10Roy Shuker, Wax Trash and Vinyl Treasures: Record Collecting as a Social Practice (Burlington, VT: Ashgate, 2010). See especially Chapters 2–4. 11Ivan Karp, “Introduction,” in Museums and Communities: The Politics of Public Culture, eds., Ivan Karp, Christine Mulen Kreamer, and Steven D. Levine (Washington: Smithsonian, 1992), 7. 12Kiri Miller, Traveling Home: Sacred Harp Singing and American Pluralism (Urbana: University of Illinois Press, 2008), 144–45. 8See Robert Cantwell, When We Were Good: The Folk Revival (Cambridge, MA: Harvard University Press, 1996); Paola Igliori, ed. American Magus, Harry Smith: A Modern Alchemist (New York: Inanout Press, 1996); David A. Jannsen and Edward J. Whitelock, Apocalypse Jukebox: The End of the World in American Popular Music (New York: Soft Skull Press, 2009); Greil Marcus, Invisible Republic: Bob Dylan’s Basement Tapes (New York: H. Holt and Co., 1997); Kevin M. Moist, “Collecting, Collage, and Alchemy: The Harry Smith Anthology of American Folk Music as Art and Cultural Intervention,” American Studies 48, no. 4 (Winter 2007): 111–27; and John Street, Music and Politics (Malden, MA: Polity Press, 2012). https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press er, Wax Trash and Vinyl Treasures: Record Collecting as a Social Practice (Burlington, VT: Ashgate, 2010). See pters 2–4. Smith’s Decisions: Collecting and Curating the Anthology Harry Smith’s work as creator of the Anthology of American Folk Music began, as museum collections do, with the act of collecting. Scholars have begun to explore record collecting as a cultural activity. In Wax Trash and Vinyl Treasures: Record Collecting as a Social Practice, Roy Shuker offers a compelling ethnographic overview of these activities, examining how and why collectors pursue this hobby. Of particular importance to Smith’s collecting, Shuker details how collectors are driven both by an interest in nostalgia and history and by personal aesthetics in their collecting, and how collectors in turn shape the economics of the music industry and help construct canons by instilling value upon certain art- ists.10 These perspectives resonate with how museum scholars have theorized collecting. Ivan Karp finds that the act of collecting is “intimately tied to ideas about art, science, taste, and heritage. Hence [it is] bound up with assertions about what is central or peripheral, valued or useless, known or to be discovered, essential to identity or marginal.”11 This aspect of articulating social and cultural values was especially borne out in the early work of music collectors. Heritage and cultural values have long shaped music collection in the twentieth century. As early as 1899, folk music collecting promoted white rural culture as a form of racial purity. William Goodell Frost noted that rural whites played the role of “living ancestors” particularly because of their lack of contact with African Americans; John Fox Jr. would similarly praise their patriotism in contrast with that of immigrants, African Americans, and Native Americans 2 years later.12 Cecil Sharp, one of the most prominent collectors of Anglo-American folk culture, deemed that the folk singers he Journal of the Society for American Music 181 encountered in the United States had “one and all entered at birth into the full enjoyment of their racial heritage.”13 At the same time, African Americans during the Harlem Renaissance similarly turned to Black musics as a source of racial pride, and considered their own musical traditions as a folk culture. W. C. Handy and James Weldon Johnson both sought to position blues and ragtime respectively as folk, rather than commercial, music.14 Nevertheless such attitudes of racial purity were mythologized; the reality of folk performance featured a freer interchange of music across racial lines. Smith’s Decisions: Collecting and Curating the Anthology Historian Karl Hagstrom Miller notes that Black Americans enjoyed square dance tunes, while white Americans enjoyed the blues just as much as the more stereotypical converse.15 Intricately entwined with folk collectors’ emphasis on racial heritage and purity was a rejection of the commercial. The early twentieth century saw not only a rise in immigration, but also a rise in urbanization, as rural Americans migrated to urban centers, drawn by job opportunities from booming industrialization. Folk collectors and anthropologists like Cecil Sharp, Jean Toomer, and Zora Neale Hurston all were suspicious of industrialism’s corruptive power and the threat of cultural elimination it posed, and saw folk music collection as a safeguard for cultural heritage.16 In his own collection of cowboy songs, John Lomax rejected traditional songs that sounded too redolent of popular music, while also including newly written songs that fit his preconception of traditional-sounding music but excising the authors to maintain the illusion of their timelessness.17 Although commercial produc- ers of folk music—the same producers who made the recordings Harry Smith used—did not share this suspicion, they nevertheless sought to capitalize on folk music’s novelty by making it distinct from Tin Pan Alley songs. Record scouts would censor repertoire and shape the sound to conform to expecta- tions on the basis that they already had better ensembles performing contemporary hits.18 Likewise, Jeff Todd Titon suggests, performers might self-censor themselves based on their own perceptions of what record companies might want to hear.19 As with race, Karl Hagstrom Miller refutes the idea that folk artists shunned the commercial, detailing how both Black and white artists performed popular Tin Pan Alley music of the day.20 A generation later, record collecting emerged in the 1940s and 1950s as a subcultural discourse that was shaped by the same values of racial purity and anti-commercialism. In this instance, however, collectors were predominantly white, Ivy League-educated fans of jazz and blues. Smith’s contempo- raries—Charles Edward Smith, Charles Frederick Ramsey Jr., Samuel Charters, and James McKune, among others—collected and traded records, debated their merits, and eventually reissued historic jazz and blues recordings and published books on the subjects. Their output remains problematic, however. Although their intentions to shed light on important and influential Black musicians who had been unfairly eclipsed by more popular and successful white artists were laudable, they neverthe- less trafficked in and perpetuated essentializing myths. 13David E. Whisnant, All That Is Native and Fine: The Politics of Culture in an American Region (Chapel Hill: University of North Carolina Press, 1983), 120. 14Karl Hagstrom Miller, Segregating Sound: Inventing Folk and Pop Music in the Age of Jim Crow (Durham, NC: Duke University Press, 2010), 250–51. 15Miller, Segregating Sound, 77–79. 16For Cecil Sharp’s position, see Benjamin Filene, Romancing the Folk: Public Memory and American Roots Music (Chapel Hill: University of North Carolina Press, 2000), 22; for Toomer’s and Hurston’s, see Marybeth Hamilton, In Search of the Blues (New York: Basic Books, 2008), 16. 17Miller, Segregating Sound, 86. 18Karl Hagstrom Miller, Segregating Sound, 234. 19Jeff Todd Titon, Early Downhome Blues: A Musical and Cultural Analysis (Chapel Hill: University of North Carolina Press, 1994), 215–16. 20Miller, Segregating Sound, 216. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Smith’s Decisions: Collecting and Curating the Anthology Historian Marybeth Hamilton has examined how collecting strategies were “shaped by mythologies of race… the venerable vision of a pure and uncorrupted black voice,” whereas critic John Dougan has suggested that romanticizing poverty was “guided by the collectors’ bohemian refusal of what they saw as the egregious commerciality of rock https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 182 Dan Blim and roll, an aesthetic distinction which allowed them to wed the collecting of obscure vernacular music with their anti-consumerist ethics.”21 This was the world in which Harry Smith traveled, and he corresponded with many other prom- inent collectors of the time. Nevertheless, Smith differed from them, rejecting the mythology of racial purity and the idealization of the non-commercial. He did this through his roles as curator of the Anthology—first, by collecting and selecting tracks; second, by his sequencing of his selections, akin to the curation of a display, laying out each artifact in a narrative sequence; and third, by his labeling of artifacts, seen in his elaborate liner notes (Figure 1). Smith breaks down racial segregation in the Anthology, mixing tracks and deliberately obscuring racial lines: White as well as Black artists play the blues; a white and a Black artist each sing about John Henry; and Furry Lewis, a Black artist, performs a white-associated ballad about Casey Jones.22 In an interview, Smith directly critiqued the practices of record collectors he saw as continuing this segregated practice through their emphasis on racial purity: Before the Anthology there had been a tendency in which records were lumped into blues catalogs or hillbilly catalogs, and everybody was having blindfold tests to prove they could tell which was which. That’s why there’s no such indications of that sort (color/racial) in the albums. I wanted to see how well certain jazz critics did on the blindfold test. They all did horribly. It took years before anybody discovered that Mississippi John Hurt wasn’t a hillbilly.23 Although recordings were sold in a strictly segregated fashion, as “hillbilly” and “race records,” Smith never used race as a means of organizing and sequencing the tracks. Indeed, his liner notes squarely denounce the practice. 21Marybeth Hamilton, “Searching for the Blues: James McKune, Collectors, and a Different Crossroads,” in Listen Again: A Momentary History to Popular Music, ed. Eric Weisbard (Durham, NC: Duke University Press, 2007), 39; John Dougan, “Objects of Desire: Canon Formation and Blues Record Collecting,” Journal of Popular Music Studies 18, no. 1 (April 2006): 47. 22For a fuller delineation of the racial associations and history of Casey Jones, see Nick Tosches, Country: The Twisted Roots of Rock ‘n’ Roll (New York: Da Capo Press, 2009), 198–99. Tosches likewise finds the same evidence of racial crossing in Henry Thomas, who appears twice on the Anthology. See Tosches, Country, 195–98. 23Harry Smith, “A Rare Interview with Harry Smith” by John Cohen, Sing Out! 19, no. 1 (1969). Reprinted in Igliori, American Magus, Harry Smith, 134. 24Smith, “Foreword,” liner notes for Harry Smith, Anthology of American Folk Music, Folkways Records, 1952; Smith, “Bibliography,” liner notes for Harry Smith, Anthology of American Folk Music. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 24Smith, “Foreword,” liner notes for Harry Smith, Anthology of American Folk Music, Folkways Records, 1952; Smith, “Bibliography,” liner notes for Harry Smith, Anthology of American Folk Music. 25Smith, “A Rare Interview with Harry Smith,” 126. Henry Lee is not the first ballad in Child’s collection, but presumably was the lowest numbered Child Ballad within Smith’s collection. Smith’s Decisions: Collecting and Curating the Anthology In the foreword, he remarks, “Unfortunately these unpleasant terms are still used by some manufacturers,” and in the bibliography, he reproduces several catalog images of race records, stating, “The advertising of these envelopes gives a good idea of the companies [sic] attitude toward their artists.”24 Similarly, Smith embraced the modern and commercial as central to folk music. Although all of the tracks on the album were commercial recordings (rather than ethnographic field recordings), the def- inition of “folk music” was expansive enough to include, among other things, a novelty jazz band (“Moonshiner’s Dance Part One”), a track from a Hollywood Western (“The Lone Star Trail”), and a recent ballad about the Titanic (“When That Great Ship Went Down”). Smith also signals his fas- cination with the commercial in his liner notes. Some notes are constructed to mimic newspaper head- lines and telegraphs, a nod to contemporaneous commercial methods of communication. The notes are also filled with images of catalog advertisements, emphasizing the commercial selling of folk music in the recorded era (Figure 2). In assembling the Anthology, Smith instead focused on two criteria for inclusion: Unusual and exotic sounds, and historical importance. In one interview, he held that: The Anthology was not an attempt to get all the best records (there are other collections where everything is supposed to be beautiful), but a lot of these were selected because they were odd—an important version of the song, or one which came from some particular place. For example, there were things from Texas included that weren’t very good. There was a Child Ballad, Henry Lee. It’s Journal of the Society for American Music 183 Figure 1. Cover page to Harry Smith’s liner notes for Anthology of American Folk Music. Printed with permission from Smithsonian Folkways Recordings. Figure 1. Cover page to Harry Smith’s liner notes for Anthology of American Folk Music. Printed with permission from Smithsonian Folkways Recordings. Figure 1. Cover page to Harry Smith’s liner notes for Anthology of American Folk Music. Printed with permission from Smithsonian Folkways Recordings. not a good record but it had to go first in the set because it was the lowest numbered Child Ballad. Then there were good performances. The Brilliancy Medley occurs to me. You couldn’t get a representative cross-section of music into such a small number of records. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press ps://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Smith’s Decisions: Collecting and Curating the Anthology Instead, they were selected to be ones that would be popular among musicologists, or possibly with people who would want to sing them and maybe would improve the version. They were basically picked out from an epistemological, musicological selection of reasons.25 not a good record but it had to go first in the set because it was the lowest numbered Child Ballad. Then there were good performances. The Brilliancy Medley occurs to me. You couldn’t get a representative cross-section of music into such a small number of records. Instead, they were selected to be ones that would be popular among musicologists, or possibly with people who would want to sing them and maybe would improve the version. They were basically picked out from an epistemological, musicological selection of reasons.25 25Smith, “A Rare Interview with Harry Smith,” 126. Henry Lee is not the first ballad in Child’s collection, but presumably was the lowest numbered Child Ballad within Smith’s collection. Dan Blim 184 Figure 2. Example of Harry Smith’s liner notes for Anthology of American Folk Music. Printed with permission from Smithsonian Folkways Recordings. Figure 2. Example of Harry Smith’s liner notes for Anthology of American Folk Music. Printed with permission from Smithsonian Folkways Recordings. Unlike collectors of blues and jazz who were concerned with finding the best performances and most authentic renditions, Smith’s criteria saw value in a number of tracks that might have otherwise been excluded. Unlike collectors of blues and jazz who were concerned with finding the best performances and most authentic renditions, Smith’s criteria saw value in a number of tracks that might have otherwise been excluded. Smith’s sequencing and liner notes further elucidate these aims. Volume One, Ballads, is organized chronologically, from the earliest Child Ballads to ballads that dealt with modern disasters: Train acci- dents, the sinking of the Titanic, and Depression-era farm failures. The liner notes from this volume simultaneously chronicle the histories of the ballads and the U.S. historical events that inspired them. Smith essentially narrates a history of the United States through folk music, positioning folk music as an integral tool for capturing history. In Volume Two, Social Songs, the volume is essentially divided in half, the first dealing with secular music and the second with sacred music. In the sacred half, Smith again uses history as a guiding structure, from the old lined out hymns to the more recent communal singing of sacred harp tunes and hymns with orchestral backing. In the secular half, however, Smith’s attention is on geography, noting with care the regional styles heard in each track, giving particular attention to rhythm and instrumentation. The result is a sort of musical travelogue through space rather than time. Finally, Volume Three, Songs, eschews a clear organization, instead intermixing styles Journal of the Society for American Music 185 and subject matter to suggest a large diversity of American folk music. Blues, jug band tunes, and Louisiana Acadian music all reappear, as do a number of artists from the first two volumes in a collage of different sounds. Likewise, the liner notes sometimes call attention to historical attributes, and sometimes geographic style as on the first two volumes. Rock critic Greil Marcus has read the third volume as highlighting a form of alienation and strangeness, a politically charged opportunity to call attention to the marginalized voices absent from American mythology. 26Smith, “A Rare Interview with Harry Smith,” 120. 27Michael Baxandall, “Exhibiting Intention: Some Preconditions of the Visual Display of Culturally Purposeful Objects,” in Exhibiting Cultures: The Poetics and Politics of Museum Display, eds., Ivan Karp and Steven D. Levine (Washington: Smithsonian Institution Press, 1991), 40. 28Kirshenblatt-Gimblett, Destination Culture, 25. 29Sharon Macdonald, “Collecting Practices,” in A Companion to Museum Studies, ed. Sharon Macdonald (Malden, MA: Blackwell Publishing, 2006), 82. 30Tony Olmsted, Folkways Records: Moses Asch and his Encyclopedia of Sound (New York: Routledge, 2003), 12. He writes, “Part of the charge in the music on the Anthology of American Folk Music…comes from the fact that, for the first time, people from isolated, scorned, forgotten, disdained communities and cultures had the chance to speak to each other and to the nation at large.”26 What is perhaps most striking about Smith’s work here is the complex, cumulative effect of all three albums. Rather than focus on purity—a common emphasis among Smith’s contemporaries—or a single, unbroken narrative, Smith lays out several stories about American folk music, then abandons narrative entirely in the third volume. The essence of American folk music comes across via the diversity of the collection and its purposeful sequencing, or exhibition, in ways that highlight attributes through juxta- position. In this regard, the collection is not unlike museum exhibits that similarly lay out disparate arti- facts, helping the viewer to draw connections. Michael Baxandall praises such exhibitions, noting, “The juxtaposition of objects from different cultures signals to the viewer not only the variety of such systems but the cultural relativity of his own concepts and values.”27 For listeners in the 1950s, or now, the music asks us to refigure our ideas of American folk music, confronted by the collection’s stylistic diver- sity, racial mixing, and engagement with commercialism. Barbara Kirshenblatt-Gimblett goes even fur- ther, claiming that individual artifacts in museums “were never intended to hold up to scrutiny as singular creations. Moreover, they are at their most documentary when presented in their multiplicity, that is, as a collection.”28 Indeed, the original recordings used in the tracks were commercially produced, intended as entertainment, and marketed either to migrant listeners as a means to reconnect with a familiar regional tradition, or to new audiences as novelties; they were never intended as keepsakes or as markers of a national cultural heritage. However in Smith’s hands, the acts of collecting and reis- suing them indelibly transmuted them into historical documents of American folk music in all its diver- sity. “In forming collections,” Sharon Macdonald writes, “museums recontextualize objects” and imbue them with distinct cultural value.29 The same is true for the Anthology, as tracks are transformed from contemporary music meant for entertainment to historical markers of music history, from regional tra- ditions to a national symbol, disparately gathered and arranged to tell a story, or rather, several stories of American folk music history. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 26Smith, “A Rare Interview with Harry Smith,” 120. Smithsonian’s Decisions: Preserving and Restoring the Anthology and global folk music were “helping Americans to feel at home with themselves and with their global neighbors.”34 To be sure, Asch and Smith did not always get along, in part due to Asch’s business-mindedness and Smith’s capricious interests, penchant for distractions, and completist approach to documentation. However in Asch, Smith found a producer who shared many of his own aims. The Smithsonian acquired the Folkways catalog in 1987 and it continues Asch’s and Smith’s social vision. According to the Smithsonian Folkways website, the label is “dedicated to supporting cultural diversity and increased understanding among people” and seeks “to strengthen people’s engagement with their own cultural heritage.”35 Prior to the Anthology, the Smithsonian reissued a number of recordings in keeping with this mission of diversity, including African-American music (Sing For Freedom: The Story of the Civil Rights Movement Through Its Songs (1990)), Native American music (Creation’s Journey: Native American Music (1992)), global musics (Mbuti Pygmies of the Ituri Rainforest (1992)), and American folk music (Smithsonian Folkways American Roots Collection (1996)). The Smithsonian’s decision to reissue the Anthology was buoyed by this mission and shaped by a confidence in its success. Archivist Jeff Place stated in 1997 that the Anthology had been the most requested item from the Smithsonian’s catalog since 1988, when he joined the organization.36 Confidence was also likely boosted by the success of Columbia Records’ reissue Robert Johnson: The Complete Recordings to immense critical and, more surprisingly, commercial success: By 1994, the Robert Johnson reissue had gone platinum.37 Envisioning the Anthology reissue, Marketing Director Brenda Dunlap confirms they saw great potential for the reissue: “Given the requests we’ve received over the Web, and handwritten postcards and that sort of thing, I do see a lot of interest in it. We have kids at radio stations asking about it as well, which is always a good sign, so there is a new gen- eration of interest there, too.”38 When asked what sort of audience she would be targeting, Dunlap responded, “We’ll be doing mailings to academics at all different levels, American Folk Society mem- bers, ethnomusicologists. …In terms of retail, a lot of the folk stores . . . are perfect outlets for it. Places like Best Buy and Borders Books tend to carry a lot of Smithsonian Folkways, and they’ll probably have some available. Smithsonian’s Decisions: Preserving and Restoring the Anthology Although Smith was the curator of the Anthology, its function as a public statement on U.S. folk music was no doubt amplified by its reissue by the Smithsonian. Arguably, this process began with the orig- inal release on Folkways Records, a label with a national audience (albeit niche) and political attitudes. Folkways founder Moses Asch and Smith shared a philosophical approach to folk music. Both first encountered U.S. folk music through his parents: Asch’s father supplied him with books about cow- boys and their songs, which he said “guided me through life because [Teddy Roosevelt, in the intro- duction to John Lomax’s collection] said that folklore and songs are the cultural expression of people. So here I had these books and was able to show that we had this kind of uniqueness to our culture which was not just a melting pot, but were part of a whole bunch of other things.”30 Asch described https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Dan Blim 186 his Folkways catalog as a “mosaic” and included music that Smith found appealing, including jazz, avant-garde art music, and anthropologic field records of music from around the world, and of course the reissuing of older records of which the Anthology was a central part.31 Asch felt artists should con- trol all aspects of the album design, from theme and selection to cover photos and liner notes. The cover and liner notes Asch felt were particularly important, partly because they distinguished the label at the time, and partly because he was extraordinarily sensitive to the educative possibilities of these albums, adhering to the philosophy “music is music in context” and orienting his releases toward museums, libraries, and schools.32 Asch also remained sympathetic to artists with politically sensitive material, envisioning albums as a sort of “living newspaper.”33 Thus, just as Smith hoped to see the United States changed by music, so too Asch sought to use his label to further social causes. To sup- port civil rights for African Americans, Asch issued albums with the music and poetry of the move- ment and coverage of the Nashville sit-in, and to further global and national harmony, he felt that his devotion to U.S. 31Richard Carlin, Worlds of Sound: The Story of Smithsonian Folkways (New York: Harper Collins, 2010), 223. 32Olmsted, Folkways Records, 43. 33Olmsted, Folkways Records, 67. 34Olmsted, Folkways Records, 44. 35“Mission and History,” Smithsonian Folkways Recording, accessed June 18, 2020, https://folkways.si.edu/mission-and-his- tory. Bolded language appears in the original source. 36Chris Morris, “Smithsonian to Reissue Its Classic Folk Anthology Set,” Billboard, June 21, 1997, 8. 37Bill Holland, “Johnson’s Son Gets Right,” Billboard, November 1, 2003, 12. 38Morris, “Smithsonian to Reissue Its Classic Folk Anthology Set,” 8. 39Morris, “Smithsonian to Reissue Its Classic Folk Anthology Set,” 8. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 40Fundraising flyer, undated, untitled folder, Moses and Frances Asch Collection. 41Albin Zak has effectively unsettled this claim that the 1950s were an era of musical sameness. See Albin Zak, I Don’t Sound Like Nobody: Remaking Music in 1950s America (Ann Arbor: University of Michigan Press, 2010). 42Paraphrased from the Smithsonian’s Mission and Vision, accessible at “Purpose and Vision,” Smithsonian, accessed June 18, 2020, http://www.si.edu/About/Mission. 43For a fuller explanation of copyright and reissues, see Tim Brooks, “How Copyright Law Affects Reissues of Historic Recordings,” ARSC Journal 36, no. 2 (Fall 2005): 183–203. 44Olmsted, Folkways Records, 66–68. Asch’s whole career, in fact, amounts to a stand against big companies, after early failures with popular music bankrupted him, he retained a very small profit margin and focused exclusively on music larger corporations would not offer. 45Carlin, Worlds of Sound, 74. Smithsonian’s Decisions: Preserving and Restoring the Anthology I think collectors will be a large part of this.”39 Although hoping to find a new gen- eration of listeners, the Anthology reissue was largely aimed at the extant one, especially those likely to already be aware of it: Academics, collectors, and committed folk enthusiasts. Journal of the Society for American Music 187 A fundraising flyer to help with the $146,000 estimated price tag of the reissue further elucidates the principles that guided the reissue.40 The flyer again targets the academic aim of the reissue, particularly through the language of canonization and scholarship. It labels the anthology “perhaps the most influ- ential set of records in the history of recorded sound” and describes a “digitally remastered” box set wherein Smith’s notes “will be framed by an historical essay, expanded song annotations, testimonials by literary and musical figures, and archival photographs.” The flyer promises it “will be a landmark educational tool for generations.” Likewise, the flyer is designed to appeal to novelty and rarity, includ- ing the aforementioned “digitally remastered” tracks and the true if somewhat misleading claim that “This historic collection of sound has not been available commercially for almost half a century.” Furthermore, the flyer adopts an aim of social change by reminding readers of the Smithsonian’s mis- sion to provide “models for facilitating public dialogue among diverse ethnic, racial, and regional groups” and contextualizes the original as a fight against consolidation of record companies in the 1950s, which were “producing musical sameness.”41 In other words, the flyer—indeed the process of reissuing the Anthology—is best understood as an extension of the Smithsonian’s mission of pre- serving heritage, expanding access, fostering education, and championing diversity.42 Perhaps the biggest change from Asch’s release was that the tracks had to be licensed under copy- right; a long, difficult process of retracing the conglomeration of numerous now-defunct recording companies and seeking copyright permissions that represented over 40 percent of the total cost, and which largely benefitted Sony.43 When these records were made, record companies sought out ballads and other folk material that was not subject to copyright; performers were usually given a lump sum, that is if they were paid at all, and the recording company held the rights to the recording. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 45Carlin, Worlds of Sound, 74. Smithsonian’s Decisions: Preserving and Restoring the Anthology Peter Reiniger, the sound engineer, began by examining the master tapes held by the Smithsonian and documenting for each the crackles, clicks, splices, dropouts, skips, distortions, and noises from the wind or passing vehicles, or inabilities to discern instruments. Two tracks by Dave Lunsford, numbers 51 and 63, were simply directed to an extant reissue on CD. Only three tracks, numbers 7, 8, and 27, merited a simple “pretty good,” whereas for twenty-one tracks Reiniger suggested an alternate source be sought. A second, unsigned list directing the reader to locate sources almost exactly replicates Reiniger’s original list. A third list, also unsigned, notes that alternate sources should be found for nine tracks and should be considered for eleven tracks, only partially overlapping with Reiniger’s recommendations (Table 1). In other words, what merited an alternate source was somewhat subjective. Using Reiniger’s notes, I can find no clear pattern to gauge what sonic defects prompted a track’s rejection in favor of an alternate source. The reissue ultimately used alternate sources for twenty-seven tracks, only partially overlapping with these lists, suggesting some desired alternate sources may have proven impossible to find, while others may have fortuitously presented themselves. It was fitting that the reissue relied on the availability of clean copies among collectors, the same strategies Smith employed in building his own collection. Striving for the cleanest sound possible aligns with what Asch’s believed: “With my records, I think if I am documenting a thing, then I want as good quality as possible, because the person 20 years from that time should be able to reconstruct what I recorded.”47 Nevertheless Asch opposed any electronic manipulation, resisting the move to stereo as much as possible. So, although the fundraising flyer and reviews continually touted the digital remas- tering of the Anthology, the Smithsonian appears to have ultimately hewed somewhat closely to Asch’s philosophy. Reiniger comments in the reissue, “We have consistently adhered to the idea that it is far better to listen to some noise with the music than to eliminate all the noise and a good part of the audio spectrum with it,” though he also admits that pitch justification was used to account for the var- iance of turntable speeds, and that other technical enhancements may have been employed. Smithsonian’s Decisions: Preserving and Restoring the Anthology The Depression left companies with large stockpiles of unsold records, which became the cheap source for Smith’s collecting habit as stores tried to clear out space, and during World War II, the records we melted down for shellac, rendering what was on the record economically inert. Once the recordings found new listeners, Asch skirted musicians’ unions to record during the strike in 1942, and later used Folkways to return ownership of the nation’s folk music to the nation, using subsidiary labels to help protect him from charges of piracy. Asch went through several legal battles following his reis- sues, but successfully argued that the public had a right to hear this music and that if record companies were no longer willing to make these recordings available, they could not block his efforts to make the music available to the public.44 Victor records disputed his reissue of Woody Guthrie’s Dust Bowl Ballads after it became profitable, but Asch insisted, “cultural property belongs to all and is limited to individual ownership only in so far as the copyright of the material is subjected to and limited to. …[R]ecords do not carry this copyright.”45 y py g For the reissue, the necessity for copyright was simply a matter of fact. A memo from March 7, 1996 elucidates the confusing path to obtaining it: MCA’s inability to clear tracks has to do with the ambiguity of their ownership of the Paramount catalog, and we are looking into potential alternative repertoire owners. BMG’s delay seems to come from the amount of research involved in clearing this quantity of tracks. Many of these tracks are obscure and may not have been used since they were licensed for the original release of this album in 1952. Although these tracks were legally licensed for the original issue of this 188 Dan Blim package, today’s licensing administrators may not feel confident licensing material without the proper contractual proof. Also, catalogs constantly change hands.46 The modern necessities behind the licensing of the reissue make the commentary in the aforemen- tioned fundraising flyer about musical conglomerations even more pointed, although once in produc- tion the Smithsonian rarely made any similar references, perhaps not wishing to jeopardize Sony’s cooperation with the project. Once licensed, the tracks had to be located and in many cases cleaned up. 46Memo from Ed Rahn to Amy Horowitz, March 7, 1996, folder 2951, Moses and Frances Asch Collection. 47Olmsted, Folkways Records, 15. 48Emily Chivers Yochim and Megan Biddinger, “It Kind of Gives You that Vintage Feel’: Vinyl Records and the Trope of Death,” Media, Culture, Society 30, no. 2 (March 2008): 183–95. Vanessa Chang examines a similar effect in the course of sam- pling through vinyl, identifying a particular sonic aura of historicity that emerges particularly in the light of reissues. See Vanessa Chang, “Records that Play: The Present Past in Sampling Practice,” Popular Music 28, no. 2 (May 2009): 153–54. 49Christina Baade and Paul Aitken, “Still ‘In the Mood’: The Nostalgia Aesthetic in a Digital World,” Journal of Popular Music Studies 20, no. 4 (December 2008): 361. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 46Memo from Ed Rahn to Amy Horowitz, March 7, 1996, folder 2951, Moses and Frances Asch Collection. 47 Smithsonian’s Decisions: Preserving and Restoring the Anthology p y p y The decision to leave in the noise no doubt appealed to vinyl enthusiasts, collectors and tradition- alists among them, who regularly criticized digitization for its compression and diminishment of the richness of the sound. However sound quality would not be the only thing lost. The presence of that noise confirms the historicity of these recordings—that what you are hearing is not a new CD box set at all, but an old recording, a vinyl LP or even the noisier shellac of the original 78s. Scholars have noted the continued championing of vinyl over digital as an exercise in nostalgia and authenticity, per- ceived as communing with or listening to the dead, physically preserving a historical item.48 In their study of reissues of Glenn Miller’s “In the Mood,” Christina Baade and Paul Aitkin found that many listeners found the presence of “‘flaws’ of ‘pop and crackle’ enhanced their connection with the past” on those recordings.49 This effect is akin to what museologist Peter van Mesnch calls “patina”; the “his- torical process of interaction between the object and its environment” that “creates an atmosphere of Journal of the Society for American Music 189 Table 1. Smithsonian’s Decisions: Preserving and Restoring the Anthology Tracks selected for replacement with alternate sources on the reissue of Anthology of American Folk Music Track A B C D A Lazy Farmer Boy X Bandit Cole Younger X X (X) Brilliancy Medley X Country Blues X X Drunkard’s Special X Dry Bones X X East Virginia X X X Expressman Blues X Fatal Flower Garden X Frankie X (X) Georgia Stomp X Got the Farm Land Blues (X) Home Sweet Home X I Wish I Was a Mole in the Ground X X James Alley Blues X John Hardy Was a Desperate Little Man (X) John the Baptist X X X John the Revelator (X) King Kong Kitchie Kitchie Ki-Me-O X La Danseuse X X Moonshiner’s Dance (part 1) X X X My Name is John Johanna X X Newport Blues X Old Country Stomp X X (X) X Old Lady and the Devil X X X Peg and Awl X X X Poor Boy Blues X X (X) X Present Joys X X X Prison Cell Blues X X X Rabbit Foot Blues X X X Rocky Road X Sail Away Ladies X Saut Crapaud X See That My Grave is Kept Clean X Single Girl, Married Girl X X Spike Driver Blues X X (X) X Stackalee X X Sugar Baby X The Coo Coo Bird X X (X) X (Continued) 1. Tracks selected for replacement with alternate sources on the reissue of Anthology of American Folk Music https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 190 Dan Blim Dan Blim Table 1. (Continued.) Track A B C D The Mountaineer’s Courtship X X (X) X When That Great Ship Went Down X White House Blues X X (X) X A, notes from sound engineer Peter Reiniger; B, first unsigned list; C, second unsigned list (“(X)” for “also considering”); D, CD reissue. from sound engineer Peter Reiniger; B, first unsigned list; C, second unsigned list (“(X)” for “also considering”); D, CD reissue authenticity and historicity.”50 Reiniger’s work directly parallels that of a museum restorationist. As Johnathan Djabarouti observes, “the relationship between restoration and authenticity in conservation is traditionally related back to the notion of patina,” where “old things are perceived as having more inherent value.”51 Of course, recordings are distinct from many traditional artistic works in that recordings lack a distinct and unique original, which is especially true in the act of reissuing. Smithsonian’s Decisions: Preserving and Restoring the Anthology By retain- ing the patina of auditory imperfections, much like the decay of an old building, the reissue grants an aura of authenticity often lacking in reproductions, as Walter Benjamin famously theorized in his essay The Work of Art in the Age of Mechanical Reproduction. The effect for the listener effectively mirrors the palimpsest effect listeners of the original had: The feeling that what you are hearing is from a distant and strange past whose traces remain buried under the present, the performers long dead. Recall again author William Gay remarked that the voice of Dock Boggs on the Anthology was “so dissociated it seems to be coming not just from some other time but from outside time itself, from beyond the pale, a voice half-filtered through a mouthful of graveyard dirt,” thus tying the act of listening to disembodiment, death, and decay.52 The reissue, released during the vinyl revival of the 1990s, retained the distinct sound of the record, as potent a symbol as the sepia tone of old photographs, preserving this paradoxical aesthetic imme- diacy of temporal and corporeal distance. The interest in vinyl has also been understood as a subcul- tural move in opposition to the recording industry.53 This attitude echoes the Smithsonian’s characterization of the original album as doing the same, defiantly refusing to play by the industry’s rules. However the reissue itself sought a more commercially viable approach, released not on vinyl but also on CDs, which only carried the sound of the vinyl LPs and shellac records. If the sound was designed to mimic the effect of the original, the look of the reissue was practically indistinguishable. This aesthetic was not, however, the goal from the start. An email describes the ini- tial plan as an inexpensive reissue: A set of three simple, $6, 2-CD reissues, very much in line with Asch’s mission to ensure public access to folk material.54 The production team, however, felt the Anthology demanded a box set treatment. To be sure, the high retail price of the resulting box set, $79, helped to offset the extraordinary cost of licensing all the tracks, something a non-profit public organization would have trouble shouldering. However, the price tag also reflects the shift of their tar- get demographic from new audiences to audiophiles, collectors, and educational institutions. 50Peter van Mensch, “Methodological Museology; Or, Toward a Theory of Museum Practice,” in Objects of Knowledge, ed. Susan Pearce (Atlantic Highlands, NJ: Athlone Press, 1990), 150. 51Johnathan Djabarouti, “Imitation and Intangibility: Postmodern Perspectives on Restoration and Authenticity at the Hill House Box, Scotland,” International Journal of Heritage Studies 28, no. 1 (2021): 3. 52William Gay, Time Done Been Won’t Be No More: Collected Prose, ed. J. M. White (Johannesburg: Wild Dog Press, 2010), 170. 53David Hayes, “‘Take Those Old Records off the Shelf’: Youth and Music Consumption in the Postmodern Age,” Popular Music and Society 29, no. 1 (2006): 51–68. 54Email from Michael Maloney to Rani Singh, January 10, 1997, untitled folder, Moses and Frances Asch Collection. 55Minutes from Meeting on Reissue of Harry Smith Anthology, untitled folder, Moses and Frances Asch Collection. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Smithsonian’s Decisions: Preserving and Restoring the Anthology Instead of talking about the U.S.A., this becomes a window onto this part of the world, without political bound- aries of any kind, and thus could be from any time period, 1952…1927–32…or even centuries before.”56 From this, we can glean that the original image was neither the Depression-era photo, nor the monochord, but at heart a map of the United States, playing up the nationalist aspect of the Anthology reissue. Nevertheless there must have been concerns, and the solution was to create a United States without borders, something I have argued was very much at the heart of Smith’s vision, as well as a sense of timelessness coupled with the modern technology of radio, a central tension in the Anthology. A second image retains the radio waves, and scatters around them stars and circles. Stowell notes it “has one foot in Harry Smith’s mystical world of the Celestial Monochord, and the other in Smithville…. What look like stars in some sort of celestial diagram are actually towns and cities from a map of the Southeast (or in this case, from a Crossroads CD label). The radio waves here are emanating from people in the villages, towns, and cities that could be anywhere, at any time.” Here, the mythology of the South, central to Smith’s vision of the folk, is present, if abstractly, while the concept of time- lessness is again emphasized. Versions three and four feature the monochord image, one without and one with radio waves. Stowell preferred the one with the radio waves because it made “a direct con- nection between the ancient and the modern, and by extension between the myths and traditions on the Anthology and modern listeners.” The fifth design abstracted from the fourth the circles of radio waves and the circles emanating from the monochord—precisely what he liked about the rela- tionship in the fourth design. Nevertheless he also felt he should add text “much like the type used in the booklet,” presumably because he feared the design was too abstract.57 The selection of the original does not appear to have been made by Stowell, whose enthusiasm for that design was muted—possibly because he, as a graphic designer, had very little to do with it. Smithsonian’s Decisions: Preserving and Restoring the Anthology Minutes from a production meeting confirm that the decision was guided not just by economics, but also by a more emotional attachment to the Anthology as a collector’s item, as production team members won- dered, “How will we maintain the integrity of the original work if we don’t do it all at once in a 12 × 12 box set?”55 I draw particular attention to the characterization of the Anthology as a “work,” implying it 191 Journal of the Society for American Music was not simply a collection of tracks but a fully realized whole, placing the emphasis on Harry Smith as the visionary artist. Maintaining integrity, as it would turn out, meant replicating the look of the original boxes of LPs, material, size, and all. The outside label replicates Smith’s original cover: A copy of a celestial mono- chord engraved by Johann Theodor de Bry taken from a publication by Robert Fludd. The simple addi- tion of a frame around it is suggestive, elevating the cover image, and by extension the whole Anthology, to the realm of a museum piece to be preserved. Like any museum piece, Smith’s cover was effectively restored, having been replaced in the 1960s by Irwin Silber, who selected a 1935 pho- tograph by Ben Shahn of a rehabilitation client in Boone County, Arkansas. It was Shahn’s image that was used in the fundraising flyer, perhaps to resonate with the flyer’s more political claims, or perhaps simply to cater to a familiarity with the more commonplace image that had fronted the album for three decades. Returning to the original was not the obvious choice, however. Five covers, including the selected original, were proposed by designer Scott Stowell (Figure 3). The first depicts an outline of North America, with radio waves emanating from the center of the image, from the iconic heartland (rather than the South). Stowell writes, “This solution keeps the things that we liked about the original version (including the general concept) while making the map a much more non-political image. 56Ellipses appear in the original. 57Fax from Scott Stowell, undated, untitled folder, Moses and Frances Asch Collection. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press p pp g 57Fax from Scott Stowell, undated, untitled folder, Moses and Frances Asch Collection. 56Ellipses appear in the original. Smithsonian’s Decisions: Preserving and Restoring the Anthology As such, I would venture the image was included on request from someone at the Smithsonian, a strong enough recommendation that Stowell visually or textually referenced the monochord in four of the five designs. Indeed, Stowell’s designs effectively bring together the past—Harry Smith’s original celestial monochord—and the present day, characterized by the technological radio waves and geographic allu- sions to the United States, mirroring the intermingling of past and present, regional and national/inter- national that Smith’s Anthology undertook. Only the first, which was a continuation of some unretained earlier design plan, made no reference to the monochord. Nevertheless it was the third, which made no reference to the modern American present, that succeeded through its historical accu- racy and continuity, and the perceived purity of Smith’s vision. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 192 Dan Blim Five suggested cover designs for the reissue of Anthology of American Music. Printed with per an Folkways Recordings. 3. Five suggested cover designs for the reissue of Anthology of American Music. Printed with perm onian Folkways Recordings. Figure 3. Five suggested cover designs for the reissue of Anthology of American Music. Printed with permission from Smithsonian Folkways Recordings. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press ontinued. Journal of the Society for American M Journal of the Society for American Music 193 Figure 3. Continued. bl h d l b b d ontinued. Figure 3. Continued. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 194 Dan Blim Figure 3. Continued. Figure 3. Continued. Figure 3. Continued. This preference for Smith’s original vision, elevated to a museum piece, was evident nowhere more clearly than the booklet. The original booklet was perhaps the hallmark of the Anthology, totally unlike anything else. As the producers take pains to note, its inclusion in the reissue was a precise facsimile: Enclosed is a facsimile of Harry Smith’s original handbook, which he composed, designed and laid out himself. There have been several reprintings since the original edition, each with changes in art, copy, and design. This reproduction is faithful to Harry’s creation. It has been produced with the same reprographic methods in use at the time of the original edition: film negatives were shot with a photostat camera from a printed copy of the 1952 handbook because the original mechanical boards had been lost. 58Unsigned note, Supplemental Notes to Anthology of American Music, Smithsonian Folkways SFW 40090, 1997, compact disc, 34. ttps://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Smithsonian’s Decisions: Preserving and Restoring the Anthology We did no retouching or cleaning up and made no changes to the original art or copy. Offset plates were then made from these negatives, and paper was selected to match the original. The result is a reproduction which is as close as possible to the original artifact.58 The language here exceeds the language regarding the audio transfer, proudly touting the absence of any retouching or alterations, the careful selection of the smallest details, and seems to comply with the goal of maintaining “the integrity of the original.” The language here treats the reissue itself as a work of art. It makes explicit the level of craft in its construction, validating Smith’s artistic vision and the careful work of the Smithsonian simultaneously. Although the Smithsonian’s reissue was not a single artwork, as it was a mass-produced recording, https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Journal of the Society for American Music 195 their marketing of it as a high-priced, elaborate collectible for connoisseurs rather than a cheaper, sim- pler (and more accessible) version, the reissue is positioned closer to an original work of art than, say, the mass-marketed posters one might buy in a museum gift shop. In other words, reissuing is rendered as a culturally distinct process from mere reproduction. y p p Accompanying the original booklet in the box set are “supplemental notes” crafted by Jeff Place in a strikingly similar aesthetic: Tracks are arranged in vertical columns left to right, each introduced with a large number on the left, with label information next to it. Both Smith and Place create similar keys to the annotations in their introductions. Place even scatters a few images throughout the notes—cer- tainly not to the extent of Smith’s original notes, but enough to recall it. Place’s notes, all meticulously researched, largely focus on performers that Smith wrote little or nothing about, or corrects Smith’s various erroneous dates and instrumentation. In this regard, Place most directly takes on the role of curator, labeling the artifacts and providing critical historical information, even when doing so under- cuts the authority of Smith’s original. Producing side-by-side notes creates a dual narrative that pre- serves Smith’s narrative while deepening the listener’s understanding of the tracks’ history prior to Smith. Moreover, Place recognizes a crucial dilemma for the curator: A mission to educate and a desire to let Smith’s work speak for itself. Susan M. Smithsonian’s Decisions: Preserving and Restoring the Anthology Pearce reminds us that “the relationship of the profes- sional curator to the public, and the balance of power within such a relationship raises questions about ‘whose history?’ and ‘produced for whom?’”59 In many ways Place cedes his own authority to Smith, downplaying his role as merely “updat[ing] Smith’s notes as necessary.” In this regard, his relationship to the public is somewhere between historian and preservationist, in many ways unwilling to dramat- ically recontextualize Smith’s history, despite its limitations. The result is something of a paean to Smith, even as Place describes these notes as a way to move not back but forward: “Personal portraits, social landscapes and historical perspectives intended to lead you on your own to find out more. … Hopefully, the new Anthology will evolve with new channels for exchanging information and will serve as a model for presenting important audio recordings to an interested public.”60 Although Place’s comment invokes the individual tracks as “important audio recordings,” the per- formances seem secondary to the elevation of Smith as the true author here. Reverting to the original cover design, carefully reprinting Smith’s liner notes, and even minimizing the corrections to Smith’s errors in those notes—all of this suggests Smith’s Anthology is a sort of Urtext, especially considering the production team’s commitment to “the integrity of the original.” This approach to reissuing is quite different from Smith’s, and different from the original recording process as well. The original recordings were hardly definitive recordings, edited by producers and but one of many versions of each song. Smith’s approach to reissuing took a heavy and overt editorial hand; the Smithsonian, by contrast, sought to minimize their imprint. Moreover, although Smith used the Anthology to illuminate folk prac- tices of the early twentieth century, using juxtaposition and fragments to defy narratives, the Anthology as reissued by the Smithsonian emerges primarily as a document of the 1950s folk revival, seen in the language of their flyer and in their discussions. It fits into existing narratives about that time—a snap- shot of a moment of social upheaval, and a singular creative work by a single figure. That approach guided the process of collecting additional notes about the Anthology’s cultural impact as an object. 59Susan M. Pearce, “Museum Studies in Material Culture: Introduction,” in Museum Studies in Material Culture, ed. Susan M. Pearce (New York: Leicester University Press, 1989), 9. 60Jeff Place, Supplemental Notes to Anthology of American Folk Music. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press y 60Jeff Place, Supplemental Notes to Anthology of American Folk Music. Smithsonian’s Decisions: Narrating History She says Mike Seeger will talk with Pankake and ask him to revise the notes. The first question is which volume do these notes go with?62 Here, the desired tone for the notes is similarly scholarly and contextual, with even less room for per- sonal reflection than Seitel’s, which allowed for anecdotal tales. An early list of names of potential con- tributors demonstrates producers cast a wide net, endeavoring to cover the anthology from multiple angles (Appendix 3B). Musicians made up the largest share, covering a wide range of styles and gen- erations: Bono, Laurie Anderson, Michael Stipe, Loretta Lynn, Ornette Coleman, Patti Smith, Johnny Cash, Pete Seeger, Oscar Brand, Phil Spector, Beck, Doc Watson, Yoko Ono, and Bob Dylan. Colleagues and collaborators with Smith like Moses Asch, Peter Bartok, Jon Pankake, Allen Ginsberg, Sam Charters, and Ralph Rinzler were naturally included, as were a variety of prominent artists and intellectuals: Thomas Pynchon, Robert Frank, Sam Shepard, LaMonte Young, Dennis Hopper, and Amiri Baraka.63 pp Rani Singh, the director of the Harry Smith Archives, sent an update on the status of notes. Notes had been received from several artists: Eleven of them constituted long, multiple page remembrances, whereas others offered short anecdotes or pithy, heartfelt comments. Singh indicated that she needed to edit Taylor’s and lightly edit Rinzler’s note, and that Pankake’s eight-page contribution, by far the longest, could be edited though she didn’t want to do so. A host of other contributors had also com- mitted to the project, though they had not sent anything in, and still several others Singh remained hopeful about getting involved. Singh’s approach retained the wide variety of viewpoints that could touch upon all four of the areas recommended by Seitel: Big name performers to draw attention to the Anthology’s impact; others who assisted the folk movement behind the scenes through production and management like Young, Rooney, and Pearl; colleagues to offer a deeper account into Smith like Asch, Rinzler, Saunders, and Ginsberg; and a number of scholars and writers who could provide the appropriate historical and cultural context—Kahn, Cantwell, Sante, Heylin, and Ivey, who would soon chair the National Endowment for the Arts. Singh was also particular about the ordering of the notes. Smithsonian’s Decisions: Narrating History Perhaps the most active role the producers of the Anthology reissue played was in the additional notes that accompanied the box set. In a memo from editor Peter Seitel from October 17, 1996, he urges that the notes should cover four principal areas: (1) What does this musical snapshot taken in 1928–32, the Harry Smith Collection, represent? How is our picture affected by the means of recording at the time—the technical equipment https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 196 Dan Blim and the social organization/economic practices of the music industry at the time? Who are these people and what is their relationship to the music that they sing? and the social organization/economic practices of the music industry at the time? Who are these people and what is their relationship to the music that they sing? and the social organization/economic practices of the music industry at the time? Who are these people and what is their relationship to the music that they sing? (2) What was the impact of this collection on American music?—told both anecdotally…and based on the evidence of styles and repertoires of subsequent musicians. … (2) What was the impact of this collection on American music?—told both anecdotally…and based on the evidence of styles and repertoires of subsequent musicians. … y p q (3) What does this collection represent in the context of Harry Smith’s work? … (4) What can be added to the annotations of individual songs in terms of their significance or their history from 1952 to the present?61 (4) What can be added to the annotations of individual songs in terms of their significance or their history from 1952 to the present?61 Seitel maps out an ambitiously thorough and scholarly assessment of the Anthology and its historical contexts. This appears to have held some sway early on. An undated memo remarks that: Seitel maps out an ambitiously thorough and scholarly assessment of the Anthology and its historical contexts. This appears to have held some sway early on. An undated memo remarks that: We have notes from Jon Pankake (which appear to be for volume 4). Note in the file from Kate Rinzler saying that they are too personal and need to be focused to include comments on the early recording industry and the anthology’s impact on it in the early 50s. 61Memo from Peter Seitel, October 17, 1996, untitled folder, Moses and Frances Asch Collection. 62Unauthored memo, undated, folder 2951, Moses and Frances Asch Collection. 63Unauthored memo, undated, untitled folder, Moses and Frances Asch Collection. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Smithsonian’s Decisions: Narrating History In her plan, the set opened with her own contribution as director of the archives, followed by critic Pankake, fellow record collector Kemnitzer, musicians Stampfel and Von Schmidt, musician-scholar Cohen, scholar Cantwell, musician Fahey, friend and collaborator Ginsberg, and ending with Pirtle. The placement of the contribution by Moses Asch, she said, still needed to be decided, if it was to be included at all. There’s a Smith-like flow to the arrangements: The two scholars together in the middle, musicians on either side, and the two poets at the end. Furthermore, her insistence upon Pirtle to close was meaningful, for Pirtle’s rather touching note tells of how he reintroduced Smith to the album in his later years and https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 197 Journal of the Society for American Music how Smith had been moved to tears by the experience, providing a logical conclusion to the narrative shaped here. Smaller pieces were to be scattered throughout the document—as many as could fit.64 The final result was much smaller in scope than either Seitel or Singh had hoped. No doubt this was a practical move to keep costs down and maintain a manageable size. Nevertheless it also bears the distinct imprint of a careful editorial hand. Although some recollections were scattered throughout, as Singh had envisioned, only four were “short”; indeed, six authors who had contributed longer works were excerpted and placed in the margins of multiple consecutive pages, much like magazine articles that are continued on later pages in thin columns. The prioritizing of longer works may have stemmed from wanting to recognize the effort that went into crafting them, or possibly from agreements made with the authors, but it also speaks to the scholarly and documentary aims the col- lection had, giving the volume some heft. 67Chuck Pirtle, [Liner Note], Supplemental Notes to Anthology of American Music, 52. Other contributors included were John Fahey, [Liner Note], Supplemental Notes to Anthology of American Music, 8–12; John Cohen, [Liner Note], Supplemental Notes to Anthology of American Music, 15; Elvis Costello, [Liner Note], Supplemental Notes to Anthology of American Music, 18; Peter Stampfel, [Liner Note], Supplemental Notes to Anthology of American Music, 21–25; Luc Sante, [Liner Note], Supplemental Notes to Anthology of American Music, 30–31; Dave Van Ronk, [Liner Note], Supplemental Notes to Anthology of American Music, 36; Eric von Schmidt, [Liner Note], Supplemental Notes to Anthology of American Music, 42–44; Allen Ginsberg, [Liner Note], Supplemental Notes to Anthology of American Music, 55–59. g 65Neil Rosenberg, “Notes on Harry Smith,” Supplemental Notes to Anthology of American Music, 35–37; Kemnitzer, 29–31; Jon Pankake, “The Brotherhood of the Anthology,” Supplemental Notes to Anthology of American Music, 26–28; and Asch, “The Birth and Growth of Anthology of American Folk Music as told by Moses Asch,” reprinted in Supplemental Notes to Anthology of American Music, 32–33. 66 l “ h ld d ” l l h l f h pp gy f 68Note from Sam Charter, undated, untitled folder, Moses and Frances Asch Collection. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 64Memo from Rani Singh, undated, untitled folder, Moses and Frances Asch Collection. Smithsonian’s Decisions: Narrating History The essays given pride of placement in the anthology are particularly scholarly or historically minded: Neil Rosenberg’s essay on recording practices of the 1920s and 1930s, Luis Kemnitzer’s in-depth look into the practice of record collecting, Jon Pankake’s personal yet detailed remembrance of the Anthology and the folk revival, and Moses Asch’s recollection of the album’s conception and production.65 Biggest of all was a full twenty-one pages devoted to an adapted excerpt from Greil Marcus’s chapter on the Anthology from his just-published book on Bob Dylan, Invisible Republic.66 The essays given pride of placement in the anthology are particularly scholarly or historically minded: Neil Rosenberg’s essay on recording practices of the 1920s and 1930s, Luis Kemnitzer’s in-depth look into the practice of record collecting, Jon Pankake’s personal yet detailed remembrance of the Anthology and the folk revival, and Moses Asch’s recollection of the album’s conception and production.65 Biggest of all was a full twenty-one pages devoted to an adapted excerpt from Greil Marcus’s chapter on the Anthology from his just-published book on Bob Dylan, Invisible Republic.66 Marcus’s name did not appear at all in earlier documented plans for the notes, but the decision to excerpt part of a book, rather than to devote the space to other notes, speaks again to the scholarly ambitions of the reissue. However given that other scholars like Robert Cantwell had submitted con- tributions, the selection of Marcus, a more public and famous figure, suggests that commercial appeal was also strong; Marcus offered the right combination of scholarship and fame to match the competing goals of the organizers. Other contributions scattered throughout the margins largely gave anecdotal color: Personal testimonials about the Anthology’s impact on their work and reminiscences about Smith’s eccentric behavior. It was to these margins that Pirtle’s essay, with which Singh wanted to close the notes, was inconspicuously consigned.67 p gy j p y , p Marcus’s name did not appear at all in earlier documented plans for the notes, but the decision to excerpt part of a book, rather than to devote the space to other notes, speaks again to the scholarly ambitions of the reissue. 64Memo from Rani Singh, undated, untitled folder, Moses and Frances Asch Collection. 65Neil Rosenberg, “Notes on Harry Smith,” Supplemental Notes to Anthology of American Music, 35–37; Kemnitzer, 29–31; Jon Pankake, “The Brotherhood of the Anthology,” Supplemental Notes to Anthology of American Music, 26–28; and Asch, “The Birth and Growth of Anthology of American Folk Music as told by Moses Asch,” reprinted in Supplemental Notes to Anthology of American Music, 32–33. 66Greil Marcus, “The Old, Weird America,” Supplemental Notes to Anthology of American Music, 5–25. No other note was longer than three pages. 67Chuck Pirtle, [Liner Note], Supplemental Notes to Anthology of American Music, 52. Other contributors included were John Fahey, [Liner Note], Supplemental Notes to Anthology of American Music, 8–12; John Cohen, [Liner Note], Supplemental Notes to Anthology of American Music, 15; Elvis Costello, [Liner Note], Supplemental Notes to Anthology of American Music, 18; Peter Stampfel, [Liner Note], Supplemental Notes to Anthology of American Music, 21–25; Luc Sante, [Liner Note], Supplemental Notes to Anthology of American Music, 30–31; Dave Van Ronk, [Liner Note], Supplemental Notes to Anthology of American Music, 36; Eric von Schmidt, [Liner Note], Supplemental Notes to Anthology of American Music, 42–44; Allen Ginsberg, [Liner Note], Supplemental Notes to Anthology of American Music, 55–59. 68Note from Sam Charter, undated, untitled folder, Moses and Frances Asch Collection. 69Note from Pat Conte, undated, untitled folder, Moses and Frances Asch Collection. 70Marcus, Invisible Republic, 96. 70Marcus, Invisible Republic, 96. cus, “The Old, Weird America,” Supplemental Notes to Anthology of American Music, 5–25. No other note was ree pages. Smithsonian’s Decisions: Narrating History One major factor was the Great Depression, another was radio, and a third was the increasing consolidation and monopolization of the musical mass media by Tin Pan Alley—a group of mostly white men, most of whom had no direct experience of African and European American folk musics, and many of whom had never ventured outside of New York City, but who had the business acumen to monopolize the national publishing, performance, and broadcast media with their catalogs of “hit” songs periodically refreshed by the selective appropriation of “Negro” or “Hillbilly” affects.72 Including this charge of white men might have been incongruous with the reissue, all of whose contributors were, in fact, white men. Including this charge of white men might have been incongruous with the reissue, all of whose contributors were, in fact, white men. This evasion of racial issues underscores a central paradox of the Anthology and its influence on the folk movement. The Anthology had been leveled at a segregated society, and yet the folk and avant- garde movements it fed were dominated by white men. The reissue of Anthology was never able to come to terms with its racialized history. More troubling still, it seems to deliberately skirt the issue, for one of the very few instances I could find where a contributed note was edited for inclu- sion—indeed, the only substantive edit—was Eric Von Schmidt’s account of his introduction to folk music. Schmidt’s note in the anthology ends with his description of his group of folkies as romantics, and details how he named a boat after John Hurt, how another friend hoped to find Blind Lemon Jefferson’s grave and sweep it (a reference to his song on the Anthology “See That My Grave Is Kept Clean”), and how he mourned the fact that Leadbelly had died before he met him. This is where the reissue note ends, on a moment of pathos and indebtedness. Von Schmidt’s paragraph con- tinues, however, in the original draft: “It was a mostly white, middle-class bunch of college kids calking up little discoveries on the slates of their young lives.”73 The omission of this line speaks volumes about all that the reissue does not say about race, class, and status. y The editorial excisions here seem designed particularly to avoid deeper conflicts about the legacy of Smith and the folk revival in general. Smithsonian’s Decisions: Narrating History However given that other scholars like Robert Cantwell had submitted con- tributions, the selection of Marcus, a more public and famous figure, suggests that commercial appeal was also strong; Marcus offered the right combination of scholarship and fame to match the competing goals of the organizers. Other contributions scattered throughout the margins largely gave anecdotal color: Personal testimonials about the Anthology’s impact on their work and reminiscences about Smith’s eccentric behavior. It was to these margins that Pirtle’s essay, with which Singh wanted to close the notes, was inconspicuously consigned.67 p y g More telling is what was collected but not included. The producers steered clear of anything too critical or controversial. Sam Charter pointedly asked in his note, “Why do you want to propagate the myth of Harry Smith? He wasn’t doing anything that anyone else wasn’t doing at the time.”68 Pat Conte used Smith’s Anthology to critique other anthologies, such as Henry Cowell’s 1951 Music of the World’s Peoples, which he deemed “seemingly arbitrary in selection, and lackluster in produc- tion, critiqued with vague musicology,” and thus sought to bring Smith’s revitalizing energy to future global music projects.69 Neither note was included in the final product. Although it’s unclear who edited Marcus’s text for his extended note, a few details from the full chapter were excised. In one, Marcus notes that Smith “liked to brag about killing,” which certainly might prove controversial and cast Smith in a negative light.70 In another, Marcus describes how “devotees surrounded him” https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 198 Dan Blim when he died.71 Because this detail was omitted, Smith is more easily interpreted as a forgotten or mis- understood genius, not unlike the prevailing view of the artists his album featured. These decisions serve to enhance the mythology surrounding Smith. The Smithsonian also excluded some more pointed critiques. Steven Taylor’s contribution, which was not printed, extensively discussed the racial and commercial exploitation inherent in these older recordings. Indeed, one of his critiques echoes the Smithsonian’s criticism of media conglomer- ation, albeit more strongly worded: A number of factors contributed to the virtual disappearance of the music represented on the Anthology. 71Marcus, Invisible Republic, 97. 72Note from Steven Taylor, undated, untitled folder, Moses and Frances Asch Collection. 73Note from Eric Von Schmidt, undated, untitled folder, Moses and Frances Asch Collection. 74Richard H. Kohn, “History and the Culture Wars: The Case of the Smithsonian Institution’s Enola Gay Exhibition,” The Journal of American History 82, no. 3 (December 1995): 1055. p 72Note from Steven Taylor, undated, untitled folder, Moses and Frances Asch Collection. Note from Eric Von Schmidt, undated, untitled folder, Moses and Frances Asch Collection. 74Richard H. Kohn, “History and the Culture Wars: The Case of the Smithsonian Institution’s Enola Gay Exhibition,” The Journal of American History 82, no. 3 (December 1995): 1055. y 73Note from Eric Von Schmidt, undated, untitled folder, Moses and Frances Asch Collection. //doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Note from Steven Taylor, undated, untitled folder, Moses and Frances Asch Collection. 73Note from Eric Von Schmidt, undated, untitled folder, Moses and Frances Asch Collection. 74Richard H. Kohn, “History and the Culture Wars: The Case of the Smithsonian Institution’s Enola Gay Exhibition,” T Journal of American History 82, no. 3 (December 1995): 1055. 71Marcus, Invisible Republic, 97. Smithsonian’s Decisions: Narrating History Although no documents I examined discussed the editorial pro- cess, such decisions are perhaps best understood in the context of another Smithsonian event: The can- cellation of the Enola Gay exhibit at the National Air and Space Museum in 1994. The exhibit came under intense protest from politicians and veterans groups, who claimed the exhibit’s negative tone and its ambivalence toward the action of dropping two atomic bombs on Japan was disrespectful of veterans. As Richard H. Kohn details, the Smithsonian repeatedly edited the exhibit, which only suc- ceeded at alienating historians without allaying the concerns of the veterans and politicians.74 Ultimately, the exhibition was canceled to avoid further controversy. Although the Enola Gay exhibit was perhaps foremost in the Smithsonian’s memory, it was not the only politically charged exhibition. The 1980s and 1990s saw a growing contention between political arts and the government—broadly described as “the culture wars”—including an infamous Whitney Biennial in 1993, which featured politically engaged and confrontational works by women, queer artists, and people of color, that https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Journal of the Society for American Music 199 was largely reviled by (white, male) reviewers.75 In this light, the notes for the reissue perhaps seek a balance—between the scholarly and the commercial, the historical and the personal, the insightful and the uncontroversial. Given the success which greeted it, that balance was likely found. g y Reviews of the reissue were positive, and unsurprisingly, much of the praise echoed how Marcus and the Smithsonian framed the Anthology. Although the artists heard on the discs received some attention, Smith received the lion’s share of credit for the work: David Fricke’s review for Rolling Stone even goes so far as to describe Smith as “an alchemist turning familiar, base materials into some- thing precious and enduring.”76 By suggesting that the tracks themselves were merely “materials,” rather than worthy in their own right, Fricke positions Smith as the true artist whose Anthology shaped them into a timeless work of art. Geoffrey O’Brien in the New York Times similarly calls the work a “one-man cultural revolution,” a statement that again promotes Smith over the artists.77 This attitude may be partly due to the popularity and familiarity of several artists on the Anthology, some of whom had received other reissues. Smithsonian’s Decisions: Narrating History Nevertheless this familiarity, it must be acknowledged, is due to Smith’s reissuing work, and this framing is a shift away from how Smith’s original reissue was understood, as listeners and especially musicians were drawn more to the performances, leading to performers reviving repertoire, folkies conducting oral histories with performers, and even a few performers hav- ing second careers revived thanks to the Anthology. Reviewers also repeatedly seized upon Smith’s racial mixing of artists and repertoire as bold, iconic, and political. O’Brien calls the mixing “Smith’s most utopian gesture” and Robert Cristgau in Spin con- textualizes it as preceding many of the milestones of the Civil Rights movement, even as he betrays his own racialized essentialism by admitting that he prefers the Black artists because they are “less repressed, musically and sexually.”78 Scholars have gone even further in touting the revolutionary equality of the album. In his note, Marcus reads the songs as giving voice to racially and economically marginalized figures, summing up the album’s effect as “a great victory—a victory over decades of los- ing those who did have the courage to speak out in the sociologies of their poverty—that anyone can now hear these men and women, and those they sing about, as singular, as people whose voices no particular set of circumstances could even ensure would be heard.”79 Others have followed suit; John Street reads the entire album as a journey from enslavement to freedom.80 Such readings not only echo Smith’s own racially progressive views, they also evince the “culture war” climate, heralding the anthology as a diverse vision of the United States as a utopia, albeit one whose program of “color- blindness” was broadly acceptable, unlike the racially confrontational exhibit at the 1993 Whitney Biennial or the racial critiques edited out from the notes. At the same time, certain critiques display a misreading of Smith’s vision that served the contem- porary political climate. Many read the Anthology as a McCarthy-era critique against consumerism and conformity, a position acknowledged early on in the Smithsonian’s fundraising flyer, and echoed in Marcus’s note: “The Anthology of American Folk Music was a seductive detour away from what, in the 1950s, was known not as America but as Americanism. 75A rich history of art and museum responses to identity and politics during the 1980s and 1990s can be found in Jerry Saltz and Rachel Corbett, “How Identity Politics Conquered the Art World,” Vulture, accessed June 18, 2020, https://www.vulture. com/2016/04/identity-politics-that-forever-changed-art.html. 76Fricke, “Anthology of American Folk Music edited by Harry Smith,” 101–2. 77Geoffrey O’Brien, “Recapturing the American Sound,” New York Review of Books, April 9, 1998, 45. 78O’Brien, “Recapturing the American Sound,” 47; Robert Cristgau, “Review of Anthology of American Folk Music,” Spin, October 1997, accessed via “10: ANTHOLOGY OF AMERICAN FOLK MUSIC,” Robert Cristgau, accessed June 18, 2020, https://www.robertchristgau.com/xg/cdrev/smithson-cut.php. 79Greil Marcus, “Uncle Dave Macon: Agent of Satan?,” in Harry Smith: The Avant-Garde in the American Vernacular, eds., Andrew Perchuk and Rani Singh (Los Angeles: Getty Research Institute, 2010), 185. 80Street, Music and Politics, 144. 81Marcus, “The Old, Weird America,” 7. 82Cantwell, When We Were Good, 230–31. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press 75A rich history of art and museum responses to identity and politics during the 1980s and 1990s can be found in Jerry Saltz and Rachel Corbett, “How Identity Politics Conquered the Art World,” Vulture, accessed June 18, 2020, https://www.vulture. com/2016/04/identity-politics-that-forever-changed-art.html. 83Gay, Time Done Been Won’t Be No More, 172. 84Street, Music and Politics, 148. 85O’Brien, “Recapturing the American Sound,” 46. 86Walter Benjamin, “The Work of Art in the Age of Mechanical Reproductions,” in Illuminations, trans. Harry Zohn (New York: Schocken Books, 1968), 220. 87Benjamin, “The Work of Art in the Age of Mechanical Reproductions,” 220. 88Benjamin, “The Work of Art in the Age of Mechanical Reproductions,” 221. Smithsonian’s Decisions: Narrating History That meant the consumer society, as adver- tised on TV.”81 Robert Cantwell fixed his critical ear on the penultimate track, “The Lone Star Trail,” taken from a Hollywood film, suggesting the song was nothing more than an absurd, if ingenious, intrusion “utterly out of place.”82 William Gay and John Street make Cantwell’s critique even more https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Dan Blim 200 explicit. Gay hears it as urging the listener to reconsider “the tale you have been told about a lost America,”83 and Street calls it “a deliberate impertinence that reminds us of white America’s willing- ness to airbrush out exactly the kind of pain and unabridged oppression that the preceding songs document.”84 These critiques highlight more confrontationally both the whiteness of the cowboy figure and, to a lesser degree, its blatantly commercial status, and perhaps assumedly fictionalized roots in Hollywood. Due to the rise of Marxist and cultural criticism among scholars, such claims are unsurprising; they find confirmation in Smith’s role as a progressive figure. Nevertheless, Smith’s own commentary contradicts these claims. He praised the track as “authentic ‘cowboy’ singing,” and continually embraced the commercial aspects of the music he selected. Conclusion In his review of the Smithsonian’s reissue of the Anthology, Geoffrey O’Brien reflects on the age of box-set reissues: It provides further confirmation that the CD box set is, in its reverent attention to detail, our moment’s equivalent of the medieval illuminated manuscript. It is not enough to have learned how to capture sound; there must be an appropriate monument to enclose it and keep it from escaping.85 Comparing the reissue to an illuminated manuscript suggests that the reissue is more than a simple repro- duction of a recording: Rather, it is an object of lasting value. For fans, the reissue is just that: A cherished collectible. Historical and retrospective reissues often tout their rare and unreleased tracks and photo- graphs, suggesting their collectability as unique historical items despite their broad commercial availability. g gg g y y Reissues traffic in “authenticity” in a way that complicates Walter Benjamin’s critique of reproduc- tions of artworks. Not incidentally, Benjamin also invokes the medieval manuscript in his definition of authenticity, observing that: The presence of the original is the prerequisite to the concept of authenticity. Chemical analyses of the patina of a bronze can help to establish this, as does the proof that a given manuscript of the Middle Ages stems from an archive of the fifteenth century. The whole sphere of authenticity is outside technical—and, of course, not only technical—reproducibility.86 In O’Brien’s metaphor, however, the reissue is the medieval manuscript, not the reproduction of it. How, then, do reissues promise authenticity, or “aura,” to use Benjamin’s term? To be sure, not all reissues and compilations do. Nevertheless more elaborate reissues, which gain critical praise and are usually sold at a higher price, frequently invoke this idea in fans and critics. Benjamin roots authenticity in the original’s “presence in time and place, its unique existence at the place where it happens to be.”87 For audio recordings, authenticity would seem to be an impossibility; indeed, Benjamin positions recordings as reproductions.88 However even if Benjamin might not grant reissues authenticity, fans do in ways that suggest they display original artifacts of sound just as museums exhibit original artifacts. Recording is not the object but rather the medium to display performance—a fleeting occurrence that otherwise leaves no physical object (as opposed to a classical composition, where the score is the primary object). https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Conclusion Reissues invoke their rootedness in a historical time (if not place) and cast their product as rare and collectible, offering unfamiliar or previously unreleased material. Smith did this in the Anthology, introducing records that had fallen out of the In O’Brien’s metaphor, however, the reissue is the medieval manuscript, not the reproduction of it. How, then, do reissues promise authenticity, or “aura,” to use Benjamin’s term? To be sure, not all reissues and compilations do. Nevertheless more elaborate reissues, which gain critical praise and are usually sold at a higher price, frequently invoke this idea in fans and critics. Benjamin roots authenticity in the original’s “presence in time and place, its unique existence at the place where it happens to be.”87 For audio recordings, authenticity would seem to be an impossibility; indeed, Benjamin positions recordings as reproductions.88 However even if Benjamin might not grant reissues authenticity, fans do in ways that suggest they display original artifacts of sound just as museums exhibit original artifacts. Recording is not the object but rather the medium to display performance—a fleeting occurrence that otherwise leaves no physical object (as opposed to a classical composition, where the score is the primary object). Reissues invoke their rootedness in a historical time (if not place) and cast their product as rare and collectible, offering unfamiliar or previously unreleased material. Smith did this in the Anthology, introducing records that had fallen out of the https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Journal of the Society for American Music 201 public ear—forgotten recordings by obscure artists that cast new light on U.S. folk music traditions. Reissues also employ the scholarly language of historians and preservationists through their liner notes and attention to detail. When the Smithsonian emphasized replicating Smith’s own reproduction methods, paper, and not cleaning up the images or tracks, they worked to reassure buyers that the reissue was “the real thing” by collapsing the original and the reissue. g y p g g O’Brien’s review also describes the reissue as a monument, “enclosing” the music, fixing it fast and locking it away. I find this description telling, yet I would argue that the process of reissuing is para- doxical in this regard. Reissues do have the power to fix musical history. They speak with authority in their scholarly and completist approach. Audiovisual Materials Anthology of American Music. Smithsonian Folkways SFW 40090, 1997, compact disc. Conclusion References Archival Materials Moses and Frances Asch Collection. Smithsonian Center for Folklife and Cultural Heritage. Washington, DC. Conclusion In doing so, reissues have the power to canonize artists and songs, and to shape historical narratives of music. Nevertheless as I have argued, the act of reissuing also has the potential to unfix musical history, as reissuers highlight hidden histories and marginalized voices, and juxtapose tracks in provocative ways. The very power of Smith’s anthology came in the ways it unsettled essentialized myths about folk music’s relationships with commercialism and race. However, listeners must be wary of accepting any reissue’s suggestion of a “complete” or “definitive” account of history, often part-and-parcel with claims of authenticity; as the Anthology reissuing process demonstrates, reissues are the product of interpretation and negotiation, and thus limited by the reis- suer’s perspectives, values, and biases, as well as their own commercial needs. Gyan Prakash offers a similar warning against the museum’s display of artifacts as objective: Exotic artifacts are positioned as authentic residues of myths, practices, values, and forms of orga- nization that are thought to underlie the wholeness and integrity of other cultures. Such projec- tions of authenticity and wholeness project the exhibition of exotic cultures as entirely separate from and unaffected by the structure that gathers and stages them.89 In other words, reissues have just as much to say about the values of the present moment as they do about the past. In other words, reissues have just as much to say about the values of the present moment as they do about the past. The Anthology of American Folk Music highlights the act of reissuing as provocative and meaningful. The very fact that Smith emerged as a canonic figure of U.S. folk music because of his reissue attests to this power. Reissues, including the Anthology, become a means of narrating U.S. history. With each step, the narrative changes. The original folk songs and ballads on the Anthology recounted historical events as part of a flexible oral folk tradition. The original Anthology unveiled a complex narrative of musical evo- lution and diversity in the United States. Moreover, the CD reissue narrated its own history of the folk revival of the 1950s. Reissues remain potent sources for music historians because of their interpretive work and their potential to tell multiple histories. As a rare example of a reissue whose process has been documented, the Anthology offers a blueprint for further work to interrogate the histories reissues curate and construct through sound. Secondary Materials Baade, Christina and Paul Aitken. “Still ‘In the Mood’: The Nostalgia Aesthetic in a Digital World.” Journal of Popular Music Studies 20, no. 4 (December 2008): 353–77. 89Gyan Prakash, “Museum Matters,” in Museum Studies: An Anthology of Contexts, ed. Bettina Messias Carbonell (Malden, MA: Blackwell Publishers, 2004), 210. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press “Johnson’s Son Gets Right.” Billboard, November 1, 2003. y ll. “Johnson’s Son Gets Right.” Billboard, November 1, 2 g Igliori, Paola, ed. American Magus, Harry Smith: A Modern Alchemist. New York: Inanout Press, 1996. Igliori, Paola, ed. American Magus, Harry Smith: A Modern Alchemist. N Jannsen, David A. and Edward J. Whitelock, Apocalypse Jukebox: The End of the World in American Popular Music. New York: Soft Skull Press, 2009. Karp, Ivan. “Introduction.” In Museums and Communities: The Politics of Public Culture, edited by Ivan Karp, Christine Mulen Kreamer, and Steven D. Levine, 11–24. Washington: Smithsonian Institution Press, 1992. Kirshenblatt-Gimblett, Barbara. Destination Culture: Tourism, Museums, and Heritage. Berkeley: University of California Press, 1998. Kohn, Richard H. “History and the Culture Wars: The Case of the Smithsonian Institution’s Enola Gay Exhibition of American History 82, no. 3 (December 1995): 1036–63. Macdonald, Sharon. “Collecting Practices.” In A Companion to Museum Studies, edited by Sharon Macdonald, 81–97. Malden, MA: Blackwell Publishing, 2006. g Marcus, Greil. Invisible Republic: Bob Dylan’s Basement Tapes. New York: H. Holt and Co., 1997. Marcus, Greil. “Uncle Dave Macon: Agent of Satan?” In Harry Smith: The Avant-Garde in th Andrew Perchuk and Rani Singh, 175–86. Los Angeles: Getty Research Institute, 2010. Marcus, Greil. “Uncle Dave Macon: Agent of Satan?” In Harry Smith: The Avant-Garde in the American Vernacular, edited by Andrew Perchuk and Rani Singh, 175–86. Los Angeles: Getty Research Institute, 2010. McLeod, Kembrew. “Confessions of an Intellectual (Property): Danger Mouse, Mickey Mouse, Sonny Bono, and Winding Path as a Copyright Activist-Academic.” Popular Music and Society 28, no. 1 (February 2005): 79– Miller, Karl Hagstrom. Segregating Sound: Inventing Folk and Pop Music in the Age of Jim Crow. Durham, NC: Duke University Press, 2010. Miller, Kiri. Traveling Home: Sacred Harp Singing and American Pluralism. Urbana: University of Illinois Press, 2008. Moist, Kevin M. “Collecting, Collage, and Alchemy: The Harry Smith Anthology of American Folk Music a Intervention.” American Studies 48, no. 4 (Winter 2007): 111–27. Morris, Chris. “Smithsonian to Reissue Its Classic Folk Anthology Set.” Billboard, June 21, 1997. O’Brien, Geoffrey. “Recapturing the American Sound.” New York Review of Books, April 9, 1998, 45–51. Olmsted, Tony. Folkways Records: Moses Asch and his Encyclopedia of Sound. New York: Routledge, 2003. Pearce, Susan M. “Museum Studies in Material Culture: Introduction.” In Museum Studies in Material Culture, e M. Pearce, 1–10. New York: Leicester University Press, 1989. Plasketes, George. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Dan Blim 202 Baxandall, Michael. “Exhibiting Intention: Some Preconditions of the Visual Display of Culturally Purposeful Objects.” In Exhibiting Cultures: The Poetics and Politics of Museum Display, edited by Ivan Karp and Steven D. Levine, 33–41. Washington: Smithsonian Institution Press, 1991. Benjamin, Walter. “The Work of Art in the Age of Mechanical Reproductions.” In Illuminations, translated by Harry Zohn, 217–52. New York: Schocken Books, 1968. Brooks, Tim. “How Copyright Law Affects Reissues of Historic Recordings.” ARSC Journal 36, no. 2 (Fall 2005 Cantwell, Robert. When We Were Good: The Folk Revival. Cambridge, MA: Harvard University Press, 1996. Carlin, Richard. Worlds of Sound: The Story of Smithsonian Folkways. New York: Harper Collins, 2010. Chang, Vanessa. “Records that Play: The Present Past in Sampling Practice.” Popular Music 28, no. 2 (May 20 Records that Play: The Present Past in Sampling Practice.” Popular Music 28, no. 2 (May 2009): 143–59. Clague, Mark. “Portraits in Beams and Barlines: Critical Editions and the Art of Notation.” American Music 23, no. 1 (April, 2005): 39–68. Cristgau, Robert. “Review of Anthology of American Folk Music.” Spin, October 1997. Accessed June 18, 2020. https://www.rob- ertchristgau.com/xg/cdrev/smithson-cut.php. Djabarouti, Johnathan. “Imitation and Intangibility: Postmodern Perspectives on Restoration and Authenticity at the Hill House Box, Scotland.” International Journal of Heritage Studies 28, no. 1 (2021): 1–18. Dougan, John. “Objects of Desire: Canon Formation and Blues Record Collecting.” Journal of Popular Music Studies 18, no. 1 (April 2006): 40–65. Erlewine, Stephen Thomas. “Why the Death of Compilation Albums and Reissues Is Worth Mourning.” Pitchfork. May 2, 201 https://pitchfork.com/features/article/9887-why-the-death-of-greatest-hits-albums-and-reissues-is-worth-mourning/. Filene, Benjamin. Romancing the Folk: Public Memory and American Roots Music. Chapel Hill: University of N Press, 2000. Romancing the Folk: Public Memory and American Roots Music. Chapel Hill: University of North Carolina Fricke, David. “Anthology of American Folk Music edited by Harry Smith.” Rolling Stone, September 18, 1997 Time Done Been Won’t Be No More: Collected Prose. Edite Hamilton, Marybeth. “Searching for the Blues: James McKune, Collectors, and a Different Crossroads.” In Listen Again: A Momentary History to Popular Music, edited by Eric Weisbard, 26–49. Durham, NC: Duke University Press, 2007. Hamilton, Marybeth. In Search of the Blues. New York: Basic Books, 2008. Hamilton, Marybeth. In Search of the Blues. New York: Basic Books, 2008. Hayes, David. “Take Those Old Records off the Shelf”: Youth and Music Consumption in the Postmodern Age.” Popular Music and Society 29, no. 1 (2006): 51–68. y Holland, Bill. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Cite this article: Blim, Dan. “Historical Records: Reissuing as Curatorial Practice in Harry Smith’s Anthology of American Folk Music.” Journal of the Society for American Music 17, no. 2 (May 2023): 178–203. https://doi.org/10.1017/S1752196323000093. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press “Like a Version: Cover Songs and the Tribute Trend in Popular Music.” Studies in Popular Cu (1992): 1–18. Plasketes, George, ed. Play It Again: Cover Songs in Popular Music. Burlington, VT: Ashgate, 2010. Prakash, Gyan. “Museum Matters.” In Museum Studies: An Anthology of Contexts, edited by Bettina Messias Carbonell, 208–15. Malden, MA: Blackwell Publishers, 2004. Saltz, Jerry and Rachel Corbett. “How Identity Politics Conquered the Art World.” Vulture. Accessed June 18, 2020. https:// www.vulture.com/2016/04/identity-politics-that-forever-changed-art.html. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Journal of the Society for American Music 203 Shuker, Roy. Wax Trash and Vinyl Treasures: Record Collecting As a Social Practice. Burlington, VT: Ashgate, Sinnreich, Aram. Mashed Up: Music, Technology, and the Rise of Configurable Culture. Amherst: University of Massachusetts Press, 2010. Smith, Harry. “A Rare Interview with Harry Smith.” By John Cohen. Sing Out! 19, no. 1 (1969). Reprinted in Igliori, American Magus, Harry Smith, 125–45. Smithsonian Folkways Recording. “Mission and History.” Accessed June 18, 2020. https://folkways.si.edu/mission-and-history. Street, John. Music and Politics. Malden, MA: Polity Press, 2012. Titon, Jeff Todd. Early Downhome Blues: A Musical and Cultural Analysis. Chapel Hill: University of North 1994. Tosches, Nick. Country: The Twisted Roots of Rock ‘n’ Roll. New York: Da Capo Press, 2009. van Mensch, Peter. “Methodological Museology; or, toward a Theory of Museum Practice.” In Objects of Knowledge, edited by Susan M. Pearce, 141–57. Atlantic Highlands, NJ: The Athlone Press, 1990. Methodological Museology; or, toward a Theory of Mus van Mensch, Peter. “Methodological Museology; or, toward a Theory of Museum Practic Susan M. Pearce, 141–57. Atlantic Highlands, NJ: The Athlone Press, 1990. Walser, Robert. “Review of The Poetics of Rock: Cutting Tracks, Making Records by Albin J. Zak.” Journal of Musicological Society 60, no. 1 (Spring 2007): 253–57. Whisnant, David E. All That is Native and Fine: The Politics of Culture in an American Region. Chapel Hill: University of North Carolina Press, 1983. Woods, Alyssa. “Violence and the Negotiation of Musical Meaning in Rock, Pop, and Rap Cover Songs.” Master’s thesis, University of Ottawa, 2004. Yochim, Emily Chivers and Megan Biddinger. “It Kind of Gives You that Vintage Feel’: Vinyl Records and the Trope of Death.” Media, Culture, Society 30, no. 2 (March 2008): 183–95. Zak, Albin. I Don’t Sound Like Nobody: Remaking Music in 1950s America. Ann Arbor: University of Michiga Zak, Albin. I Don’t Sound Like Nobody: Remaking Music in 1950s America. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Dan Blim is an associate professor of music at Denison University. He has published on U.S. music topics, including the political reception of the score for Robert Altman’s Nashville, commemoration in John Adams’s On the Transmigration of Souls, political ideology in the music at the 2016 U.S. Presidential Inauguration, and narrative realism in filmic adaptations of Broadway musicals. https://doi.org/10.1017/S1752196323000093 Published online by Cambridge University Press Ann Arbor: University of Michigan Press, 2010. Dan Blim is an associate professor of music at Denison University. He has published on U.S. music topics, including the political reception of the score for Robert Altman’s Nashville, commemoration in John Adams’s On the Transmigration of Souls, political ideology in the music at the 2016 U.S. Presidential Inauguration, and narrative realism in filmic adaptations of Broadway musicals. Dan Blim is an associate professor of music at Denison University. He has published on U.S. music topics, including the political reception of the score for Robert Altman’s Nashville, commemoration in John Adams’s On the Transmigration of Souls, political ideology in the music at the 2016 U.S. Presidential Inauguration, and narrative realism in filmic adaptations of Broadway musicals. Cite this article: Blim, Dan. “Historical Records: Reissuing as Curatorial Practice in Harry Smith’s Anthology of American Folk Music.” Journal of the Society for American Music 17, no. 2 (May 2023): 178–203. https://doi.org/10.1017/S1752196323000093.
https://openalex.org/W4297696625	https://dergipark.org.tr/tr/download/article-file/85951	Turkish	null	Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine	DergiPark (Istanbul University)	2,002	cc-by	12,266	VÂSIT KENTİNİN KURULUŞU VE İLK SAKİNLERİ ÜZERİNE M. Mahfuz SÖYLEMEZ * 1 İlhan Tekeli, “Tarih Yazımında Gündelik Yaşam Tarihçiliğinin Kavramsal Çerçevesi Nasıl Genişletilebilir”, Tarih Yazımında Yeni Yaklaşımlar, Küreselleşme ve Yerelleşme, İstanbul 2000, s. 54 Foundation of Vâsıt and its early inhabitants Vâsıt, a very important city bearing a great mission in the history of Islam, has been founded by Amevid’s governor of province of Irak called Yusuf es- Sekafî in Irak next to recent Kut-Amâra’s territory. It has become a center governance since its foundation and therefore it has some characteristics of a typical Islamic city. For instance, there are a masjid (mosque), daru’l imâre (center of governance) and market place at the center of the city. On the other hand, Only soldiers from Syria, Kufa and Basra, and Turks from Buharas were settled there during its foundation period. Also, it has been let Aramians who were from a different religion to be settled there in the last period of Amevids and early period of Abasids. Interestingly, there were not any struggles between these inhabitants from different religious sides in Vâsıt. In contrast, there was a peace-keeping process could be evaluated a civilized relations among them. Key words: Islamic city, Vasıt, Haccac b. Yusuf, Islamic History * Yrd. Doç.Dr. Gazi Üniversitesi Çorum İlahiyat Fakültesi * Yrd. Doç.Dr. Gazi Üniversitesi Çorum İlahiyat Fakültesi 1 İlhan Tekeli, “Tarih Yazımında Gündelik Yaşam Tarihçiliğinin Kavramsal Çerçevesi Nasıl Genişletilebilir”, Tarih Yazımında Yeni Yaklaşımlar, Küreselleşme ve Yerelleşme, İstanbul 2000, s. 54 3 Haccâc Irak’a gelir gelmez yaptığı ilk konuşmada katı bir yönetim tercih ettiğini ilan etti. Haccâc’ın bu konuşması ile ilgili geniş bilgi için bkz. Zübeyr b. Bekkâr (ö.256/870), el- Ahbâru’l-Muveffekiyyât, tahk.: Samî Mekkî, Bağdat 1972, ss. 96-104; Ebû’l-Ferec İsfahanî (ö.356/966), el-Eğanî, tahk.: Abdullah Ali Muhanna, Beyrut 1995, c. XIV, ss. 237-238; İbn Abdirabbih, İkdu’l-Ferid, tahk.: Ahmed Emin, Ahmed ez-Zeyn, İbrahim el- Ebyarî, Abdusselam Harun, Kahire 1968, c. IV, s. 119-120; İbnu’l-Cevzî, el-Muntazam fi Tevârihi’l-Mulûk ve’l-Ümem, tahk.: Süheyl Zekkâr, Beyrut 1995, c. IV, s. 299; İbnu’l-İbrî, Tarihu Muhtasar ed-Duvel, Beyrut 1958, s. 113; Muhammed b. Abdullah eş-Şiblî ed- 2 Bkz. Lawrence I. Conrad, “Wasıt”, Dictionary of the Middle Ages, edit.: Joseph R. Strayerl, New York 1989, vol. XII, p. 574; Beşir Yusuf Fransis, “el-Mezâhiru’l-Fenniyye fi Avâsimi’l- Iraki’l-İslâmiyyeti’l-Kadime ala Davi’l-İstikşâfâti’l-Hadise”, Sümer, Bağdad 1948, c. IV, sayı: 1, s. 106; R.Darley-Doran, “Wâsit”, The Encyclopedia of İslâm, (new edition) Leiden 2001, vol: XI, p. 165 Key words: Islamic city, Vasıt, Haccac b. Yusuf, Islamic History Şehir tarihçiliği, mekanı ve mekanda yaşayan insanın sergüzeştini ele alan bir tarihçilik türüdür. Oldukça eski bir geçmişe sahip olmasına rağmen bu asrın başlarına kadar hak ettiği ilgiyi görememiş, daha çok amatör tarihçilerin çalışmalarıyla sınırlı kalmıştır. Yirmibirinci yüzyılla bir- likte bu alana ciddi anlamda bir yönelişin olduğu görülmektedir. Söz ko- nusu yönelimin arkasında tarihi küçük ünitelerin oluşturduğu, bütünün aydınlatabilmesi için parçaların keşfi ve birleştirilmesi gerektiğini savunan anlayışın yaygınlaşması ve de postmodernitenin etkisiyle büyük anlatılar ve sosyo-ekonomik açıklamalara dayanan makro tarihin önemini yitirmeye başlaması bulunmaktadır.1 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 148 İslam tarihine makro tarih perspektifinden bakılıp hakkında genel değerlendirmeler yapılmış olan tarihi dönemlerin başında Emeviler gel- mektedir. Üzerinde çokça konuşulduğu halde hala bütünüyle ortaya ko- nulmaktan uzak olan bu dönemin doğru bir şekilde tespit edilebilmesi için parçaların ayrı ayrı çalışılması ve daha sonra da bunların bir araya getirile- rek bütün hakkında doğru tahliller yapılması gerekmektedir. Özellikle de bu dönemdeki kentler ve orada yaşayanların sahip oldukları birikimin orta- ya konması dönemin hakikate en yakın resminin çizimi için büyük bir ö- neme sahiptir. İşte bu anlayıştan hareketle, Emevilerin Irak valisi Haccâc b. Yusuf tarafından askerî bir kent olarak kurulmuş olan ve aynı zamanda o döne- min en önemli kentlerinden biri olan Vâsıt şehrini ele almak istiyoruz. I- rak’ın idarî merkezi olmak üzere kurulan ve İslâm alemindeki ilk Türk kit- leyi de barındıran bu kent, Bağdat’ın 148/765’te inşasıyla gerileme süre- cine girmiş, miladî 15. Yüzyılda, kenarında kurulduğu Dicle Irmağının ya- tağının değişmesi ile birlikte, önemini tamamen yitirmiştir. Nitekim miladî 17. yüzyılda küçük bir kasabaya dönüşmüş, 19. Yüzyılda ise metruk, ha- rabe bir şehir haline gelmiştir. Vâsıt kalıntıları bu gün Güney Irak’ta Kut- Amâra’ya 70 km. mesafede bulunmaktadır. Bu kalıntılar, Minâre harabeleri olarak bilinmektedir.2 Temel kaynaklara dayanarak hazırlamaya çalıştığımız bu yazıda ön- ce şehrin kuruluş sebeplerini daha sonra da sırasıyla, kuruluşu, fizikî yapı- sı, ilk sakinleri ve son olarak da “Vâsıt merkezli” oluşmuş olan apokrif ri- vayetleri değerlendirmek istiyoruz. 4 Bkz. İbnu’l-Fakîh, Kitabu’l-Buldân, tahk.: Yusuf el-Havî, Beyrut 1996, s. 266; İbnu’l-Esîr, el Kâmil fi't-Târih, çev.: Ahmet Ağırakça, İstanbul 1991, c. IV, s. 419. Taberî, Haccâc’ın amillerinin kendisine, Zimmîlerin İslâm dinini benimseyip şehirlere yerleşmeleri sebepiyle harac gelirlerinin düştüğünü yazmaları üzerine, Haccâc’ın şehirlere yerleşmiş olan Zimmî- leri çıkarmalarını emrettiğini, Basra’dan bu insanların zorla çıkarıldıklarını, gidecek yerleri olmadığı için bir süre şehrin dışında konaklamak zorunda kaldıklarını ve sürekli “ey Mu- hammed” diyerek feryat ettiklerini, Basralı kurrânın da onları desteklediklerini ve kendileri ile beraber ağlaştıklarını, Abdurrahman’ın Basra’ya gelmesiyle her iki kitlenin de ona katıl- dığını söylemektedir. Bkz. Taberî, Tarihu'l-Ümem ve'l Mulûk, Beyrut 1987, c. VII, s. 278- 279 Dımeşkî, Mehâsinu’l-Vesâil fi Ma’rifeti’l-Evâil, tahk.: Muhammed Altuncu, Beyrut 1992, s. 203 5 Bkz. Müberred, el-Kâmil fi'l-Luğa ve'l-Edeb ve'n-Nahv ve't-Tasrif, thk Muhammed Ahmed ed-Dalî, Beyrut 1993, c. II, s. 493 7 Bunların en ciddisi Haccâc’ı uzun süre uğraştıran Şebîb hadisesidir. Bu hadise ile ilgili geniş bilgi için bkz. Halife b. Hayyat, Tarihu Halife b. Hayyat, tahk.: Süheyl Zekkâr, Bey- rut, 1993, ss. 210-212, Yakubî, Tarih, c. II, s. 274 vd. 9 Bkz. İbn Kuteybe, el-İmâme ve’s-Siyâse, Kum 1363, c. II, s. 33; Mesudî, Murûcu'z-Zeheb ve Me'âdinu'l-Cevher, Kum 1984, c. III, s. 138; İbnu’l-Cevzî, Muntazam, c. IV, s. 365 1. Vâsıt’ın Kuruluş Sebepleri Vâsıt, siyasî mülahazalar ile inşa edilmiş bir askerî şehirdir. Şehrin inşasına sebep olan bir dizi siyasî ve askerî hadise gerçekleşmiştir. Bu hadiseler ele alınmadan şehrin kuruluş sebeplerini izah etmek mümkün olmadığından bunlara kısaca değinmenin yararlı olacağını düşünüyoruz. Şehrin kuruluş sürecini hızlandıran hadiseler Emevî halifesi Abdulmelik b. Mervan (65-86/685-705) tarafından Irak valiliğine atanan Haccâc b. Yusuf es-Sekafî (75-95/694-713)’nin buraya gelişi ile başlamak- tadır.3 Irak’ı sıkıyönetime dayalı bir idare anlayışı ile yöneten Haccâc, yö- Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 149 rede giderek güçlenmekte olan Emevî muhaliflerini devletin yanına çeke- medi. Aksine, onların Emevilere karşı daha da bilenmelerine sebep oldu. Hele haraç gelirlerinde düşüşü gerekçe göstererek çiftçilikle uğraşan Sevâd yerlilerinin şehirlere yerleşmelerini yasaklaması ve buralara yerleşip mal mülk edinmiş olan kimi insanları zorla memleketlerine geri gönderme- si, Irak’ın çoğunluğunu oluşturan mevalî ve zimmîlerin onun karşısında yer almasına sebep oldu.4 Müberred’in de ifade ettiği gibi Haccâc, şehirlerden çıkardığı bu insanların yerine Arapları yerleştirdi, geldikleri yerlere dönme mecburiyetinde bırakılan insanların ellerine köylerinin isimlerini yazdırdı, izinsiz olarak şehirlere girmelerini yasakladı.5 Onun bu sert politikalarından bıkanların bir kısmı memleketlerini terkederek Hicaz’a sığınmak zorunda kaldılar.6 Bu gayri memnun unsur tarafından, bölgede silahlı gruplar dahi teşekkül ettirildi. Genelini Haricilerin oluşturduğu bu gruplar, irili ufaklı onlarca münferit hadise meydana getirmiş7 olmalarına rağmen, bir araya gelmeyi başaramadıklarından dolayı Hicri 81 yılına kadar geçen sürede Haccâc ve Emevler için ciddi bir tehlike oluşturamadılar. Hicri 81 yılı söz konusu kitle için bir dönüm noktası olmuştur. Bu yılın başında Haccâc’ın, Sicistân valisi Abdurrahman b. Muhammed b. el-Eş’as ile arası açılmış, neticede Haccâc, Abdurahman’ı görevden aldı.8 Fakat Abdurahman bunu kabul etmeyerek kendisine bağlı birliklerle Haccâc’a isyan etti ve9 kendisini bir umut ışığı olarak gören yönetim muhaliflerini etrafında toplamayı ba- şardı. rede giderek güçlenmekte olan Emevî muhaliflerini devletin yanına çeke- medi. Aksine, onların Emevilere karşı daha da bilenmelerine sebep oldu. Hele haraç gelirlerinde düşüşü gerekçe göstererek çiftçilikle uğraşan Sevâd yerlilerinin şehirlere yerleşmelerini yasaklaması ve buralara yerleşip mal mülk edinmiş olan kimi insanları zorla memleketlerine geri gönderme- si, Irak’ın çoğunluğunu oluşturan mevalî ve zimmîlerin onun karşısında yer almasına sebep oldu.4 Müberred’in de ifade ettiği gibi Haccâc, şehirlerden çıkardığı bu insanların yerine Arapları yerleştirdi, geldikleri yerlere dönme mecburiyetinde bırakılan insanların ellerine köylerinin isimlerini yazdırdı, izinsiz olarak şehirlere girmelerini yasakladı.5 Onun bu sert politikalarından bıkanların bir kısmı memleketlerini terkederek Hicaz’a sığınmak zorunda kaldılar.6 Bu gayri memnun unsur tarafından, bölgede silahlı gruplar dahi teşekkül ettirildi. 6 Bkz. Yakubî, Tarihu Ya’kubî, Beyrut 1992, c. II, s. 290 8 Bkz. Halife b. Hayyat, s. 217; Mesudî, et-Tenbîh ve’l-İşrâf, Beyrut 1981, s. 287 6 Bkz. Yakubî, Tarihu Ya’kubî, Beyrut 1992, c. II, s. 290 12 Abdurrahman Basra mescidinde halka yaptığı konuşmada Haccâc ile Abdulmelik’in dini değerlerden uzaklaştıklarını, dine önem vermez hale geldiklerini, bunların görevden uzak- laştırılmasının dinin emrettiği en önemli ödevlerden biri olduğunu söylemiş ve destek is- temiştir. Haccâc’ın uygulamalarından bezmiş olan Basralıların hemen hemen tamamı Abdurrahman’ın yanında yer almaya karar vermiş ve onu destekleyeceklerini ilan etmiş- lerdir. Bkz. Taberî, c. VII, s. 238; İbnu’l-Esîr, c. IV, s. 419 10 Bkz. Taberî, c. VII, s. 237; Mesudî, Tenbih ,s. 287; İbnu’l-Cevzî, Muntazam, c. IV, s. 367 11 Bkz. Taberî, c. VII, s. 237; İbnu’l-Esîr, c. IV, s. 419 Çorum İlahiyat Fakültesi Dergisi, 2002/2 1. Vâsıt’ın Kuruluş Sebepleri Genelini Haricilerin oluşturduğu bu gruplar, irili ufaklı onlarca münferit hadise meydana getirmiş7 olmalarına rağmen, bir araya gelmeyi başaramadıklarından dolayı Hicri 81 yılına kadar geçen sürede Haccâc ve Emevler için ciddi bir tehlike oluşturamadılar. Hicri 81 yılı söz konusu kitle için bir dönüm noktası olmuştur. Bu yılın başında Haccâc’ın, Sicistân valisi Abdurrahman b. Muhammed b. el-Eş’as ile arası açılmış, neticede Haccâc, Abdurahman’ı görevden aldı.8 Fakat Abdurahman bunu kabul etmeyerek kendisine bağlı birliklerle Haccâc’a isyan etti ve9 kendisini bir umut ışığı olarak gören yönetim muhaliflerini etrafında toplamayı ba- şardı. İsyanın hızla yayılmakta olduğunu gören Haccâc, Basra’dan aldığı takviye güçler ile Huzistan’da bulunan Abdurrahman’ın üzerine yürüdü. Tuster yakınlarında kendi öncü kuvvetleri ile Abdurrahman’ın öncü birlikle- ri karşı karşıya geldiler. Bu ilk karşılaşmada kuvvetleri büyük bir hezimete Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 150 uğrayınca,10 Haccâc Basra’ya geri dönmek zorunda kaldı ve ez-Zaviye’de karargah kurdu,11 onu takip etmekte olan Abdurrahman ise, hiçbir direnme ile karşılaşmaksızın Irak’ın idarî merkezlerinden biri olan Basra’ya girdi, bölge halkının da desteğini alarak12 önemli bir askerî güce ulaştı. Suri- ye’den takviye güçler alan Haccâc ile Abdurrahman’ın kuvvetleri bu bölge- de yeniden karşı karşıya geldiler. Günlerce süren savaşta Abdurrahman yenildi ve beraberindekiler ile birlikte Kûfe’ye doğru çekildi.13 uğrayınca,10 Haccâc Basra’ya geri dönmek zorunda kaldı ve ez-Zaviye’de karargah kurdu,11 onu takip etmekte olan Abdurrahman ise, hiçbir direnme ile karşılaşmaksızın Irak’ın idarî merkezlerinden biri olan Basra’ya girdi, bölge halkının da desteğini alarak12 önemli bir askerî güce ulaştı. Suri- ye’den takviye güçler alan Haccâc ile Abdurrahman’ın kuvvetleri bu bölge- de yeniden karşı karşıya geldiler. Günlerce süren savaşta Abdurrahman yenildi ve beraberindekiler ile birlikte Kûfe’ye doğru çekildi.13 Muhaliflerin Kûfe’ye yönelmeleri üzerine Basra’yı kuşatan Haccâc, beş gün süren şiddetli çarpışmanın sonucunda şehre hakim olmayı başar- dı. Ancak Basra halkının desteğini yitirmiş olması buranın icbarî merkez olarak kullanılmasını sakıncalı hale getirmişti.14 Abdurrahman’ın Basra ve çevresinde elde etmiş olduğu bu başarılar Kûfe’ye ulaşınca Haccâc’a karşı pusuda beklemekte olan muhalifleri İbn Nâciye’nin liderliğinde harekete geçtiler ve şehirde bulunan Haccâc’ın vekili ile Suriye kökenli 4000 kişilik askeri gücü çıkardılar.15 Böylece Kûfe de hiç direnmeden Abdurrahman’ın tarafına geçmiş oldu.16 Iraklı mevalî17 ve Araplardan oluşan Abdurrahman’ın kuvvetleri ile Haccâc’ın kuvvetleri hicri 82 yılında Kûfe yakınlarında bulunan Deyr’u Cemâcim’de karşı karşıya geldiler. Tari- he Deyru Cemâcim savaşı olarak geçen ve günlerce sürmüş olan bu savaş, Haccâc lehine sonuçlandı.18 Abdurrahman Savaş meydanını tekederek 13 Yardım talebinde bulunmak üzere Kûfelilere de mektup yazmıştır. 1. Vâsıt’ın Kuruluş Sebepleri Dineverî tarafından kaydedilmiş olan bu mektupların metni şöyledir: “Ey insanlar bu zalimi ve yaptıklarını görmüyor musunuz? Sünnetin yok edildiğini, ahkamın ortadan kaldırıldığını, münkerin i- kame edildiğini, öldürmelerin yaygınlaştırıldığını görmüyor musunuz? Allah için harekete geçiniz. Benimle beraber Haccâc’a isyan ediniz. Susmanız asla caiz değildir.” Bkz. Dineverî, Ahbâru’t-Tıvâl, tahk.: Abdulmünim Âmir-Cemalettin eş-Şeyyâl, Kahire 1960, s. 317 13 Yardım talebinde bulunmak üzere Kûfelilere de mektup yazmıştır. Dineverî tarafından kaydedilmiş olan bu mektupların metni şöyledir: “Ey insanlar bu zalimi ve yaptıklarını görmüyor musunuz? Sünnetin yok edildiğini, ahkamın ortadan kaldırıldığını, münkerin i- kame edildiğini, öldürmelerin yaygınlaştırıldığını görmüyor musunuz? Allah için harekete geçiniz. Benimle beraber Haccâc’a isyan ediniz. Susmanız asla caiz değildir.” Bkz. Dineverî, Ahbâru’t-Tıvâl, tahk.: Abdulmünim Âmir-Cemalettin eş-Şeyyâl, Kahire 1960, s. 317 14 Bkz. İbn Kesîr, el-Bidâye ve’n-Nihâye, Büyük İslâm Tarihi çev.: Mehmet Keskin, İstanbul 1995, c. IX, s. 71. Taberî, Haccâc’a karşı Basralıların Abdurrahman b. Abbas b. Rebia b. el-Hâris b. Abdulmuttalib komutasında savaştıklarını söylemektedir. Bkz. Taberî, c. VII, s. 240 15 Bkz. İbnu’l-Esîr, c. IV, s. 421 15 Bkz. İbnu’l-Esîr, c. IV, s. 421 16 Bkz. Yakubî, Tarih, c. II, s. 278; 16 Bkz. Yakubî, Tarih, c. II, s. 278; 17 Abdurrahman’ın askerî harcamalarının büyük bir kısmını, Irak’ın en zengin sınıfını oluştu- ran mevalî üstlenmiştir. Konu ile ilgili geniş bilgi için bkz. Belâzûrî, Kitâbu Cumel min Ensâbi’l-Eşrâf, tahk.: Süheyl Zekkâr-Riyâd Ziriklî, Beyrut 1996, c. VII, s. 389; Taberî, c. VII, ss. 277-278; el-Askerî, Kitâbu’l-Evâil, Beyrut 1987, ss. 231-232 18 Deyru Cemâcim savaşı ile ilgili geniş bilgi için bkz. Yakubî, Tarih, c. II, s. 278; Taberî, c. VII, s. 241 vd.; İbnu’l-Cevzî bu savaşın 100 gün sürdüğünü ve toplam 81 karşılaşmanın meydana geldiğini söylemektedir. Bkz. İbnu’l-Cevzî, Muntazam, c. IV, s. 383 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 151 Sicistan’a kaçmak zorunda kaldı, bir süre sonra da burada öldürüldü veya intihar etti.19 Sicistan’a kaçmak zorunda kaldı, bir süre sonra da burada öldürüldü veya intihar etti.19 Deyru Cemâcim’den sonra Irak’ın iki idarî merkezinden biri olan Kûfe’ye yerleşen Haccâc, kısa sürede divanlar oluşturarak muhaliflerini tespit ettirdi.20 Hayatta olanları teker teker getirterek, küfrünü ikrar etme- ğe zorladı; küfre girdiklerini kabul etmeyenleri kılıçtan geçirdi,21 savaşta esir edilenleri öldürdü, mallarına el koyarak taraftarları arasında bölüştür- dü.22 Hatta nakledildiğine göre Kûfe ile Basra’yı yıkmayı dahi düşündü.23 Bütün bunlar halkın tepkisine yol açtı. 19 Bkz. Dineverî, s. 320; Taberî, c. VII, s. 266; Mesudî, Murûc, c. III, s. 141; Tenbih, s. 287; İbnu’l-Cevzî, Muntazam, c. IV, s. 386 20 Bkz. Veki’, Ahbâru'l-Kudât, Beyrut, trs. , c. II, s. 408; İbn Kesîr, Bidâye, c. IX, s. 84 21 Bkz. Belazurî, Ensâb, c. VII, s. 391; Taberî, c. VII, s. 262; İbn Abdirabbih, c. II, s. 464 22 Mesudî, bu hadiseden haberdar olan Abdulmelik’în Haccâc’a mektup yazarak, öldürülmüş olan esirlerin diyetinin ödenmesini, malları müsadere edilenlerin zararlarının tazmin edil- mesini emrettiğini, ancak Haccâc’ın bu emre uymadığını söylemektedir. Bkz. Murûc, c. III, s. 141 23 Ömer b. Abulaziz (ö.101/720)’in ifadesine göre, Haccâc bu hadisenin bitiminden hemen sonra Kûfe ve Basra’yı yıkmayı, halkını geldikleri yere geri göndermeyi dahi düşündü. An- cak danışmanlarından Ebû Münebbih’in uyarısını dikkate alarak bu kararından vazgeçti. Bkz. İbnu’l-Fakîh, s. 260 24 İbnu’l-Fakîh ve Yâkût konu ile ilgili şunları söylemektedirler. “savaşın bitiminde Haccâc, Kûfe’ye yerleşti, fakat burada nefret ile karşılandı. Dolayısıyla güvendiği bir adamına yeni bir şehir kurması için uygun bir yer bulmasını emretti” Bkz. İbnu’l-Fakîh, 263; Yâkût, Bul- dân, c. V, s. 401 25 Haccâc’ın bu uygulaması daha sonra Türkistân illerinde yaygınlık kazanacaktır. 26 Bkz. Taberî, c. VII, s. 281; İbnu’l-Cevzî, Muntazam, c. IV, s. 387; İbnu’l-Esîr, c. IV, ss. 444-445 27 Bkz. Taberî, c. VII, s. 281 23 Ömer b. Abulaziz (ö.101/720)’in ifadesine göre, Haccâc bu hadisenin bitiminden hemen sonra Kûfe ve Basra’yı yıkmayı, halkını geldikleri yere geri göndermeyi dahi düşündü. An- cak danışmanlarından Ebû Münebbih’in uyarısını dikkate alarak bu kararından vazgeçti. Bkz. İbnu’l-Fakîh, s. 260 24 İbnu’l-Fakîh ve Yâkût konu ile ilgili şunları söylemektedirler. “savaşın bitiminde Haccâc, Kûfe’ye yerleşti, fakat burada nefret ile karşılandı. Dolayısıyla güvendiği bir adamına yeni bir şehir kurması için uygun bir yer bulmasını emretti” Bkz. İbnu’l-Fakîh, 263; Yâkût, Bul- dân, c. V, s. 401 25 Haccâc’ın bu uygulaması daha sonra Türkistân illerinde yaygınlık kazanacaktır. 19 Bkz. Dineverî, s. 320; Taberî, c. VII, s. 266; Mesudî, Murûc, c. III, s. 141; Tenbih, s. 287; İbnu’l-Cevzî, Muntazam, c. IV, s. 386 20 Bkz. Veki’, Ahbâru'l-Kudât, Beyrut, trs. , c. II, s. 408; İbn Kesîr, Bidâye, c. IX, s. 84 21 Bkz. Belazurî, Ensâb, c. VII, s. 391; Taberî, c. VII, s. 262; İbn Abdirabbih, c. II, s. 464 22 Mesudî, bu hadiseden haberdar olan Abdulmelik’în Haccâc’a mektup yazarak, öldürülmüş olan esirlerin diyetinin ödenmesini, malları müsadere edilenlerin zararlarının tazmin edil- mesini emrettiğini, ancak Haccâc’ın bu emre uymadığını söylemektedir. Bkz. Murûc, c. III, s. 141 Ö â 1. Vâsıt’ın Kuruluş Sebepleri Halk ona içten içe kin besler oldu.24 Kendisinden nefret edildiğini bildiği için de Kûfe’nin ancak Suriyeli askerler ile elde tutulabileceğine karar veren Haccâc, Abdurrahman hadisesinde onu desteklemek üzere gelmiş olan Suriyelileri, Kûfelilerin evlerine dağıt- tı.25 Kûfelilerin evlerine yerleştirilen Suriyeliler, içkiler içip ev ahalisine sarkıntılık ediyor, huzursuzluk çıkarıyorlardı. Nitekim bu hadiselerden biri- nin sonucunda evin sahibi, Suriyeli askerlerden birini öldürmüş, olay Haccâc’a intikal etmişti. Vakayı soruşturan Haccâc, hadisenin doğru oldu- ğunu anlamış ve Suriyeli asker için diyet ödenmesinin gerekmediğine hükmetmekle kalmamış, bu uygulamayı kaldırarak26 şehirde oluşmuş olan infialin önüne geçmeyi de hedeflemiştir. Taberî’nin ifadesine göre; Haccâc, Kûfe’de bir ay ikamet ettikten sonra, Suriyeli askerleri buradan çıkarıp şehrin dışında kurduğu karargâha yerleştirmiştir.27 Yaşanan bu hadiseler Haccâc’ın yeni bir şehir kurma düşüncesinin gelişmesini sağlamıştır. Şehrin kuruluşunu ele almadan önce, Vâsıt isminin kökeni ile ilgili tartışmalara kısaca değinmek istiyoruz. Kaynaklarımızın önemli bir kısmı anılan şehre bu adın Kûfe ve Basra ile eşit mesafede bulunmasından ötü- 25 Haccâc’ın bu uygulaması daha sonra Türkistân illerinde yaygınlık kazanacaktır. 26 Bkz. Taberî, c. VII, s. 281; İbnu’l-Cevzî, Muntazam, c. IV, s. 387; İbnu’l-Esîr, c. IV, ss. 444-445 27 Bkz. Taberî, c. VII, s. 281 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 152 rü verildiği kanaatindedirler.28 Bunlara göre şehre, söz konusu isim bizatihi Haccâc tarafından verilmiştir. Nitekim Haccâc, burayı kurduktan sonra Halife Abdulmelik b. Mervan’a gönderdiği mektupta Kûfe, Basra ile Cebel arasında yeni bir şehir kurduğunu ve adını Vâsıt koyduğunu ifade etmiş- tir.29 İbnu’l-Fakih ve Yakut’a göre, Haccâc, gönderdiği mektupta şu ifade- leri kullanmıştır. “Ben yer yüzünün ortasında Kûfe, Basra ile Cebel bölge- sine aynı mesafede bulunan bir şehir kurdum ve adını da Vâsıt koydum.”30 rü verildiği kanaatindedirler.28 Bunlara göre şehre, söz konusu isim bizatihi Haccâc tarafından verilmiştir. Nitekim Haccâc, burayı kurduktan sonra Halife Abdulmelik b. Mervan’a gönderdiği mektupta Kûfe, Basra ile Cebel arasında yeni bir şehir kurduğunu ve adını Vâsıt koyduğunu ifade etmiş- tir.29 İbnu’l-Fakih ve Yakut’a göre, Haccâc, gönderdiği mektupta şu ifade- leri kullanmıştır. “Ben yer yüzünün ortasında Kûfe, Basra ile Cebel bölge- sine aynı mesafede bulunan bir şehir kurdum ve adını da Vâsıt koydum.”30 Yukarıdaki görüşe katılmayan el-Vâsıtî ise, bu şehrin, adını Vâsıtu’l- Kasap isimli ve aynı yerde bulunan kadim bir kentten aldığı kanaatinde- dir.31 Nitekim o, Hz. Osman (ö.35/656)’ın Said b. el-As (ö.59/678-679)’ı Kûfe, Vâsıtu’l-Kasab ve Basra’ya vali olarak atadığını, Said’in Muaviye (ö.60/680) dönemine kadar bu görevde kaldığını, Muaviye, hilâfete geldiği zaman bu üç şehrin valiliği için Abdu’l-A’la b. 28 Bkz. Belazurî, Futûhu’l-Buldân, çev.: Mustafa Fayda, Ankara 1987, s. 415; Makdisî, s. 106; İbnu’l-Fakîh, s. 260; el-Bekrî, Mu’cem ma İsta’cem min Esmâi’l-Bilâd ve’l-Mevâdi’, tahk.: Mustafa es-Sakka, Beyrut 1983, cc. III/IV, s. 1363; Yâkût, Buldân, c. V, s. 400, el- Yafu’î, Mirâtu’l-Cinân ve İbretu’l-Yakzân, Kahire 1993, c. I, s. 194; Himyerî, Kitâbu’r- Ravdu’l-Mi’târ fi Haberi’l-Aktâr, tahk.: İhsân Abbâs, Beyrut 1980, s. 599 Yakubî’ye göre Vâsıt, Kûfe, Basra ve Ahvaz’a elli fersah mesafede olduğu gibi 148 yılında kurulmuş olan Bağdat’a da aynı mesafede idi. Bkz. Yakubî, Buldân, s. 101 28 Bkz. Belazurî, Futûhu’l-Buldân, çev.: Mustafa Fayda, Ankara 1987, s. 415; Makdisî, s. 106; İbnu’l-Fakîh, s. 260; el-Bekrî, Mu’cem ma İsta’cem min Esmâi’l-Bilâd ve’l-Mevâdi’, tahk.: Mustafa es-Sakka, Beyrut 1983, cc. III/IV, s. 1363; Yâkût, Buldân, c. V, s. 400, el- Yafu’î, Mirâtu’l-Cinân ve İbretu’l-Yakzân, Kahire 1993, c. I, s. 194; Himyerî, Kitâbu’r- Ravdu’l-Mi’târ fi Haberi’l-Aktâr, tahk.: İhsân Abbâs, Beyrut 1980, s. 599 Yakubî’ye göre Vâsıt, Kûfe, Basra ve Ahvaz’a elli fersah mesafede olduğu gibi 148 yılında kurulmuş olan Bağdat’a da aynı mesafede idi. Bkz. Yakubî, Buldân, s. 101 29 Himyerî, s. 599 30 İbnu’l-Fakîh, ss. 260, 262; Yâkût, Buldân, c. V, s. 401. Aynı bilgiler Belazurî’de de bulun- maktadır. Bkz. Belazurî, Futûh, ss. 415-416 31 Bkz. el-Vâsıtî, s. 38; Himyerî, s. 599 32 Bkz. el-Vâsıtî, ss. 35-36 33 Bkz. M. Mahfuz Söylemez, Bedevîlikten Hadarîliğe Kûfe, Ankara 2001, s. 177 34 İbn Sa'd, Tabakâtü'l- Kubrâ, Beyrut, 1957, c. V, s. 34; Halife, Tarih, s. 168. Mes'ûdî, Murûc, c. II, s. 345 34 İbn Sa'd, Tabakâtü'l- Kubrâ, Beyrut, 1957, c. V, s. 34; Halife, Tarih, s. 168. Mes'ûdî, Murûc, c. II, s. 345 30 İbnu’l-Fakîh, ss. 260, 262; Yâkût, Buldân, c. V, s. 401. Aynı bilgiler Belazurî’de de bulun- maktadır. Bkz. Belazurî, Futûh, ss. 415-416 38 Irakta kurulan bu şehir dışında birkaç Vâsıt daha vardır. Batnu Merr ve Vâdiu’n-Nahle bir yerleşim birimi, Belh’e bağlı bir köy, Habur’da Karkisya’nın yakınlarında bulunan bir köy, Bağdat’a üç fersah mesafede bulunan Duceyl’de bir başka köy, Hişam b. Abdulmelik tara- fından kurulmuş olan Vâsıtu’l-Rekka, Hille’nin yakınında Muteyrebâz’a bağlı bir köy, Mu- sul’a bağlı bir köy, Mekke’de bir bölge, Endülüs’te Kabra’ya bağlı bir yerleşim birimi, Necid’de bir bölge, Yemen’de Zebîd sahillerinde bulunan bir başka yerleşim yeri de bu i- simle anılmaktaydı. Geniş bilgi için bkz. Yâkût, Buldân, Vâsıt maddesi 1. Vâsıt’ın Kuruluş Sebepleri Abdullah’ı görevlendirdiğini, Abdulmelik b. Mervan’ın ise bu üç kentin valiliğine Haccâc’ı getirdiğini söy- lemektedir.32 Görüldüğü gibi el-Vâsıtî’nin bu rivayetine göre Vâsıtu’l-Kasab zaten eski bir yerleşim birimidir. Dahası Kûfe ve Basra ile birlikte anılacak kadar da önemli bir şehirdir. Ancak rivayet yakından incelendiğinde bir çok yanlışı bir arada barındırdığı görülecektir. Birincisi, Hz. Ömer ve Hz. Os- man döneminde Irak’ın Kûfe bölgesinden fethedilen yerleri Kûfe’den, Bas- ra bölgesinden fethedilen yerleri ise Basra’dan yönetilmekteydi. Dolayısıyla Kûfe ve Basra’nın yönetimleri hiçbir zaman birleştirilmemiştir.33 İkincisi: Said b. el-As Hz. Osman tarafından Kûfe valiliğine atanmış ve yine onun tarafından görevden alınıp yerine de Ebû Musa el-Eş’arî getirilmiştir.34 Ü- çüncüsü: Hz. Osman’dan sonra hilâfete Hz. Ali gelmiş, o da burayı merkez edinmiştir. Said’in Hz. Ali ile beraber Kûfe’yi yönetmesinin mümkün olma- dığı gerçeğini düşündüğümüzde rivayetin geçersizliği zaten ortaya çık- maktadır. Bu kadar açık bilgilere rağmen el-Vâsıtî’nin neden böyle bir kanaat belirttiğine gelince; Vâsıt, Dicle’nin batı yakasında, Sasanîlerin Suristan yani Orta Mezopotamya bölgesindeki önemli idarî merkezlerden biri olan Çorum İlahiyat Fakültesi Dergisi, 2002/2 153 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine kadim Kesker kentinin tam karşısında kurulmuştu.35 Doğudaki Kesker ile Batıdaki Vâsıt’ı birbirinden sadece Dicle nehri ayırmaktaydı. Haccâc, Vâsıt’ı kurduktan bir süre sonra bu iki şehri birbirine bağlayan bir köprü inşa etti. Böylece Kesker ile Vâsıt kısa bir süre sonra tek bir şehir haline geldiler. Nitekim hicri dördüncü asır coğrafyacılarından Makdisî burayı bir tek şehir saymakta ve şöyle demektedir: “Vâsıt büyük bir yerleşim yeridir. İki cami- si bulunmaktadır. Dicle’nin her iki tarafını da kapsamaktadır. Şehrin iki yakası arasında ise köprü mevcuttur.”36 İbn Havkal ve Himyerî de Vâsıt’ın Kesker ile tek şehir haline geldiğini, şehrin hem doğu ve hem de batı ya- kasında cuma namazının kılındığı camilere sahip olduğunu ifade etmişler- dir.37 Bu iki şehrin birleşmesinden bir süre sonra Kesker ismi unutulmuş Dicle’nin doğu yakasındaki şehir de, batı yakası ile birlikte “Vâsıt” olarak anılmıştır. Vâsıt kentinin inşa edilmiş olduğu bu dönemde, İslâm âleminde muhtelif yerlerinde bu isme sahip başka yerleşim birimleri de bulunmak- taydı.38 Dolayısıyla Haccâc tarafından kurulmuş olan bu şehri söz konusu yerleşim birimlerinden ayırmak icap ediyordu. Bunun için de buraya Irak’ın Vâsıt’ı veya Haccâc’ın Vâsıt’ı denmesinin yanı sıra kurulduğu yerden ötürü Vâsıtu’l-Kasab dendiği de bilinmektedir.39 Dolayısıyla el-Vâsıtî’nin belirttiği kadim kent Kesker şehrinden başka bir yer değildir. Bu kent de yukarıda da ifade ettiğimiz gibi Vâsıt’ın kurulduğu yerde olmayıp Dicle nehrinin öte tarafında bulunmaktaydı. 39 Bkz. Belazurî, Futûh, s. 415; Bkz. İbnu’l-Fakîh, s. 263; Yâkût, Buldân, c. V, s. 400; Himyerî, s. 599 41 Bkz. el-Vâsıtî, Tarihu Vâsıt, tahk.: Korkis Avvâd, Beyrut 1986, s. 38 36 Makdisî, Ahsenu’t-Tekâsim fi Ma’rifeti’l-Ekâlim, tahk.: Muhammed Mahzûm, Beyrut 1987, s. 106 1. Vâsıt’ın Kuruluş Sebepleri el-Vâsıtî, yaklaşık olarak bir asır sonraya ait olan bu kullanımı daha erken bir döneme yerleştirmiştir. 35 Yakubî, Buldân, s. 101; Selman el-Muaddidî, “Hitatu Medineti Vâsıt fi Asri’l-Abbasî”, Sü- mer, c. XXXIV, sayı: 1-2, Bağdat 1978, s. 181; Beşir Yusuf Fransis, s. 106 40 Bkz. İbnu’l-Fakîh, s. 263; Yâkût, Buldân, c. V, s. 401 37 Bkz.İbn Havkal (ö.367/977), Suretu’l-Arz, Beyrut trs. , s. 214; Himyerî, s. 599 40 Bkz. İbnu’l-Fakîh, s. 263; Yâkût, Buldân, c. V, s. 401 42 İbnu’l-Fakîh ile Yâkût, Asmaî’ye dayandırdıkları bir haberde şöyle demektedirler. “Haccâc bir şehir kurmak istediğinde, yer seçmeleri amacıyla yetkililer gönderdi. Bunlar Aynu’t- Temr ile körfez arasını baştanbaşa dolaştıktan sonra, Haccâc’a döndüler ve kendisine ‘Ha- vasının hafifliğinden ve çölün girişinde olmasından dolayı senin seçtiğin bu bölgeden daha iyi bir yer bulamadık’ dediler.” Bkz. İbnu’l-Fakîh, s. 262; Yâkût, Buldân, c. V, s. 401 46 Yakubî, şehrin h 82, Taberî, İbn Fakîh, İbnu’l-Esîr, İbnu’l-Kesîr, İbnu’l-İbrî, İbn Verdî, ve Yâkût ise 83 yılında kurulduğu kanaatindedirler. Bkz. Yakubî, Tarih, c. II, s. 279; İbnu’l- Fakîh, s. 264; İbn İberî, s. 113; İbnu’l-Verdî, c. I, s. 240; Yâkût, Buldân, c. V, s. 401 45 Haccâc şehir yeri olarak ilkin yine Kesker şehrine oldukça yakın olan es-Sin bölgesini seçmiş, çevrede bir takım çalışmalar dahi başlatmış, kanallar kazdırmıştı. Ancak çevreyi incelemek üzere gönderdiği uzmanların Vâsıt’ın kurulduğu yeri tavsiye etmeleri üzerine, buradan vazgeçmiştir. Bkz. Belazurî, Futûh, s. 416; İbnu’l-Fakîh, s. 262; Yâkût, Buldân, c. V, s. 401 44 Bkz. Yakubî, Vâsıt ile Basra, Bağdat ve Musul arasında gemilerin gidip geldiklerini söyle- mektedir. Bkz. Yakubî, s. 38 47 Kazvinî, Asâru’l-Bilâd ve Ahbâru’l-İbâd, Beyrut trs. , s. 478 43 Bkz. M. Mahfuz Söylemez, s. 177 43 Bkz. M. Mahfuz Söylemez, s. 177 2. Kentin Kurulduğu Yer ve Kuruluş Tarihi Yukarıda kısaca zikrettiğimiz siyasî hadiselerden sonra Haccâc, yeni bir şehir kurmaya karar verince, şehrin kurulacağı yeri tespit etmek üzere görevliler tayin etti. Bunlara, inşa edilecek olan şehrin nehir kenarında olmasına,40 hatta Kûfe ve Basra’nın ortasında bulunmasına dikkat etmele- rini özellikle belirtti.41 İbnu’l-Fakih ve Yâkût geniş bir coğrafya taranarak Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 154 Vâsıt’ın kurulacağı yere karar verildiğini belirtiyorlarsa da,42 şehrin inşası için Kûfe ile Basra’nın -o dönemdeki- idarî sınırının seçilmiş olması, tayin edilen görevlilerin Haccâc’ın emirlerine sıkı sıkıya uyduklarını ortaya koy- maktadır. Bilindiği gibi Kûfe Sevâdı denilen ve buradan yönetilen bölge Vâsıt’ın kurulduğu yerden başlayıp Musul’un Cuha bölgesine kadar uzanır- ken, güneyinden körfez sahiline kadar uzanan bölge ise Basra’dan yöne- tilmekte idi.43 Ayrıca Vâsıt’ın kurulacağı bu bölge, nehir taşımacılığının en rahat yapılabileceği bir noktada olmasının yanında,44 ticaret güzergahı üzerinde de yer alacağı için stratejik bir öneme de sahipti. Keza Huzistan, Cebel ve Horasan’ı, Irak’ın eş idarî merkezleri olan Kûfe ve Basra’ya bağ- layan yolların geçtiği kavşakta da bulunuyordu.45 Vâsıt’ın isminde olduğu gibi kuruluş tarihi hakkında da ihtilâflar bu- lunmaktadır. Bahşel el-Vâsıtî dışındaki kaynaklar şehrin hicri 82 veya 83 yılında kurulduğunu söylerken,46 muahhar kaynaklardan Kazvinî ise 84 yılında kurulduğunu iddia etmektedir.47 Bu farklılığın sebebi şehrin 82 yılı- nın son aylarında planlanmış olması ve 83 yılının ilk aylarında ise inşaata başlanmasıdır. Dolayısıyla kimi kaynaklar “82 yılında kuruldu” derken, diğerleri ise 83 yılında kurulduğunu söylemektedirler. Kazvinî de bu iddia- sında yalnız kalmaktadır. İddiasında yalnız kalanlardan bir başkası ise el- Vâsıtî’dir. el-Vâsıtî, Haccâc’ın şehrin inşasına 75 yılında başladığını ve 78 yılında bitirdiğini ileri sürmektedir.48 Ancak onun bu görüşü, yukarıda da belirttiğimiz gibi- hiçbir kaynak tarafından desteklenmemiştir. Dahası, erken dönem İslâm kaynaklarının tamamına yakınında önemli bir yer tut- muş olan, yukarıda da özet bir şekilde sunduğumuz, Abdurrahman hadise- si ile ilgili verilen bilgilerin hiçbirinde Vâsıt’tan bahsedilmiyor olması, bu hadisenin cereyan ettiği hicri 80-82 yıllarında adı geçen şehrin var olmadı- ğını katiyetle ortaya koymaktadır. 48 Bkz. el-Vâsıtî, s. 38 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 155 Şehrin bitirildiği tarih ise 86 yılı olarak kaydedilmektedir.49 Kanaa- timize göre bu tarih, şehrin bitirildiği zamandan ziyade yerleşilmeye baş- lanma tarihini ifade etmektedir. Nitekim inşaata harcanan para ile ilgili bilgi veren kaynaklar Irak’ın 5 yıllık gelirinin buraya harcandığını söyle- mektedirler. Bu da şehir inşaatının 5 yıl sürmüş olduğunu göstermektedir. 49 Bkz. İbnu’l-Fakîh, s. 264; Beşir Yusuf Fransis, s. 106 50 Bkz. el-Vâsıtî, s. 38 51 Yâkût burada en-Nil ve ez-Zab adında iki kanalın kazıldığını söylemektedir. Bkz. Yâkût, Buldân, s. 401 52 Bkz. İbnu’l-Fakîh, s. 269 53 Bkz. Belazurî, Futûh, s. 416 54 Kûfe’de pazarlar Fırat nehri ile Cuma Camii’nin arasında, bir başka ifade ile merkezi unsur- ların doğusunda iken bu unsurlar Vâsıt’da da aynı yerde konumlandırılmıştır. 2. Kentin Kurulduğu Yer ve Kuruluş Tarihi Bu bilgilerden, Vâsıt’ın, 86 yılında iskâna açılmış olmasına rağmen, inşaa- tın daha sonra da devam ettiği sonucunu çıkarmak mümkündür. 49 Bkz. İbnu’l-Fakîh, s. 264; Beşir Yusuf Fransis, s. 106 53 Bkz. Belazurî, Futûh, s. 416 55 Konu ile ilgili geniş bilgi için bkz. Belazurî, Futûh, s. 416; İbnu’l-Fakîh, s. 263; Yâkût, Buldân, c. V, s. 402 56 Himyerî, s. 599 57 Bkz. Yâkût, Buldân, c. V, s. 402 58 Bkz. İbnu’l-Fakîh, s. 262 59 Bkz. Muaddîdî, ss. 182-183. Âdil Necm ise rakamlardaki küsuratı tümleyerek caminin ebatlarının 104x104 metre olduğunu söylemektedir. Bkz. Âdil Necm “el-Muduni’l-İslâmiyye fi Karni’l-Evveli’l-Hicri”, Âdâbu’r-Rafidîn, Musul 1981, c. XIII, s. 600 60 Bkz.Âdil Necm, s. 600 61 Yılmaz Can, İslâm Şehirlerinin Fiziki Yapısı, Ankara 1995, s. 110 3. Şehrin Kuruluşu Şehir yeri tespit edildikten sonra, kurulacağı alan Dâverdân adındaki dihkandan 10.000 dirheme satın alındı.50 Büyük bir kısmı bataklık ve sazlık olan bölgenin yerleşime müsait hale getirilmesi için büyük gayretler sarfedildi. Önce çevrede geniş kanallar açılmak suretiyle bataklıklar kuru- tuldu, arkasından çevre düzenlemesi yapılarak yeşillendirildi. Dicle’den açılan kanallar ile içme ve sulama suyu tedarik edildi.51 Şehrin mimarlığını, kimliğini tam olarak tespit edemediğimiz, Zubiâ adında birinin yaptığı söylenmektedir.52 Aynı belirsizlik inşaatta çalıştırıldığı ifade edilen işçiler için de geçerlidir. Nitekim kaynaklarımızda bu işçilerin nereli oldukları ve nereden getirildikleri ile ilgili bilgilere rastlanmamakta- dır. Sadece Belâzurî’de bunlar ile ilgili bir bilgi bulunmaktadır. Onun verdiği bilgi de “kaçmamaları için birbirlerine zincirlerle bağlandıkları”ndan iba- rettir.53 Fırat’ın kenarına kurulmuş olan Kûfe ile Dicle’nin kenarına kurul- muş olan Vâsıt’ın merkezî unsurlarındaki benzerlik bir yana, bu unsurların bulunduğu yerlerin dahi aynı olması gerek mimarın gerekse inşaatta görev almış olanların önemli bir kısmının Kûfeli olma ihtimalini akla getirmekte- dir.54 Haccâc’ın şehir yerini tespit etmek için Kûfe’den görevliler göndermiş olması da bu ihtimali kuvvetli bir hale getirmektedir. Tipik bir İslâm kenti olarak tasarlanmış olan Vâsıt’ın merkezinde cami, hükümet konağı, pazar ve meydan yer alıyordu. Diğer İslâm kentle- rinde olduğu gibi yolların tamamı bu merkeze açılmaktaydı. Şehir, merkezî unsurlardan başlanarak inşa edilmiştir. Önce cami ve hükümet sarayı olan Kubbetu’l-Hadra ile şehri kuşatan surlar ve surları kuşatan hendekler ya- pılmıştır. Şehrin inşasında kullanılan malzemenin önemli bir kısmı o çevreden elde edilmiş olmasına rağmen, ahşap malzeme, özellikle de saray ve bü- yük caminin kapıları Sevâd’ın Zendeverd, Derkara, Daru Vesât, Deyru Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 156 Masircesân ve Şerâbit gibi şehirlerinden getirilmiştir. Rızası alınmadan, zorla ellerinden alınan bu malzemeden dolayı, halkın tepki gösterdiği ve canları ile mallarına eman verilmiş olduğu halde yapılan bu hukuksuz uy- gulamayı şikayet ettikleri, ancak Haccâc’ın bu tepkiyi ve şikayeti hiç ciddi- ye almadığı kaydedilmektedir.55 a. Mescit: Vâsıt’ın fizikî yapısına gelince; yukarıda da ifade ettiğimiz gibi şehir, merkeze mescit alınarak inşa edilmiştir. Başka bir ifade ile şehri oluşturan unsurlar mescit esas alınarak konumlandırılmıştır. Dolayısıyla şehir merke- zînin kuşkusuz en önemli unsurunu mescit oluşturmuştur. Kaynaklarımızda zaman zaman Mescidu Haccâc olarak da isimlendirilen bu mabed56 ile ilgili ayrıntılı bilgiye sahibiz. Oldukça geniş olarak inşa edilmiş olan mescit, 200x200 zira’ idi.57 Mescidin içinde büyükçe bir de maksure mevcuttu. İbnu’l-Fakih, el-Vedâh b. Ata’dan aktararak bu maksurenin içerisinde 40 kişinin rahatlıkla gizlenebildiğini söylemektedir.58 İslâm döneminin erken mimari örneklerinden biri olan bu mescidin kalıntıları günümüze kadar gelmiştir. Irak Arkeoloji Müdürlüğü (Müdüriyyetu’l-Âsâri’l-Ammeti’l- Irakiyye) tarafından burada yapılan kazılarda temelleri ortaya çıkarılmıştır. Buna göre mescit kare biçimindeydi, güney ve kuzey uzunluğu 104 metre 30 cm., batı ve doğu uzunluğu ise 103 metre 30 cm. idi, yapı malzemesi olarak tuğla ve sarı kils kullanılmıştı.59 Duvarların kalınlığı 2.5 metre idi. Âdil Necm, burası ile Kûfe ve Basra mescitleri arasında bir benzerlik kur- maktadır. Hatta kullanılan malzemenin dahi aynı olduğu ifade edilmekte- dir.60 Bu durum, Vâsıt Kûfe örnek alınarak inşa edilmiş olduğu şeklindeki yukarıdaki kanaatimizi destekler mahiyettedir. 61 Yılmaz Can, İslâm Şehirlerinin Fiziki Yapısı, Ankara 1995, s. 110 b. Dâru’l-İmâre Vâsıt’ın ikinci merkezi unsuru Dâru’l-İmâre’dir. Dâru’l-İmâre ile mescid birlikteliği hemen hemen her İslâm şehrinde görülmektedir.61 Bu bütünlüğün arkasında İslâm kültüründeki din ve dünya birlikteliğini simge- leyen sembolik anlam yatmaktadır. Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 157 Sahip olduğu yeşil kubbeden dolayı kimi kaynaklarımızda Kubbetu’l- Hadra olarak da isimlendirilmekte olan Vâsıt’ın Dâru’l-İmâre’si, Muaviye’nin Şam’daki sarayı örnek alınarak inşa edilmiştir. Bu durum az da olsa Suriye mimarî anlayışının da Vâsıt’ı etkilediğini göstermektedir. 400x400 zira’, yani yaklaşık olarak 200 metre kare genişliğinde olan bu bina, dönemin en büyük yapılarından biri olarak kaydedilmektedir.62 Nite- kim Dâru’l-İmâre neredeyse mescidin iki katı büyüklüğündedir. İslâm mi- marî tarihinde önemli bir kırılma olan bu durum, Emevîlerin din anlayışın- daki farklılığı göstermekte ve simgesel olarak bu hanedanın dünyayı dine öncelediğini de gözler önüne sermektedir. Zira Ka’be ile ilgili fıkhî tartış- maların tamamında görüldüğü gibi, İslâm aleminde Emevîlere gelinceye kadar sivil yapıların mabedlerden daha yüksek ve daha ihtişamlı yapılma- masına özen gösterilmiştir. Hatta bunu günah olarak telakki edenler de bulunmaktaydı. Söz konusu gelenek ilk defa Haccâc tarafından kurulan bu bina ile yıkılmıştır. Tarihi süreçte sarayların mescitlerden daha büyük ve ihtişamlı yapılması Abbasîler döneminde kurulmuş olan Bağdat ve Samarra da doruk noktasına ulaşmıştır.63 Bağdat’ın kuruluşu esnasında Mansur’un sarayına da örneklik etmiş olan bu muhteşem yapıdan günümüze bazı bölümler ulaşmıştır. 1936 yı- lından itibaren Irak Arkeoloji Müdürlüğü tarafından yapılan kazılarda te- mellerinin bir kısmı ortaya çıkarılmıştır. Bu temeller Dâru’l-İmâre’nin bü- tününü aydınlatmaya imkân vermese de büyüklüğünü tespit etme imkânı sağlamaktadır. Buna göre saray 203x203 metre idi.64 Binaya adını veren kubbenin yapıdaki yerinin tercih edilen görüşe göre binanın tam ortasında idi.65 62 Yâkût, Buldân, c. V, s. 402 63 Bkz. Âdil Necm, s. 599 64 Beşir Yusuf Fransis, s. 107; Âdil Necm, s. 600 65 Muaddîdî, s. 185 66 Kûfe örneği için bkz. M. Mahfuz Söylemez, ss. 53-55 c. Meydan Şehir merkezinin üçüncü temel unsuru ise meydandır. Yakut’un ver- diği bilgilerden anladığımıza göre Vâsıt meydanı, sarayı kuşatır bir şekilde düşünülmüştür. Meydanın Dâru’l-İmâre’yi kuşatır bir şekilde düşünülmüş olması bu şehri daha önce kurulmuş olan İslâm kentlerinden ayıran bir başka özelliktir.66 Nitekim Kûfe, Basra ve Fustat’ta Dâru’l-İmârelerin mey- dana açılan tek ana giriş kapısı bulunmakta idi. Vâsıt’ta ise saraya, her biri ayrı bir alana açılan dört giriş kapısı düşünülmüştür. Bu da meydanın mes- cit ve Dâru’l-İmâre’yi kuşatmasına sebep olmuştur. Yakut, meydanın ge- nişliğini de vermektedir. Onun aktardığı bilgilere göre meydanın, demirci- ler çarşısına uzanan kısmı 300x300 zira’, kasaplar çarşısına uzanan kısmı Çorum İlahiyat Fakültesi Dergisi, 2002/2 158 Yrd. Doç. Dr. M. Mahfuz Söylemez 300x330 zira’,67 atların eğitilmekte olan bölgeye uzanan kısmı 200x200 zira’,68 ipekçiler ile Havuza kadar uzanan bölümü ise 300x300 zira’ idi.69 300x330 zira’,67 atların eğitilmekte olan bölgeye uzanan kısmı 200x200 zira’,68 ipekçiler ile Havuza kadar uzanan bölümü ise 300x300 zira’ idi.69 d. Pazar/Çarşı Vâsıt kurulurken pazar yeri de merkezî unsurun olmazsa olmazı olan mescit, saray ve meydanın bir parçası olarak düşünülmüş, bu temel un- surların doğusuna yani şehrin Kesker tarafına yerleştirilmişti.70 Bu pazar ile ilgili en geniş bilgiler el-Vâsıtî tarafından aktarılmaktadır. Onun verdiği bilgiye göre gıda satıcıları, bezzâzlar ve attârlar çarşısı, pazarın sağ tara- fında, depocuların olduğu bölgeye kadar uzanıyordu. Bakkallar, sakatatçı- lar, meyve satıcıları çarşının güney tarafında, depocular sokağına kadar olan bölgeye yerleşmişlerdi. Depocular çarşısında ise esnafın mallarının korunmakta olduğu dükkanların yanı sıra gündelikçi olarak çalışmakta olan işçilerin bulundukları yerler ile zanaat erbabı olan esnaf da yerleşmişti. Bunların çalıştıkları işyerleri Dicle nehrine kadar uzanmaktaydı.71 Daha önce kurulmuş olan kentlerde olduğu gibi burada da her meslek erbabına ayrı ayrı yerler tahsis edilmiş, bunların birbirlerine karışmalarına müsaade edilmemiştir. Rivayetlere göre; Haccâc bu konu üzerinde titizlikle durmak- taydı. Bunun tek istisnası ise sarraflardır. Sarrafların hepsi bir yerde top- lanmamıştı. Haccâc’ın emri gereğince her çarşıda bir sarraf bulunmaktay- dı.72 Dönemin sarraflarının aynı zamanda senet tahsili yaptıkları yani gü- nümüzdeki bankalara benzer bir işlev yüklenmiş oldukları dikkate alındı- ğında bunun sebebi kendiliğinden ortaya çıkmaktadır. 67 Yâkût, Buldân, c. V, s. 402 68 Bkz. Yâkût, Buldân, c. V, s. 402 69 İbnu’l-Fakîh, s. 265 70 Bkz. Makdisî, s. 106 71 el-Vâsıtî, s. 39. Ayrıca bkz. Âdil Necm, s. 598 72 el-Vâsıtî, s. 39. İbnu’l-Fakîh’de de, eksik olmakla beraber, benzer bilgiler bulunmaktadır. Bkz. s. 265 73 el-Vâsıtî, s. 39 74 Massignon, Hitatu’l-Kûfe ve Şerhu Haritâtuhâ, Arapça’ya çev.: Taki Muhammed el- Misba’î, tahk.: Kamil Süleyman el-Cebburî, Necef, 1979, s. 66 74 Massignon, Hitatu’l-Kûfe ve Şerhu Haritâtuhâ, Arapça’ya çev.: Taki Muhammed el- Misba’î, tahk.: Kamil Süleyman el-Cebburî, Necef, 1979, s. 66 75 Muaddîdî, s. 186 76 İbn Kuteybe, Uyûnu’l-Ahbâr, tahk.: Ahmed Zeki el-Adevî, Kahire 1930, c. III, s. 250 77 Bkz. Makdisî, s. 106 78 Bkz. M. Stcek, “Vâsıt”, İA, İstanbul 1993, c. XIII, s. 222 79 Muaddîdî, s. 183 80 Bkz. Benjamin of Tudele, Rihletu Bünyamin, İbranice’den Arapça’ya tcr. Azra Haddad, Bağdat 1346/1945, s. 149 f. İskele Vâsıt kentinin hem Haccâc tarafından inşa edilmiş olan batı kısmın- da hem de sonradan burayla bütünleşmiş olan Kesker’de gemilerin yanaş- tıkları iki iskele bulunmaktaydı.77 Daha sonraları burada bir de tersanenin kurulduğu bilinmektedir. Dicle nehrinde kullanılmak üzere bu tersanede inşa edilmiş olan gemiler büyük bir üne sahipti. Bu gün hâlâ Irak’ta bu tür gemilere Vâsıtiyye deniliyor olması bunun en büyük delili olarak değerlen- dirilmektedir.78 e. Cadde ve Sokaklar Vâsıt’tın cadde ve sokakları hakkında ayrıntılı bilgiye sahip değiliz. Bu konu ile ilgili sadece el-Vâsıtî’de bazı bilgiler yer almaktadır. Onun ver- diği bilgilere göre Haccâc, sarayını inşa edince buraya dört de kapı yaptır- mış, bu kapıların açıldığı ana caddeler inşa etmiştir. Yapılmış olan bu cad- delerin genişliği seksen zira’ yani yaklaşık kırk metre idi.73 Bu caddelere daha küçük cadde, sokak veya yollar açılmak suretiyle, dörde bölünmüş olan daire tamamlanıyordu. Kendisinden yarım asır önce inşa edilmiş olan Kûfe’nin ana caddelerinin geceleri aydınlatılmış olması,74 Vâsıt caddelerinin de aydınlatılmış olabileceğini düşündürmektedir. 67 Yâkût, Buldân, c. V, s. 402 68 Bkz. Yâkût, Buldân, c. V, s. 402 69 İbnu’l-Fakîh, s. 265 70 Bkz. Makdisî, s. 106 71 el-Vâsıtî, s. 39. Ayrıca bkz. Âdil Necm, s. 598 72 el-Vâsıtî, s. 39. İbnu’l-Fakîh’de de, eksik olmakla beraber, benzer bilgiler bulunmaktadır. Bkz. s. 265 73 el-Vâsıtî, s. 39 74 Massignon, Hitatu’l-Kûfe ve Şerhu Haritâtuhâ, Arapça’ya çev.: Taki Muhammed el- Misba’î, tahk.: Kamil Süleyman el-Cebburî, Necef, 1979, s. 66 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 159 Vâsıt’ın caddelerinden tespit edilebilenler şunlardır: Sikketu’l-Arab, Sikketu Muhammed b. Halid, Sikketu Berîd, Sikketu Ehli Buhara, Derbu’l- Havd, Derbu’l-Harbiyye, Derbu’l-Hatîb, Derbu’d-Divân, Derbu’ş-Şa’ranî, Derbu’s-Sâğe, Derbu Hazzazîn. Bu caddeler içerisinde en büyüğü Derbu Hazzazîn olup cami ve Daru’l-İmâreden başlayıp büyük çarşının güney batısına uzanır, sonra pazarın güney doğusuna dönerdi, oradan da Dicle nehrine kadar uzanırdı.75 Bu caddelerden bir başkası olan Duşâb sokağı ise muhtemelen pazara yakın bir yerde bulunuyordu.76 Caddelerden bazıları oraya yerleşenlerin isimleri, kimisi ise şahıs i- simleri ile anılmıştır. Buhara kökenli Türklerin adıyla anılan bir caddenin olması bu kitlenin toplu olarak adı anılan cadde çevresinde oturduklarını göstermektedir. g. Kilise ve Havralar Şehrin kuruluşu esnasında buraya Yahudi ve Hristiyanların yerleş- mesine izin verilmediği için ilk zamanlarda şehirde havra ve kilise de bu- lunmamaktaydı. Kaynaklarımızda varlığına işaret edilen havra ve kiliseleri değerlendiren Muaddidî, gayr-i müslimlere ait bulunan mabetlerin Abbasî- ler döneminde inşa edilmiş olduğu sonucuna varmaktadır.79 Zamanla Vâsıt’a önemli bir Yahudî ve Hristiyan kitlenin yerleştiği de bilinmektedir. Nitekim Endülüslü ünlü Seyyah Bünyamin Tuleytulî’nin buraya 566/1170 yılında yaptığı seyahat esnasında şehirde 10.000 civarında Yahudi nüfus barınmaktaydı. Cemaatin başında da R.Nidân adında bir Yahudî bulunu- yordu.80 Hristiyan nüfusun ise bunun birkaç katı olduğu tahmin edilmekte- dir. Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 160 h. Mezarlık Vâsıt’ı Hz. Ömer (ö.23/644) döneminde inşa edilen diğer garnizon kentlerden ayıran bir başka özelliği de mezarlığıdır. Diğer askerî kentlerde, özellikle Kûfe’de, her mahalle müstakil bir şehir olarak düşünülmüş ve mahallelerin merkezine adına cibâne denilen ve aynı zamanda meydan olarak da kullanılıp önemli günlerde toplanılan mezarlıklar yapılmıştı.81 Bu durum toplumsal ayrışmayı daha da keskin hale getiriyordu. Vâsıt’ta ise tek bir mezarlık bulunmaktaydı.82 Hangi kabile veya boya mensup olursa olsun herkes cenazesini aynı mezarlığa gömmek durumunda idi. Şehirde tek mezarlığın varlığı etnisiteler arasındaki kaynaşmanın önemli bir gös- tergesidir. I. Köprü Dicle nehrinin Vâsıt ile Kesker’i ikiye böldüğünü yukarıda söylemiş- tik.83 Bu iki kent arasıda ilk köprü bizzat Haccâc tarafından inşa edilmiş- tir.84 Bu ilk köprünün asma köprü şeklinde olduğu rivayet edilmektedir.85 Muaddîdî’nin tespitine göre söz konusu asma köprünün uzunluğu 680 zira’ yani 340 metre civarında idi.86 Vâsıt’ta yapılan kazılardan anlaşıldığına göre daha sonraki dönemde taştan bir köprü de inşa edilmiştir.87 Ancak bu köprünün mimarî özellikleri hakkında maalesef yeterli bilgiye sahip değiliz. 89 Bkz. Kazvinî, s. 100. Kalkaşandî ise burada 50.000 kişinin bulunduğunu söylemektedir. [Bkz. Kalkaşandî, Measiru’l-İnâfe fi Meâlimi’l-Hilâfe, tahk.: Abdussetar Ahmed Ferâc, Bey- rut trs. , s. 137]. İbnu’l-İbrî ise bunların 80.000 kişi olduklarını söylemektedir. Bkz. İbnu’l- İbrî, s. 113 90 Sind bölgesinde yakalanan Muhammed b. Kasım dahi buraya getirilip hapsedilmişti. Ka- sım’ın hapsedilirken aşağıdaki şiiri söylediği rivayet edilmektedir. Ben Vâsıt’ta demirlerin Çorum İlahiyat Fakültesi Dergisi, 2002/2 88 Bkz. İbn Kuteybe, el-Mearif, tahk.:Servet Ukkaşe, Mısır 1992, s. 339 81 Bkz. M. Mahfuz Söylemez, s. 70 82 el-Vâsıtî, s. 39 83 Bkz. Kazvinî, s. 478 84 el-Vâsıtî, s. 39 85 Bkz. Yakubî, Buldân, s. 101; İbn Havkal, s. 214; Himyerî, s. 599 86 Muaddîdî, s. 189 87 Bkz. Beşir Yusuf Fransis, s. 107 88 Bkz. İbn Kuteybe, el-Mearif, tahk.:Servet Ukkaşe, Mısır 1992, s. 339 89 Bkz. Kazvinî, s. 100. Kalkaşandî ise burada 50.000 kişinin bulunduğunu söylemektedir. [Bkz. Kalkaşandî, Measiru’l-İnâfe fi Meâlimi’l-Hilâfe, tahk.: Abdussetar Ahmed Ferâc, Bey- rut trs. , s. 137]. İbnu’l-İbrî ise bunların 80.000 kişi olduklarını söylemektedir. Bkz. İbnu’l- İbrî, s. 113 90 Sind bölgesinde yakalanan Muhammed b. Kasım dahi buraya getirilip hapsedilmişti. Ka- sım’ın hapsedilirken aşağıdaki şiiri söylediği rivayet edilmektedir Ben Vâsıt’ta demirlerin j. Hapishane İlk kurulduğunda Vâsıt’ın ed-Dimas adında bir tek hapishanesi bu- lunmaktaydı. Dönemin en ünlü ve de en büyük hapishanelerinden biri olarak zikredilmekte olan bu binadan88 Haccâc’ın ölümünden sonra 33.000 kişinin salıverildiği, hemen hemen her kaynakta yer almaktadır.89 Bu ra- kamı abartılı bulsak da hapishanenin büyüklüğü hakkında bir fikir verdiğini söyleyebiliriz. Siyasî mahpusların yerleştirildiği bir eyalet hapishanesi şek- linde olduğu anlaşılan ed-Dimâs’ın,90 büyüklüğü kadar korunaklı oluşundan da bahsedilmektedir.91 81 Bkz. M. Mahfuz Söylemez, s. 70 82 el-Vâsıtî, s. 39 83 Bkz. Kazvinî, s. 478 84 el-Vâsıtî, s. 39 85 Bkz. Yakubî, Buldân, s. 101; İbn Havkal, s. 214; Himyerî, s. 599 86 Muaddîdî, s. 189 87 Bkz. Beşir Yusuf Fransis, s. 107 88 Bkz. İbn Kuteybe, el-Mearif, tahk.:Servet Ukkaşe, Mısır 1992, s. 339 89 Bkz. Kazvinî, s. 100. Kalkaşandî ise burada 50.000 kişinin bulunduğunu söylemektedir. [Bkz. Kalkaşandî, Measiru’l-İnâfe fi Meâlimi’l-Hilâfe, tahk.: Abdussetar Ahmed Ferâc, Bey- rut trs. , s. 137]. İbnu’l-İbrî ise bunların 80.000 kişi olduklarını söylemektedir. Bkz. İbnu’l- İbrî, s. 113 90 Sind bölgesinde yakalanan Muhammed b. Kasım dahi buraya getirilip hapsedilmişti. Ka- sım’ın hapsedilirken aşağıdaki şiiri söylediği rivayet edilmektedir. Ben Vâsıt’ta demirlerin 90 Sind bölgesinde yakalanan Muhammed b. Kasım dahi buraya getirilip hapsedilmişti. Ka- sım’ın hapsedilirken aşağıdaki şiiri söylediği rivayet edilmektedir. Ben Vâsıt’ta demirlerin Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 161 Hapishanenin şehrin neresinde olduğu ile ilgili yeterli bilgiye sahip değiliz.92 Temel kaynaklarımızda bu hususta bir bilgi olmadığı gibi, kazılar- da da konuyu aydınlatacak bir veriye ulaşılamamıştır. Sadece İbnu’l- Cevzî’nin buraya hapsedilmiş olan İbrahim b. Yezid b. Şerik et-Teymî hak- kında bilgi verirken, hapishanenin yerin altına inşa edilmiş olduğunu söy- lemesi,93 Hacac’ın sarayının altında olabileceği ihtimalini düşündürtmekte- dir. Ancak bunu destekleyecek bilgilere ulaşılamamıştır. Haccâc öncesinde başta Kûfe olmak üzere Irak hapishanelerinde tu- tuklu bulunan mahpusların yatakları, yiyecekleri devlet tarafından tedarik edildiği gibi, biri yazlık, diğeri ise kışlık olmak üzere yılda iki elbise veril- mekte ve gayet iyi bakılmaktaydılar.94 Ancak Haccâc döneminde ed- Dimâs’taki rejim muhalifi mahpusların insanlık dışı muamelelere tabi tu- tuldukları, içine toprak karıştırılmış arpa unu ile beslenmek zorunda bıra- kıldıkları, bunun sonucunda da bir çok mahpusun hayatını yitirdiği kayde- dilmektedir.95 tutsağı, Zincirlere bağlı olsam da, Nice atlıyı titretmiş, Nicesini de öldürmüşüm. Bkz İbnu’l- Esîr, c. IV, s. 530 91 Dineverî, s. 348 92 Muaddîdî, s. 189 93 Bkz. İbnu’l-Cevzî, Sıfatu’s-safve, tahk.: Mahmud Fâhûrî, Beyrut 1979, c. III, s. 92 94 Konu ile ilgili geniş bilgi için bkz. Ebû Yusuf, Kitâbu’l-Harâc, çev.: Ali Özek, İstanbul 1973, s. 239 95 Bkz. Belazurî, Ensâb, c. VII, s. 384 96 Bkz. Muaddîdî, s. 186 99 Dineverî bu sürenin altı, Zehebî on, Philip Hitti ise on bir ay olduğu kanaatindedir. Bkz. Dineverî, 370; ez-Zehebî, Siyer A’lam en-Nubela, tahk.: Şuayb el-Arnavud-Hüseyin el- Esed Beyrut 1984-1988, c. IV, s. 57; Philip Hitti, İslâm Tarihi, çev.: Salih Tuğ, İstanbul 1997, c. II, s. 445 97 Bkz. el-Vâsıtî, s. 38; Âdil Necm, s. 598. Yâkût ise şehrin iki surunun değil de bir sur ve bu suru kuşatan iki hendeğin olduğu kanaatindedir. Bkz. Yâkût, Buldân, c. V, s. 402 98 Yakubî, Buldân, s. 101 102 Bu kapı, adını Muhammed b. Halid tarafından Haccâc’a gönderen filden almıştır. Fil gemi ile getirilmiş, adı geçen kapıdan şehre sokulunca, söz konusu giriş, bu isimle anılır olmuş- tur. Bkz. İbnu’l-Fakîh, s. 265 k. Vâsıt’ın Mahalleleri İlk kurulan askerî kentler ve aynı zamanda da Irak’ın eş başkentleri olan Kûfe ve Basra’da halk kabileler şeklinde yerleşmiş, dolayısıyla mahal- leler de bu kabilelerin adlarıyla anılmıştır. Ancak Muaddîdî tarafından tespit edilen Vâsıt mahallelerin büyük bir kısmının kabile ismi taşımıyor olması Haccâc’ın kabile sistemini esas alan bir iskân politikası gütmemiş olduğunu ortaya koymaktadır. Vâsıt’ın tespit edilebilen mahalleleri şunlardır: Mahalletu Babu’z-Zâb, Mahalletu Tahanîn, Mahalletu Berculaniye, Mahalletu Karafisiyîn, Mahalletu Varrakîn, Mahalletu Rezzâzîn, Mahalletu Benî Dalân, Mahalletu Enbariyîn, Mahalletu Zeydiyye, Mahalletu Zübeydiyye, Mahalletu Şarkiyye, Mahalletu Kasru Resâs, Mahalletu’l-Hadire ve Hristiyanların ikamet etmekte oldukları Mahalletu Burcuniyye.96 Mahalle isimleri tahlil edildiğinde, ilk dönem İslâm kentlerinin aksine, kabile isimlerinden daha çok meslek isimlerinin ön pla- na çıktığı görülmektedir. Hristiyanların Burcuniyye mahallesinde toplanmış olmaları, Abbasîler döneminde, kabilelere göre olmasa bile dinî mensubi- yetlere göre bir yerleşimin var olduğunu göstermesi açısından önem arz ettiği gibi, kuruluş döneminde olmasa bile daha sonraki dönemlerde şehre tutsağı, Zincirlere bağlı olsam da, Nice atlıyı titretmiş, Nicesini de öldürmüşüm. Bkz İbnu’l- Esîr, c. IV, s. 530 91 Dineverî, s. 348 92 Muaddîdî, s. 189 93 Bkz. İbnu’l-Cevzî, Sıfatu’s-safve, tahk.: Mahmud Fâhûrî, Beyrut 1979, c. III, s. 92 94 Konu ile ilgili geniş bilgi için bkz. Ebû Yusuf, Kitâbu’l-Harâc, çev.: Ali Özek, İstanbul 1973, s. 239 95 Bkz. Belazurî, Ensâb, c. VII, s. 384 96 Bkz. Muaddîdî, s. 186 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 162 önemli sayıda Hristiyanın yerleştiğini göstermesi açısından da önem arzetmektedir. 105 İbnu’l-Fakîh ve Yâkût’un belirttiğine göre; Haccâc saray, cami, iki hendek ve sur için 43.000.000 dirhem harcamış, Divau’l-harac katibi olan Salih’in, halife Abdulmelik’in bu pa- rayı fazla bulacağı şeklindeki uyarısı üzerine, paranın 34.000.000 dirheminin savaş gider- 101 Bkz. Zehebî, Siyer, c. IV, s. 57 104 İbnu’l-Fakîh, s. 265 103 Muaddîdî, s. 190 6. Vâsıt’ın İlk Sakinleri Yukarıda da ifade etmeye çalıştığımız gibi Vâsıt bir garnizon kent o- larak kurulduğu için buraya ilk etapta sadece Arap kökenli, hatta Suriyeli askerler ve bunların aileleri ile Türk kökenli askerler yerleştirilmişti. Daha sonraki dönemlerde sivillerin de iskanına müsaade edilmiştir. Vâsıt, henüz kurulduğu ilk yıllarda yörenin en büyük cazibe merkezi olmuş, başta Kûfe ve Basra olmak üzere bir çok kentten önemli oranda göç almıştır. Bağ- dat’ın kuruluşu ile beraber bu şehre de büyük bir göç vermiştir. 110 Vâsıt’ın nüfusunu üç başlık altında değerlendirmek mümkündür. l. Sur Irak’ın eş idarî merkezleri olan Kûfe ve Basra’nın herhangi bir sura sahip olmadıkları, dış saldırılara açık oldukları yukarıda ifade edilmişti. Bu durum Haccâc’ı yeni idarî merkezini kurarken daha titiz davranmaya itmiş- tir. Nitekim Vâsıt, iç içe iki sura sahip olarak inşa edilmiş97 ve bu özelliği ile iç içe üç suru bulunan Bağdat’a örnek olmuştur. Yakubî’nin de belirttiği gibi aşılması neredeyse imkânsız olan bu surlar,98 Kahtaba’ya yenilen Emevilerin son Irak valilerinden Yezid b. Ömer b. Hubeyre el-Fezarî’nin 20.000 kişilik bir kuvvetten oluşan Abbasî birliklerine uzun süre direnebil- mesine imkân tanımıştır.99 İki sur ile de yetinmeyen Haccâc, bu surların etrafına üç yılda tamamlanabilen bir de hendek kazdırmıştır.100 Zehebî’nin son Emevî valisi İbn Hubeyre’nin şehrin etrafına bir hendek kazdırdığını söylemesi,101 Emevîlerin son döneminde söz konusu surunun etrafındaki hendeğin tahrip olduğunu göstermektedir. Surun tam olarak kaç kapısının var olduğu, bu kapıların nerede bu- lundukları, kapılar arsındaki mesafenin ne kadar olduğunu tespit edeme- mekle birlikte Babu’l-Midmâr, Babu’z-Zâb, Babu’l-Kayrec, Babu Hallalîn, Babu’l-Fil102 ve Babu’l-Basra103 adını taşımakta olan kapıların isimlerini biliyoruz. Şehir kapılarının mimarî özelliklerine gelince; İbnu’l-Fakîh bunla- rın dukara denilen eski Fars yapımı kapılar olduğunu söylemektedir.104 İbnu’l-Fakîh’in kullandığı bu ifade ile İranlıların dukali dedikleri çift kanatlı, arkadan sürgü ile kapatılabilen kapıları kasdettiğini tahmin etmekteyiz. Şehrin inşası için 43.000.000 dirhem tutarındaki beş yıllık Irak hara- cını buraya harcayan Haccâc’ın Vâsıt’ı iki yıl gibi kısa bir sürede iskân edilebilecek hâle getirdiği rivayet edilmektedir.105 103 Muaddîdî, s. 190 105 İbnu’l-Fakîh ve Yâkût’un belirttiğine göre; Haccâc saray, cami, iki hendek ve sur için 43.000.000 dirhem harcamış, Divau’l-harac katibi olan Salih’in, halife Abdulmelik’in bu pa- rayı fazla bulacağı şeklindeki uyarısı üzerine, paranın 34.000.000 dirheminin savaş gider- Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 163 5. Vâsıt’a Bağlı Yerleşim Birimleri Şehir hakkında verdiğimiz bu kısa bilgilerden sonra şimdi de buraya bağlı olan yerleşim birimleri üzerinde duralım. Vâsıt’a bağlı yerleşim birim- leri derken şehrin çevresinde yer alan köy ve kasabaları kasdettiğimizi belirtmemiz gerekir. Bu köy ve kasabalar ile ilgili en geniş bilgiler Makdisî tarafından aktarılmaktadır. Onun verdiği bilgilere göre; Vâsıt’a bağlı yer- leşim birimleri şunlardır: Femu’s-Silh, Demerkân, Kurakale, Siyâde, Bâzibîn, es-Sikr, et-Tîb, Kurkûb, Karyetu’r-Remle, Nehru Tirî, Lehbân ve Besâmiye,106 Yine Makdisî bu yerleşim birimlerinin küçük olduğunu bunlar içerisinde en mamur olanlarının et-Tîb ile Karkûb olduklarını söylemekte- dir.107 Kazvinî, Vâsıt dolaylarında Femu’d-Dubeyl adında bir köyden bahse- derken,108 Zehebî ise Habul adında bir köyün adını vermektedir.109 Çorum İlahiyat Fakültesi Dergisi, 2002/2 leri için, geri kalan 9.000.000 dirhemin Vâsıt’ın inşası için harcandığını söylemiştir. Bkz. İbnu’l-Fakîh, s. 263; Yâkût, Buldân, c. V, s. 402 aa. Suriyeliler Kent sakinlerinin en önemli kitlesini Suriye kökenli askerler oluştur- maktaydı. Bunların kaç bin kişi oldukları tam olarak belli olmamakla birlik- te, Haccâc’ın Abdurrahman b. el-Eş’as ile yürüttüğü savaşta, kendisini desteklemek amacıyla Suriye’den gönderilen askerlerin büyük bir kısmının buraya yerleştirildiği bilinmektedir. Aynı zamanda Haccâc’ın korumasını da üstlenmiş olan bu insanlar, camide onun arkasındaki saflarda durarak na- maz kılmaktaydılar.111 Keza büyük bir ihtimal ile Daru’l-İmâre’nin hemen çevresinde ikamet etmekteydiler. leri için, geri kalan 9.000.000 dirhemin Vâsıt’ın inşası için harcandığını söylemiştir. Bkz. İbnu’l-Fakîh, s. 263; Yâkût, Buldân, c. V, s. 402 106 Bkz. Makdisî, s. 104 107 Bkz. Makdisî, s. 106 108 Bkz. Kazvinî, s. 431 109 Bkz. Zehebî, Siyer,c. IX, s. 365 110 Kazvinî, s. 453 111 el-Vâsıtî, s. 39 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Yrd. Doç. Dr. M. Mahfuz Söylemez 164 ab. Kûfe ve Basralılar Vâsıt’a yerleşen Arapların ikinci önemli kitlesini ise Kûfeliler ile Bas- ralılar oluşturmaktaydı. Emevî taraftarı askerî unsurlar olduğu anlaşılan bu insanların kaç kişi oldukları ve şehrin neresinde oturdukları hakkında ye- terli bilgiye sahip değiliz. el-Vâsıtî’nin “Haccâc Vâsıt’ı kurunca buraya Kûfe’nin ileri gelenlerini yerleştirdi ve kendilerine maksurenin sağında, Basralılara ise solunda namaz kılmalarını emretti,”112 şeklindeki ifadelerden hareketle, Kûfelilerin Daru’l-İmare’nin sağ tarafına, Basralıların ise sol tarafına yerleştirilmiş oldukları varsayılabilir. Vâsıt’ın Abbasiler döneminde İslâm âleminin önemli ilim ve irfan merkezi haline gelmesinde yukarıda anılan Kûfe ve Basralıların ciddi katkı- larının olduğu kabul edilmektedir. 112 el-Vâsıtî, s. 39 113 Belazurî, Futûh, s. 545 114 Konu ile ilgili geniş bilgi için bkz. Hasan Kurt, Orta Asya’nın İslâmlaşma Süreci (Buhara Örneği), Ankara 1998, 118. Zekeriya Kitapçı ise buraya 25-30 bin arasında Türk’ün yer- leştirildiğini, hatta sadece şehre değil Sevâd arazisinde de bunlardan oluşan onlarca köyün kurulduğunu kaynak vermeden söylemektedir. Ancak biz Vâsıt’a yerleştirilmiş olan Türk- lerin onbinler düzeyinde oldukları ile ilgili bir veriye ulaşamadığımız gibi Sevâd’a Tüklerin iskan ettirildiklerine dair bir habere de rastlayamadık. Bkz. Zekeriya Kitapçı, Saadet Asrın- da Türkler; İlk Türk Sahabe Tabii ve Tebea Tabileri, Konya 1997, ss. 179, 181. 115 Bkz. Hakkı Dursun Yıldız, İslâmiyet ve Türkler, İstanbul 2000, s. 72 116 Bkz. Belazurî, Futûh, s. 545 115 Bkz. Hakkı Dursun Yıldız, İslâmiyet ve Türkler, İstanbul 2000, s. 72 114 Konu ile ilgili geniş bilgi için bkz. Hasan Kurt, Orta Asya’nın İslâmlaşma Süreci (Buhara Örneği), Ankara 1998, 118. Zekeriya Kitapçı ise buraya 25-30 bin arasında Türk’ün yer- leştirildiğini, hatta sadece şehre değil Sevâd arazisinde de bunlardan oluşan onlarca köyün kurulduğunu kaynak vermeden söylemektedir. Ancak biz Vâsıt’a yerleştirilmiş olan Türk- lerin onbinler düzeyinde oldukları ile ilgili bir veriye ulaşamadığımız gibi Sevâd’a Tüklerin iskan ettirildiklerine dair bir habere de rastlayamadık. Bkz. Zekeriya Kitapçı, Saadet Asrın- da Türkler; İlk Türk Sahabe Tabii ve Tebea Tabileri, Konya 1997, ss. 179, 181. b. Türkler Şehre Haccâc tarafından yerleştirilmiş olan tek gayr-i Arap unsur Türklerdir. Ubeydullah b. Ziyad’ın, Buhara seferi esnasında, beraberinde getirdiği ve Basra’ya yerleştirmiş olduğu bu insanlar,113 Taberî’ye göre, 2000, Narşahî’ye göre ise 4000 kişi idiler.114 Ubeydullah’ın bunları muhafız birliğini takviye için getirdiği tahmin edilmektedir.115 Basra’da bulundukları süre zarfında şehirde patlak veren hadiselerde veya devlete karşı baş gös- teren isyanlarda yer almamış olmaları Haccâc’ın güvenini kazanmalarını sağlamış ve Vâsıt’a yerleştirilmelerinde etkili olmuştur. Kaynaklarımızda bu Türk kitle hakkında teferruatlı bilgilere rastlan- mamaktadır. Ne iş yaptıkları, şehrin neresinde ikamet ettikleri, şehrin e- konomik ve sosyal hayatındaki yerlerini maalesef bilmiyoruz. Ancak daha sonraki dönemlerde Türkler arasında bazı ilim adamlarının yetişmiş olması, şehrin kültür hayatında etkili olduklarını göstermektedir. Belazurî kendi yaşadığı dönemde Haccâc tarafından Vâsıt’a yerleşti- rilmiş olan Türklerin soyundan gelen önemli bir topluluğun burada yaşadı- ğını söylemekte ve İbn Markulî olarak şöhret kazanmış olan Hâlid eş- Şâtır’ın da bunlardan biri olduğunu belirtmektedir.116 Vâsıt’a yerleşmiş olan bu Türklerin izole bir hayat sürmedikleri, yöredeki diğer Türk gurupları ile de irtibat içinde oldukları anlaşılmaktadır. Dahası bu insanların Buhara ile 116 Bkz. Belazurî, Futûh, s. 545 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 165 ilişkilerini sürdürdükleri ve anavatanlarının İslâmlaşmasında önemli roller üstlenmiş oldukları da tahmin edilmektedir.117 Zehebî’nin Buhara Tüklerinden biri olan Kûfeli ünlü muhaddis Şureyk hakkında verdiği bilgi- lerden bunların izole bir hayat sürmedikleri açıkça ortaya çıkmaktadır. Nitekim Zehebî onun Vâsıt’a gelip gittiğini ve burada hadis dersleri verdi- ğini özellikle vurgulamaktadır.118 Muhtemelen kendisinden ders alanların önemli bir kısmı da Türk kökenli kimseler idi. Vâsıtlı Türk bilginlerden bir diğeri ise ünlü muhaddis ve fakih Yezîd b. Harun (ö.206)’dur. Ahmed b. Hanbel’in de hocası olan Yezid, döneminin en büyük fakih ve muhaddisle- rinden biri olarak kabul edilmektedir. Zehebî onun daha sonra Bağdat’a yerleştiğini ve binlerce talebe yetiştirdiğini söylerken, Yahya b. Ebî Talip de hocası olan Yezid’in ders halkasında binlerce insanın okuduğunu ilave etmektedir.119 120 İbn Fakîh konu ile ilgili şu hadiseyi aktarmaktadır: “Aramîlerden biri Haccâc’ın mutfağında çalışıyor, onun sevdiği yemekleri yapıyordu. Haccâc Aramîleri Vâsıt’tan çıkarınca, çok sev- diği bu yemek çeşitlerini de kaybetti. Bunun sebebini sorduğunda durumu kendisine bil- dirdiler. Bir adam gönderip bu şahıstan yemek yapmayı öğrenmesini istedi. Ancak aynı ta- dı yakalayamadı. Bunun üzerine aşçının sabah gelip, akşam şehirden çıkmasını emretti. Aşçı her gün yemek kapları ile gelir, yemeği pişirir, akşam da şehri terkederdi.” Bkz. İbnu’l-Fakîh, s. 266 121 el-Vâsıtî, s. 41 122 Bkz. İbn Abdirabbih, c. V, ss. 29-30 c.Sevâd’ın Yerlileri Haccâc döneminde Sevâd yerlilerinin Vâsıt’a yerleşmelerine müsaa- de edilmediğini yukarıda belirtmiştik. Kaynaklarımızın ifadesine göre bu dönemde Sevâd yerlilerinden Vâsıt’ta işi olanlar sabah gelip akşam çıkmak zorunda idiler.120 Hatta şehrin kapılarında nöbetçiler bulunmakta, söz ko- nusu kitlenin giriş çıkışlarını kontrol etmekte,121 şehre girip çıkanların belli olması için, ellerine mühür vurmaktaydılar.122 el-Vâsıtî, Haccâc tarafından uygulanmakta olan bu sistemin Emevilerin sonuna kadar devam ettiğini, Sevâd sakinlerinin ancak Abbasiler döneminde buraya yerleşebildiklerini söylüyorsa da123 İbn Fakîh ve benzeri tarihçi ve coğrafyacıların da ortaya koyduğu gibi Haccâc’ın ölümünden hemen sonra Sevâdlıların buraya yer- leşmeye başladıkları bilinmektedir.124 Sevâd yerlilerinin büyük bir kısmı yahudî, hristiyan ve mecusîlerden oluşuyordu.125 Bu kitlelerin müslümanlar ile bir arada yaşamasıyla Vâsıt, farklı din mensuplarının bir arada barış ve huzur içerisinde hayatlarını sür- dürdükleri bir şehir haline geldi. Şehirde hakim unsur olan müslümanlar ile 117 Kurt, s. 231 118 Bkz. Zehebî, Siyer, c. VIII, s. 203 119 Bkz. Zehebî, Siyer, c. IX, ss. 358-362 120 İbn Fakîh konu ile ilgili şu hadiseyi aktarmaktadır: “Aramîlerden biri Haccâc’ın mutfağında çalışıyor, onun sevdiği yemekleri yapıyordu. Haccâc Aramîleri Vâsıt’tan çıkarınca, çok sev- diği bu yemek çeşitlerini de kaybetti. Bunun sebebini sorduğunda durumu kendisine bil- dirdiler. Bir adam gönderip bu şahıstan yemek yapmayı öğrenmesini istedi. Ancak aynı ta- dı yakalayamadı. Bunun üzerine aşçının sabah gelip, akşam şehirden çıkmasını emretti. Aşçı her gün yemek kapları ile gelir, yemeği pişirir, akşam da şehri terkederdi.” Bkz. İbnu’l-Fakîh, s. 266 121 el-Vâsıtî, s. 41 122 Bkz. İbn Abdirabbih, c. V, ss. 29-30 123 el-Vâsıtî, s. 41 124 İbnu’l-Fakîh, s. 266; Yâkût, Buldân,c. V, s. 403 125 Bkz. Muaddîdî, s. 181 Çorum İlahiyat Fakültesi Dergisi, 2002/2 166 Yrd. Doç. Dr. M. Mahfuz Söylemez farklı din mensupları arasında hiçbir çatışma yaşanmadan, gayet dostane ve de medenî ilişkilerin geliştiği anlaşılmaktadır. Nitekim Zehebî’nin açıkça ortaya koyduğu gibi Vâsıtlı ünlü Muhaddis Mansur ez-Zazân’ın cenaze tö- renine Hristiyan, Yahudi ve Mecusiler, Müslümanlar ile birlikte katılmışlar, onlarla beraber göz yaşı dökmüşlerdir.126 Haccâc’ın Ârâmîlerin yani, Vâsıt’ın inşa edildiği Sevâd bölgesi yerlile- rinin neden şehre yerleşmelerine müsaade etmediği sorusuna gelince bu- nun birden fazla sebepi bulunmaktadır. 1.Haccâc askerî bir kent kurmaya çalıştığı için buraya sivillerden zi- yade asker kökenli kimselerin yerleşmesi taraftarı idi. 2.Sevâd yerlileri tarım ile uğraşıyorlardı. Haccâc, uygulamakta oldu- ğu tarım politikası gereği, bu çiftçilerin şehre yerleşmelerini istemiyordu. Irak idaresinin en önemli geliri bu arazilerden elde edilen haraç girdileri idi. c.Sevâd’ın Yerlileri Haraç, topraktan değil üründen alındığı için çiftçiliği bırakıp şehre yer- leşen şahıslar devletin önemli bir gelir kaynağından mahrum olmasına sebep oluyordu. Zaten Haccâc, Vâsıt’tan önce de aynı gerekçeler ile Kûfe ve Basra gibi şehirlere yerleşen Sevâd kökenli çiftçileri çıkarmış, köylerin- de zorunlu ikamete tabi tutmuştu. 3.Yukarıda belirttiğimiz gibi Vâsıt, Kesker’in tam karşısında yani Dic- le’nin batı yakasında kurulmuştu. Haccâc, Kesker’in nimetlerinden yarar- lanmak için iki şehri birbirine bağlayan bir köprü inşa etmiş olmasına rağ- men şehirlerin bütünleşmesini istemiyordu. Bunun için de Keskerlilerin şehre yerleşmelerine engel olmaya çalışıyordu. Böylece askerî kentin özel- liklerini korumaya gayret ediyordu. Haccâc’ın tamamen askerî bir kent olarak kalması için gayret ettiği Vâsıt, onun ölümünden kısa bir süre sonra Sevâd kökenli siviller ile doldu. Abbasiler döneminde Kesker ile bütünleşerek tek bir şehir haline geldi. Doğu yakasına Medinetu’ş-Şarkiyye, batı yakasına ise Medinetu’l-Garbiyye denildi.127 Batı tarafına yerleşen diğer din mensupları için kilise ve havralar inşa edildiği gibi Doğu tarafına yerleşen müslümanlar da burada camii ve medrese inşa ettiler.128 Buna rağmen batı Vâsıt’ın kahir ekserisini müslüman Araplar, doğu Vâsıt’ın çoğunluğunu Farslar ve Ârâmîler oluş- turmaktaydı. 128 Âdil Necm Doğu Vâsıt’taki ilk caminin hicri 261 yılında kurulduğunu söylemektedir. Bkz. Âdil Necm, s. 599 127 Bkz. Muaddîdî, s. 181 126 Bkz. Zehebî, Siyer,c. V, s. 442 126 Bkz. Zehebî, Siyer,c. V, s. 442 127 Bkz. Muaddîdî, s. 181 128 Âdil Necm Doğu Vâsıt’taki ilk caminin hicri 261 yılında kurulduğunu söylemektedir. Bkz. Âdil Necm, s. 599 127 Bkz. Muaddîdî, s. 181 128 Âdil Necm Doğu Vâsıt’taki ilk caminin hicri 261 yılında kurulduğunu söylemektedir. Bkz. Âdil Necm, s. 599 129 Bkz. el-Vâsıtî, s. 32. Bu rivayetin bir benzeri de İbnu’l-Cevzî, İbnu’l-Esîr ve Himyerî tara- fından aktarılmaktadır. Onların bildirdiğine göre; Haccâc Vâsıt’ı kurmaya karar verince, şehri inşa edeceği mekanı tespit etmek için harekete geçer. Vâsıt’ın kurulacağı yere kadar gelir. Burada bir rahibin bir yerden eliyle toprak kazıdığını ve bunu yanı başındaki Dicle Nehrine döktüğünü görür. Sebepini sorduğunda ise şu cevabı alır: “Kitabımızda yer yü- zünde Allah’ı tevhid eden bir insan bulunduğu sürece burada Allah’ın adının anılacağı bir mescidin bulunacağını okuyoruz” . Bunu duyan Haccâc, mescidi rahibin işaret ettiği yerde inşa eder.[İbnu’l-Cevzî, el-Muntazam, c. IV, s. 388; İbnu’l-Esîr, c. IV, s. 445. Bu hikaye ufak değişikliklerle Himyerî tarafından da aktarılmaktadır. Bkz. Himyerî, s. 599] Rahibin toprağı Dicle’ye neden döktüğü ile ilgili boşluk Taberî ve İbn Kesîr tarafından doldurulmak- tadır. Onların anlattığına göre; “Haccâc eşeği üzerinde Dicle nehrini aşıp gelmekte olan bir rahip görür, bu rahip Vâsıt şehrinin kurulacağı yere geldiğinde eşeği durur ve def-i hacet eder, rahip iner ve pislenen yeri kazarak Dicle nehrine atar. Haccâc ‘bu rahibi bana getirin’ der. Rahibi getirdiklerinde ona neden böyle yaptığını sorar. Rahip ‘kutsal kitaplarında, yer- yüzünde Allah’a inanan bir kimse bulunduğu sürece burada bir mescidin var olacağını gör- düklerini söyler. Bunu duyan Haccâc orada Vâsıt şehrini kurmaya karar verir, rahibin gös- terdiği yere bir mescit inşa eder.” Bkz.Taberî, c. VII, ss. 281-282; İbn Kesir, c. IX, s. 87 7. Vâsıt Kuruluşu ve Sakinleri ile İlgili Apokrif Rivayetler İslâm tarihi kaynaklarında apokrif rivayetler önemli bir yekun tut- maktadır. Bir kabileyi, boyu, kahramanı, milleti hatta ümmeti veya mez- hebi hedef alan bu kabil rivayetler, siyasî hadiselerde taraf olan kişilerce üretilmişlerdir. İdeolojik duruşa sahip olan bu insanlar kutsal metinlerden Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 167 destek alarak ve kutsalın dili ile konuşarak meşruiyet ve hak kazanma gereği hissetmişlerdir. Bu kesimler kutsal metinlerin zaman zaman lafzî anlamlarına sarılarak, kimi zaman da kendi duruşları ile çelişen nasları tevil ederek kendilerini haklı, karşıtlarını haksız duruma sokma gayreti içerisinde olmuşlardır. Dahası kendilerini haklı çıkarmak için peygamber’in hatta Yüce Allah’ın dili ile konuşmaktan, kendilerini göklere çıkarmaktan, karşıtlarını ise yerin dibine batırmaktan çekinmemişlerdir. Vâsıt ile ilgili oluşmuş olan apokrif edebiyat bunun en güzel örneğini teşkil etmektedir. Bir taraftan Haccâc’ın taraftarları, büyük ihtimalle de Vâsıtlılar, İslâm tari- hinin en acımasız yöneticilerinden biri olan Haccâc tarafından kurulmuş olan bu şehirle ilgili olumsuz kanaatleri yıkmaya çalışırken, diğer taraftan Sevâd’ın yerlileri olan zengin ve müreffeh Ârâmîleri karalamak için Hz. Peygamber’e, kutsal kitaplara veya hristiyan ve müslüman din bilginlerine akla ve mantığa sığmayan rivayetler isnat etmişlerdir. Şimdi bu rivayetlere kısaca bakalım: Vâsıt’ın kurulduğu yerin Kudüs’ten bir parça ve dolayısıyla kutsal bir belde olduğunun iddia edildiği aşağıdaki rivayet bunlardan biridir. Avâne b. el-Hakem’in Avâne el-Kelbî’den o da babasından aktararak diyor ki : “Haccâc bir şehir inşa etmek için yer ararken ben de onun yanındaydım. Merkebe binmiş bir rahip gördük. Merkebi pisledi. Rahip indi. Merkebin pisliğini elbisesi ile topladı. Haccâc rahibi çağırdı ve nedir bu yaptığın diye sordu. Rahip ‘biz kutsal kitabımızda şunu gördük. Tufan koptuğu zaman mukaddes topraklardan bir parça kopmuş ve buraya düşmüştür. İşte o toprak burasıdır. Eşeğimin pisliğinin burada olmasını uygun görmedim’ Bunun üzerine Haccâc arkadaşlarına atlarından inmelerini ve buraya şehrinin inşa edilmesini emretmiştir.”129 el-Vâsıtî bu rivayeti aktardıktan sonra, Amr b. Avd ile Ahmed b. Beşir el-Emevî’yi de dayanak yaparak, ri- vayetin doğru olduğunu belirtmektedir. “Haccâc bir şehir inşa etmek için yer ararken ben de onun yanındaydım. Merkebe binmiş bir rahip gördük. Merkebi pisledi. Rahip indi. Merkebin pisliğini elbisesi ile topladı. Haccâc rahibi çağırdı ve nedir bu yaptığın diye sordu. Rahip ‘biz kutsal kitabımızda şunu gördük. Tufan koptuğu zaman mukaddes topraklardan bir parça kopmuş ve buraya düşmüştür. İşte o toprak burasıdır. Eşeğimin pisliğinin burada olmasını uygun görmedim’ Bunun üzerine Haccâc arkadaşlarına atlarından inmelerini ve buraya şehrinin inşa edilmesini emretmiştir.”129 el-Vâsıtî bu rivayeti aktardıktan sonra, Amr b. Avd ile Ahmed b. 7. Vâsıt Kuruluşu ve Sakinleri ile İlgili Apokrif Rivayetler Beşir el-Emevî’yi de dayanak yaparak, ri- vayetin doğru olduğunu belirtmektedir. Bir başka rivayette ise Yüce Allah dahi istismar edilerek, Vâsıt’ın ku- rulmuş olduğu yere vahyedildiği iddia edilmek suretiyle değeri yükseltil- mek istenmiştir. Çorum İlahiyat Fakültesi Dergisi, 2002/2 168 Yrd. Doç. Dr. M. Mahfuz Söylemez Muharrib b. Disâr anlatıyor: “Buhtu’n-Nasr, Beytu’l-Makdis’i yıkınca yeryüzünün tamamı ağladı. En çok ağlayan da Kesker idi. Yüce Allah Kesker’e vahyetti. ‘Sende öyle bir mescit inşa edeceğim ki orada beni zikreden çok olacaktır.’ Bu mescidin Vâsıt mescidi olduğu zikredilmektedir.”130 Avâne b. el-Hakem rivayetinde olduğu gibi, İslâm tarih yazıcılığında başta Bahira hadisesi olmak üzere bir çok apokrif gaybî haberde rahiplerin kullanıldığı görülmektedir. Bu haberler, rahiplerin kutsal metinlere daya- narak hemen hemen her şeyi bildikleri kanaatinin o dönemlerde önemli bir kitle tarafında benimsenmiş olduğunu göstermektedir. Rahiplere böyle bir payenin verilmiş olmasının iki temel sebebi bulunmaktadır: Ümmi bir top- lum olan Müslüman Araplar, ehl-i kitap ile karşılaşınca onların kendilerin- den daha çok şey bildiklerini gördüler ve bu bilgi birikimlerine hayran kal- dılar. Onların “ehl-i necât” olduklarını kabul etmemekle beraber birikimle- rinden yararlandılar, ancak kimi kötü niyetli kimseler bu birikimi abartarak kötüye kullandı. Aynı şeyler bazen fırsatçı rahipler tarafından da yapıldı. Yukarıdaki iki rivayetin dayandırıldığı ravilerin de iyi seçildiği anla- şılmaktadır. Zira bu ravilerin ilki Kûfe’nin ünlü fakih ve muhaddislerinden Muhârib b. Disâr (ö.108/726) iken, diğeri de Kûfe’nin ünlü tarihçisi Avâne b. el-Hakem (ö.147/764)’dir. Adı geçen ravilerin ölüm tarihlerini esas aldığımızda haberlerin Abbasîler dönemine ait oldukları ortaya çıkmakta- dır. Muhtemelen Abbasîlerin erken döneminde Emevîlere yönelik olarak gelişen eleştirilerin bir kısmında Vâsıt da bulunmuştur. Bu apokrif haber- lerle söz konusu iddialara cevap mahiyetinde hazırlanmıştır. Siyer kitaplarında Hz. Peygamberin doğumu esnasında gerçekleştiği iddia edilen harikuladeliklerin Vâsıtlı ravileri de etkilediği ve bu şehir ile Hz. Peygamberin doğumu arasında bağ kurmalarına sebep olduğu anlaşılmak- tadır. Mesela el-Vâsıtî diyor ki “Vâsıt’ın hemen yanı başında kurulduğu Dicle’nin asıl yatağı burası değil- di. Dicle Hz. Peygamber’in doğduğu gün yön değiştirerek bu mecradan akmaya başlamıştır.” el-Vâsıtî, bu haberin bir benzerinin Salih b. Süley- man ile Ebû Hukeym’den de rivayet edildiğini söylemektedir.131 Ârâmîlerin Vâsıt’a yerleşmelerine müsaade etmeyen Haccâc’ı temize çıkarmak için ilahî bir kılıf bulunmaya çalışıldığı da görülmektedir. Nitekim Hz. Peygamber’in vefatından yaklaşık bir asır sonra kurulmuş olan Vâsıt ile ilgili söylenenler bunun en çarpıcı örneğidir. Bu kabil rivayetlerde evrensel bir peygamber yerine sanki Arap milliyetçisi her peygamber imajı ortaya çıkmaktadır. Bu ise oldukça tehlikeli bir durum doğurur. “Peygamber şöyle buyurdu: ‘Kureyş’e buğz etmeyiniz. Araplara sövme- yiniz. Mevalîyi zelil etmeyiniz. Ârâmîler ile beraber oturmayınız. Zira onlar 7. Vâsıt Kuruluşu ve Sakinleri ile İlgili Apokrif Rivayetler Buna dair birkaç örnek verirsek konu daha iyi anlaşılabilir: “Peygamber şöyle buyurdu: ‘Kureyş’e buğz etmeyiniz. Araplara sövme- yiniz. Mevalîyi zelil etmeyiniz. Ârâmîler ile beraber oturmayınız. Zira onlar 130 Bkz. el-Vâsıtî, s. 31 131 Bkz. el-Vâsıtî, s. 33 Çorum İlahiyat Fakültesi Dergisi, 2002/2 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine 169 dinin afeti, peygamberlerin katilleridirler. Bunlar şehirlere yerleşip ev inşa ettikleri ve Arapça konuşup Kur’an öğrendiklerinde, hile ve tuzaklarla in- sanlara hakim olurlar. Bu durum da İslâm’ın yüceliğini bitirir.”132 “Ârâmîler, dinde bilgi sahibi olup Arapça konuştuklarında ve Kur’an öğ- rendiklerinde onlardan kaçınız. Çünkü onlar riba ve rüşvet yiyorlar, hile yapıyorlar. Onların İslâm’dan nasibi yoktur.”133 dinin afeti, peygamberlerin katilleridirler. Bunlar şehirlere yerleşip ev inşa ettikleri ve Arapça konuşup Kur’an öğrendiklerinde, hile ve tuzaklarla in- sanlara hakim olurlar. Bu durum da İslâm’ın yüceliğini bitirir.”132 “Ârâmîler, dinde bilgi sahibi olup Arapça konuştuklarında ve Kur’an öğ- rendiklerinde onlardan kaçınız. Çünkü onlar riba ve rüşvet yiyorlar, hile yapıyorlar. Onların İslâm’dan nasibi yoktur.”133 Bir başka haberde de şöyle denilmektedir: “Rivayet edildiğine göre; Ebû Hureyre bir arkadaşına, bir gün demiş ki ‘Ben seni çok seviyorum. Ama nereli olduğunu bilmiyorum’ Adam Sevâdlı olduğunu söyleyince, Ebû Hureyre “Benden uzak dur. Resulullah’ın şöyle dediğini duydum. ‘Sevâdlılar peygamberin katilleri ve dinin afetleri, her çağda zalimlerin yardımcıları, riba yiyiciler ve heva ehlidirler.”134 Rivayette geçen “Her çağda zalimlerin yardımcılarıdırlar” ifadesi ile muhtemelen Sevâd yerlilerinin Abdurrahman b. el-Eş’as’a verdikleri destek kasdedilmektedir. Hz. Ömer Resulullah’ın şöyle buyurduğunu aktarmaktadır. Hz. Ömer Resulullah’ın şöyle buyurduğunu aktarmaktadır. “Şu üç grup, insanların en şerlileridir: Şeytan er-Recim, Kızgınlığını ve zulmünü gizleyen imam, Araplaşan Ârâmî.”135 “Şu üç grup, insanların en şerlileridir: Şeytan er-Recim, Kızgınlığını ve zulmünü gizleyen imam, Araplaşan Ârâmî.”135 Hz. Peygamber’e söylettirilen bu uydurma rivayetler ile de yetinme- yen raviler, Ârâmîler ile birlikte oturmamak için farklı gerekçeler de bul- muşlardır. Bu gerekçelerin kuşkusuz en vahimi Ârâmîlerin soyu ile ilgili olanlardır. Burada dinî duyarlılığın tamamen yok olduğu, Allah korkusunun ortadan kalktığı ve İslâm’ın sarih beyanları ile çelişmekten çekinilmediği görülmektedir. İbn Abbas’tan rivayet edilmiştir: Şeytan İnsan olmuş olsaydı, Aramî olurdu.”136 İbnu’l-Fakih bu rivayetin bir benzerini de Avn b. Abdullah’tan nakletmektedir. Avn’ın rivayetinin devamında “çünkü onlar emrettiklerinde insanların en kibirlileri, sindiril- diklerinde en zelilleri olurlar” ifadesi yer almaktadır.137 Yine İbnu’l- Fakih’in aktardığı bir rivayette İblis ile bir domuz cinsi münasebette bu- lundurulmuş, doğan çocuğun da Ârâmîlerin babası olduğu söylenmiştir. Gerekçe olarak da Aramîlerin sıcağa ve soğuğa dayanıklı oldukları iddia edilmiştir.138 Said b. Müseyyeb anlatıyor: Ârâmîler şeytanın evlatlarıdır. 132 İbnu’l-Fakîh, s. 270 133 İbnu’l-Fakîh, s. Çorum İlahiyat Fakültesi Dergisi, 2002/2 139 İbnu’l-Fakîh, s. 270 140 Bkz. İbnu’l-Fakîh, s. 262; Kazvinî, s. 478 7. Vâsıt Kuruluşu ve Sakinleri ile İlgili Apokrif Rivayetler 270 134 Bkz. İbnu’l-Fakîh, ss. 269-270 135 İbnu’l-Fakîh, s. 270 136 İbnu’l-Fakîh, s. 277 137 İbnu’l-Fakîh, s. 270 138 İbnu’l-Fakîh, s. 270. Ünlü müfessir Mukatil b. Süleyman’a dayandırılan bir haberde şöyle denilmektedir: “Ârâmîler, Şenas b. İblis’in soyundan gelmektedirler. Şenas, Hz. Âdem’e ait Verse adında bir keçiyi nikahladı. Bir oğlu doğdu. Buna da Nebât adını verdi.” Bkz. İbnu’l- Fakîh, ss. 269-271; Çorum İlahiyat Fakültesi Dergisi, 2002/2 170 Yrd. Doç. Dr. M. Mahfuz Söylemez Hz. Ömer’e dayandırılmış olan haberlerden birinde Hz. Peygamber’e Ârâmîlerin cennete giremeyeceğinin söylettirilmiş olması ideolojik duruşun vardığı noktayı gayet güzel ortaya koymaktadır. Hz. Ömer’den rivayet edilmiştir: “Hz. Peygamber buyurdu ki. “Ârâmîler cennete giremeyecetir”.139 Haccâc’ı sevenler kendisi ve icraatları hakkında olumlu haberler uy- dururlarken, ona muhalif olanlar da olumsuz haberler uydurmuşlardır. Simak b. Harb (ö.123/741)’a dayandırılan aşağıdaki haber oldukça entere- sandır. Simak b. Harb diyor ki: “Haccâc beni Badurya bölgesine vali olarak atadı. Günlerden bir gün ben ve arkadaşım Dicle kenarında iken nehrin diğer Simak b. Harb diyor ki: “Haccâc beni Badurya bölgesine vali olarak atadı. Günlerden bir gün ben ve arkadaşım Dicle kenarında iken nehrin diğer tarafında ata binmiş olan bir adam beni, adım ve babamın adı ile çağırdı. Dedim ki, ne istiyorsun? ‘Burada 70.000 kişi mazlum olarak öldürülecek- tir’ dedi ve bunu üç kere tekrar etti. Sonra atını Dicle’ye sürdü ve suda kayboldu. Ertesi yıl kadı beni buraya gönderdiğinde yine ata binmiş bir adam gördüm. Aynı şeyleri söyledi ve aynı yerde kayboldu.”140 Sonuç olarak Hicri 83 yılında Haccâc b. Yusuf tarafından Basra ile Kûfe arasında kurulmuş olan Vâsıt, adını da buradan almaktadır. Abdurrahman b. Muhammed b. el-Eş’as hadisesinde Irak’ın eş idarî mer- kezî olan Kûfe ve Basra’nın onu desteklemiş olması yeni bir idari merkezin kurulmasına sebep olmuştur. Merkezindeki mescit, daru’l-imâre ve mey- dan ile tipik bir İslâm kenti olan Vâsıt, bu merkezi unsurların etrafında bir daire şeklinde inşa edilmiştir. İç içe iki suru bulunan Vâsıt, iç içe üç suru bulunan Bağdat’ın önemli örneklerinden biridir. Vâsıt’ın ilk sakinleri ise Araplar ve Türklerden oluşmaktaydı. Buraya yerleştirilen Türklerin Orta Asya’nın İslâmlaşma sürecinde etkili olmuş olabilecekleri tahmin edilmek- tedir. Şehre daha sonra, kurulduğu bölgenin yerlileri olan Ârâmîler yer- leşmiştir. Çoğunluğunu gayr-i müslimlerin oluşturduğu bu insanlar ile müslüman kitle arasında gayet medenî ilişkilerin geliştiği anlaşılmaktadır. Şehrin Haccâc gibi daha çok olumsuz yönleri ile dikkat çeken bir idareci tarafından kurulmuş olması, hakkında bir çok uydurma rivayetin doğması- na sebep olmuştur. Özet Vâsıt İslam tarihinde önemli misyonlar yüklenmiş bir kent olup Emevilerin I- rak valisi Haccâc b. Yusuf es-Sekafî tarafından Irak’ta, bu günkü Kut- Amâra’ya yakın bir yerde kurulmuştur. Kurulduğu dönemden itabaren Irak’ın idarî merkezliğini de yapmış olan bu şehir, tipik bir İslam kentidir. Merkezin- de mescit, daru’l-imâre ve pazar bulunmaktadır. Buraya kuruluş döneminde sadece Suriye, Kûfe ve Basralı askerler ile Buharalı Türkler yerleştirilmiştir. Emevilerin son dönemi ile Abbasîlerin erken döneminde buraya farklı dinlere mensup olan Aramîlerin de yerleşmelerine izin verilmiştir. Farklı duruşlara Çorum İlahiyat Fakültesi Dergisi, 2002/2 171 Vâsıt Kentinin Kuruluşu ve İlk Sakinleri Üzerine sahip olan Vâsıt sakinlerinin gayet medenî ileşkileri olmuş, aralarında hiçbir çatışma yaşanmamıştır. sahip olan Vâsıt sakinlerinin gayet medenî ileşkileri olmuş, aralarında hiçbir çatışma yaşanmamıştır. 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https://openalex.org/W2803272037	https://digitalcommons.unmc.edu/cgi/viewcontent.cgi?article=1003&context=com_infect_articles	English	null	Determinants of facilitated health insurance enrollment for patients with HIV disease, and impact of insurance enrollment on targeted health outcomes	BMC infectious diseases	2,018	cc-by	673	University of Nebraska Medical Center University of Nebraska Medical Center DigitalCommons@UNMC DigitalCommons@UNMC University of Nebraska Medical Center University of Nebraska Medical Center DigitalCommons@UNMC DigitalCommons@UNMC DigitalCommons@UNMC DigitalCommons@UNMC Journal Articles: Infectious Diseases Infectious Diseases 2018 Determinants of Facilitated Health Insurance Enrollment for Determinants of Facilitated Health Insurance Enrollment for Patients With HIV Disease, and Impact of Insurance Enrollment on Patients With HIV Disease, and Impact of Insurance Enrollment on Targeted Health Outcomes. Targeted Health Outcomes. Renae Furl University of Nebraska Medical Center, rfurl@unmc.edu Shinobu Watanabe-Galloway University of Nebraska Medical Center, swatanabe@unmc.edu Elizabeth Lyden University of Nebraska Medical Center, elyden@unmc.edu Susan Swindells University of Nebraska Medical Center, sswindells@unmc.edu Tell us how you used this information in this short survey. Follow this and additional works at: https://digitalcommons.unmc.edu/com_infect_articles Part of the Infectious Disease Commons Recommended Citation Recommended Citation Furl, Renae; Watanabe-Galloway, Shinobu; Lyden, Elizabeth; and Swindells, Susan, "Determinants of Facilitated Health Insurance Enrollment for Patients With HIV Disease, and Impact of Insurance Enrollment on Targeted Health Outcomes." (2018). Journal Articles: Infectious Diseases. 4. https://digitalcommons.unmc.edu/com_infect_articles/4 This Article is brought to you for free and open access by the Infectious Diseases at DigitalCommons@UNMC. It has been accepted for inclusion in Journal Articles: Infectious Diseases by an authorized administrator of DigitalCommons@UNMC. For more information, please contact digitalcommons@unmc.edu. This Article is brought to you for free and open access by the Infectious Diseases at DigitalCommons@UNMC. It has been accepted for inclusion in Journal Articles: Infectious Diseases by an authorized administrator of DigitalCommons@UNMC. For more information, please contact digitalcommons@unmc.edu. Recommended Citation Recommended Citation Furl, Renae; Watanabe-Galloway, Shinobu; Lyden, Elizabeth; and Swindells, Susan, "Determinants of Facilitated Health Insurance Enrollment for Patients With HIV Disease, and Impact of Insurance Enrollment on Targeted Health Outcomes." (2018). Journal Articles: Infectious Diseases. 4. https://digitalcommons.unmc.edu/com_infect_articles/4 Furl, Renae; Watanabe-Galloway, Shinobu; Lyden, Elizabeth; and Swindells, Susan, "Determinants of Facilitated Health Insurance Enrollment for Patients With HIV Disease, and Impact of Insurance Enrollment on Targeted Health Outcomes." (2018). Journal Articles: Infectious Diseases. 4. https://digitalcommons.unmc.edu/com_infect_articles/4 This Article is brought to you for free and open access by the Infectious Diseases at DigitalCommons@UNMC. It has been accepted for inclusion in Journal Articles: Infectious Diseases by an authorized administrator of DigitalCommons@UNMC. 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https://openalex.org/W2249844418	http://pdf.blucher.com.br/chemicalengineeringproceedings/cobeq2014/0575-24878-164495.pdf	Portuguese	null	MODELAGEM MATEMÁTICA DO CRESCIMENTO DE Lactobacillus viridescens SOB CONDIÇÕES NÃO-ISOTÉRMICAS	null	2,015	cc-by	3,064	1 Universidade Federal de Santa Catarina, Departamento de Engenharia Química e Engenharia de Alimentos, Florianópolis/SC, Brasil 2 1 Universidade Federal de Santa Catarina, Departamento de Engenharia Química Alimentos, Florianópolis/SC, Brasil 2 Universidade Federal de Rondônia, Departamento de Engenharia de Alimentos, Ariquemes/RO, Brasil 3 Universidade Federal do Paraná, Departamento de Engenharia de Alimentos, Jandaia do Sul/PR, Brasil E-mail para contato: glaucia@enq.ufsc.br RESUMO – As bactérias ácido lácticas (BAL) estão entre os principais grupos de microrganismos responsáveis pela deterioração de produtos cárneos refrigerados, embalados a vácuo e em atmosfera modificada. O crescimento das BAL pode ser predito por modelos matemáticos que descrevem a influência de diferentes fatores ambientais (como condições não-isotérmicas) sobre o crescimento microbiano. O objetivo deste trabalho foi obter um modelo matemático capaz de predizer o crescimento de Lactobacillus viridescens em condições não-isotérmicas de cultivo em meio MRS. As curvas de crescimento foram descritas pelo modelo de Baranyi e Roberts, e a dependência do parâmetro µmax com a temperatura foi descrita pelo modelo da raiz quadrada. O modelo proposto foi validado com dados experimentais de L. viridescens, com a temperatura variando de 5 a 11 ºC, e de 6 a 10 ºC a cada 24 h. Os resultados mostraram que foi possível predizer de forma segura, confirmado através dos índices estatísticos, o crescimento de L. viridescens em meio MRS em condições não-isotérmicas. A. TREMARIN1, J. C. C. P. COSTA1,2, D. A. LONGHI1,3, A. P. R. SILVA1, C. C. M. PAGANINI1, J. B. LAURINDO1, G. M. F. ARAGÃO1 A. TREMARIN1, J. C. C. P. COSTA1,2, D. A. LONGHI1,3, A. P. R. SILVA1, C. C. M. PAGANINI1, J. B. LAURINDO1, G. M. F. ARAGÃO1 1 Universidade Federal de Santa Catarina, Departamento de Engenharia Química e Engenharia de Alimentos, Florianópolis/SC, Brasil 1 Universidade Federal de Santa Catarina, Departamento de Engenharia Química e Engenharia de Alimentos, Florianópolis/SC, Brasil 1. INTRODUÇÃO A deterioração de alimentos devido à ação de microrganismos é um problema de grande importância que pode gerar grandes perdas econômicas para as indústrias, tornando fundamental a aplicação de tecnologias para prever a vida útil e obter produtos com maior qualidade (Zuera- Consano et al., 2006; Pexara et al., 2002). O comportamento dos microrganismos está intimamente associado com a disponibilidade e qualidade do alimento para o consumo humano, bem como pelas condições de armazenamento (Sperber e Doyle, 2009). As bactérias ácido lácticas (BAL) pertencem ao grupo das bactérias Gram positivas, não formadoras de esporos, estritamente fermentativas produzindo o ácido láctico como principal produto final (Lyhs e Bjorkroth, 2008). O crescimento incontrolável de algumas espécies resulta em um 1 Área temática: Engenharia e Tecnologia de Alimentos 1 impacto negativo como contaminantes, causando a deterioração de produtos embalados a vácuo, em atmosfera modificada, além de produtos cárneos processados armazenados sob temperatura de refrigeração (Françoise, 2010). A deterioração por essas bactérias é principalmente devido à produção de metabólitos, que influenciam na aparência, textura de off-flavors no alimento, causando sabores e odores desagradáveis (Carr et al., 2002; Nychas et al., 2008). impacto negativo como contaminantes, causando a deterioração de produtos embalados a vácuo, em atmosfera modificada, além de produtos cárneos processados armazenados sob temperatura de refrigeração (Françoise, 2010). A deterioração por essas bactérias é principalmente devido à produção de metabólitos, que influenciam na aparência, textura de off-flavors no alimento, causando sabores e odores desagradáveis (Carr et al., 2002; Nychas et al., 2008). A microflora natural das carnes e produtos cárneos embalados a vácuo ou em atmosfera modificada é composta por Lactobacillus spp.. Entre as espécies predominantes a L. viridescens é considerada uma das espécies mais comuns na deterioração de produtos cárneos, produzindo peróxidos que reagem com os pigmentos da carne, causando o esverdeamento da mesma (Park et al., 2001). O conhecimento e o controle da microbiota presente na matéria-prima durante o processamento cárneo são essenciais para a garantia da qualidade microbiológica e características sensoriais do produto final. Para avaliar o crescimento microbiano de forma mais rápida e precisa, a microbiologia preditiva vem sendo amplamente utilizada pelo desenvolvimento de modelos matemáticos precisos e versáteis, aptos a descrever a evolução do crescimento microbiano em produtos alimentícios como função de condições ambientais conhecidas ou mensuráveis (Van Impe et al., 2005). 1. INTRODUÇÃO Os modelos preditivos têm o objetivo de avaliar o efeito das operações de processamento e das condições de distribuição e armazenamento na segurança e qualidade microbiológica dos alimentos (McMeekin et al., 2002). Durante o período de estocagem e distribuição dos alimentos a temperatura, na qual estes alimentos estão submetidos, pode sofrer grandes variações. Como a maioria dos modelos preditivos são desenvolvidos para condições de temperatura constante, ocorre a necessidade do desenvolvimento de modelos não-isotérmicos que possam avaliar a influência da flutuação de temperatura ao longo do tempo. Estes modelos são muito válidos para a previsão da vida útil dos alimentos, à medida que eles podem simular as condições reais nas quais os alimentos são submetidos (Koutsoumanis et. al, 2006; Cayré et al., 2003). O objetivo deste trabalho foi obter um modelo matemático capaz de predizer o crescimento de Lactobacillus viridescebs em condições não-isotérmicas de cultivo em meio MRS. 2.1. Microrganismo e preparo do inóculo A cepa de L. virisdescens (CCT 5843 ATCC 12706, Lote 22.07) foi adquirida na forma liofilizada da coleção de Culturas André Tosello (Fundação Tropical de Culturas André Tosello, Campinas, Brasil). A cepa foi reidratada conforme indicação e cultivada em caldo de Man, Rogosa e Sharpe (MRS) (Difco, Le Pont de Claix, França). Em seguida, foi estocada em microtubos de polipropileno com caldo MRS contendo 20 % de glicerol à temperatura de – 24 ºC para posterior utilização. Para o preparo do inóculo, a cepa congelada a – 24 ºC foi reativada em caldo MRS a 30 ºC em estufa (Dist, Florianópolis, Brasil), por 18 h. Área temática: Engenharia e Tecnologia de Alimentos 2 2 2.2. Condições de crescimento não-isotérmico Em estudos anteriores foi avaliada a influência de diferentes temperaturas isotérmicas sobre os parâmetros de crescimento de L. viridescens, obtidos pelo modelo primário de Baranyi e Roberts (Baranyi e Roberts, 1994). Mediante a obtenção destes parâmetros de crescimento foram estudados alguns perfis de temperatura não-isotérmico. Os experimentos foram realizados em frascos de 500 mL, com volume útil de 320 mL e com 1 % (v/v) de inóculo. A concentração inicial dos experimentos foi de aproximadamente 103 UFC.mL-1 e o pH inicial de 6,0 (pH-metro V620, Analion, Ribeirão Preto, Brasil). Os frascos foram incubados em estufa (Dist, Florianópolis, Brasil) em dois perfis de temperatura que variaram entre 5-11 °C e 6- 10 °C a cada 24 h, até que as curvas de crescimento atingissem a fase estacionária. A temperatura mantida dentro das estufas de incubação foi registrada por um datalogger (Testo 174, Lenzkirch, Alemanha) com registro de temperatura e tempo a cada 5 minutos. 2.3. Construção das curvas de crescimento Para a construção das curvas de crescimento utilizou-se o método clássico de contagem de células viáveis para determinar o crescimento de L. viridescens ao longo do tempo até que a mesma atingisse a fase estacionária. No plaqueamento foi utilizado Agar MRS (Difco, Le Pont de Claix, França) com dupla camada, em placas de Petri estéreis descartáveis, incubadas a 30 ºC por 48 h em estufa (Dist, Florianópolis, Brasil). A contagem foi realizada a partir das placas com 30 a 300 colônias, sendo a contagem expressa em UFC.mL-1. 2.5. Validação do modelo não-isotérmico de L. viridescens A capacidade preditiva dos modelos não-isotérmicos de crescimento foi analisada pela aplicação dos índices estatísticos: coeficiente de determinação (R²) (Equação 7), raiz do erro médio quadrático (RMSE) (Equação 8), fator bias (Equação 9) e fator exatidão (Equação 10) (Ross, 1996).             n i observado n i predito observao média valor valor valor R 1 2 1 2 2 1 (7)   p n valor valor p n RSS predito observado       2 RMSE (8)          n Valor / Valor log observado predito 10 bias fator (9)            n Valor / Valor log observado predito 10 exatidão fator (10) RESULTADOS E DISCUSSÃO (7) (8) (9) (10) ção do crescimento de L. viridescens em condições não-isotérmicas Os modelos primário de Baranyi e Roberts (Baranyi e Roberts, 1994), Equações (1) e (2), e secundário de Ratkowsky et al. (1982), Equação (3), foram utilizados para predizer o crescimento microbiano em condições não-isotérmicas Nas Equações (1), (2) e (3), y(t) é o logaritmo natural da concentração celular (y = ln(N)) no tempo t; Q(t) é o estado fisiológico das células no tempo t, µmax é a velocidade máxima específica de crescimento; e ymax é o logaritmo natural da concentração celular máxima.     max ) ( max ) ( exp 1 1 1 ) ( y t y e dt t dy t Q           (1) max ) (   dt t dQ (2) ) 33 ,1 ( 029 ,0 max   T  (3)     max ) ( max ) ( exp 1 1 1 ) ( y t y e dt t dy t Q           (1) max ) (   dt t dQ (2) ) 33 ,1 ( 029 ,0 max   T  (3) (1) (2) (3) (3) As Equações (4) e (5) apresentam as condições iniciais que possibilitam a solução das equações diferenciais (1) e (2), sendo que y0 é o valor do logaritmo natural da concentração celular inicial, e Q0 As Equações (4) e (5) apresentam as condições iniciais que possibilitam a solução das equações diferenciais (1) e (2), sendo que y0 é o valor do logaritmo natural da concentração celular inicial, e Q0 Área temática: Engenharia e Tecnologia de Alimentos 3 Área temática: Engenharia e Tecnologia de Alimentos Área temática: Engenharia e Tecnologia de Alimentos 3 3 3 é o valor do estado fisiológico inicial das células. é o valor do estado fisiológico inicial das células. 0 ) 0 ( y y  0 )0( Q Q  0 ) 0 ( y y  0 )0( Q Q  (5) 3. RESULTADOS E DISCUSSÃO A partir dos modelos primários e secundários estabelecidos pelas Equações (1), (2) e (3) foi possível predizer o crescimento de L. viridescens sob condições de temperaturas que variam ao longo do tempo dentro da faixa de temperaturas estudadas (4 °C a 30 °C). As Figuras 1 e 2 apresentam os resultados da simulação do crescimento microbiano nas condições não-isotérmicas dos perfis 5-11 °C e 6-10 °C, respectivamente. Para ambos os perfis de temperatura, as curvas de crescimento não-isotérmicas preditas pelos modelos matemáticos superestimaram o crescimento de L. viridescens com relação aos dados experimentais, principalmente na fase exponencial de crescimento. Área temática: Engenharia e Tecnologia de Alimentos Área temática: Engenharia e Tecnologia de Alimentos 4 4 Figura 1 – Curva de crescimento de Lactobacillus viridescens sob condições não-isotérmicas predita pelo modelo matemático (linha contínua) e obtida experimentalmente (pontos) no perfil de temperatura 5-11 ºC (linha tracejada). Figura 1 – Curva de crescimento de Lactobacillus viridescens sob condições não-isotérmicas predita pelo modelo matemático (linha contínua) e obtida experimentalmente (pontos) no perfil de temperatura 5-11 ºC (linha tracejada). Figura 2 – Curva de crescimento de Lactobacillus viridescens sob condições não-isotérmicas predita pelo modelo matemático (linha contínua) e obtida experimentalmente (pontos) no perfil de temperatura 6-10 ºC (linha tracejada). Figura 2 – Curva de crescimento de Lactobacillus viridescens sob condições não-isotérmicas predita pelo modelo matemático (linha contínua) e obtida experimentalmente (pontos) no perfil de temperatura 6-10 ºC (linha tracejada). Os valores dos índices estatísticos de comparação entre os dados experimentais e a curva de predição do modelo matemático são apresentados na Tabela 1. Os valores encontrados para os índices estatísticos indicam que o modelo avaliado fornece predições seguras do crescimento de L. viridescens em meio de cultivo, pois o valor do fator bias para ambos os perfis de temperatura foi maior que um. Tabela 1 – Valores dos índices estatísticos R2, RMSE, fator bias e fator exatidão da comparação entre os dados experimentais e a curvas de predições do modelo matemático para os perfis de temperatura 5-11 °C e 6-10 °C. 3. RESULTADOS E DISCUSSÃO 5 Área temática: Engenharia e Tecnologia de Alimentos 5 Perfil de Temperatura Índices Estatísticos R2 RMSE Fator Bias Fator Exatidão 5-11 °C 0.916 1.652 1.758 2.013 6-10 °C 0.931 1.539 1.748 1.900 As variações bruscas entre as temperaturas dos experimentos (entre 5 e 11 °C, e entre 6 e 10 °C) podem ter levado o microrganismo a apresentar fases de adaptação intermediárias, as quais tem sido reportadas na literatura por outros autores (Swinnen et al., 2005; Longhi et al., 2013). Uma tentativa de predizer o crescimento de L. plantarum com variações bruscas na temperatura foi conduzida por Zwietering et al. (1994) usando o modelo de Gompertz. Os autores adicionaram termos empíricos para descrever as fases de adaptação intermediárias apresentadas pelo microrganismo, sendo que a modificação no modelo aumentou a capacidade preditiva em 73% dos casos. Entretanto, nos casos em que a temperatura do experimento foi próxima da temperatura mínima de crescimento do microrganismo (como é o caso do presente estudo), as predições com o modelo modificado não foram satisfatórias. Desta forma, a construção de modelos matemáticos que consigam predizer o crescimento microbiano para mudanças bruscas de temperatura próximas da temperatura mínima de crescimento continuam sendo um desafio para os pesquisadores. Alguns trabalhos na linteratura também tem apresentado problemas de predição ao avaliar o crescimento de outros microrganismos com variação brusca de temperatura próximo da temperatura mínima de crescimento, por exemplo, o crescimento de Pseudomonas spp. em peixe refrigerado entre 5 e 15 °C (Corradini e Peleg, 2005), e o crescimento de Listeria monocytogenes em leite pasteurizado entre 4 e 12 °C, e entre -2 e 6 °C (Xanthiakos et al., 2006). 4. CONCLUSÃO O modelo matemático proposto foi validado com os dados experimentais de L. viridescens para os perfis de variação de temperatura entre 5 e 11 ºC, e entre 6 e 10 ºC a cada 24 h. Os resultados mostraram que foi possível predizer de forma segura (fator bias maior que um) o crescimento de L. viridescens em meio MRS em condições não-isotérmicas. CARR, F. J.; CHILL, D.; MAIDA, N. The lactic bacteria a literature survey. Critical Reviews in Microbiology, v. 28, n. 4, p. 281-370, 2002. CAYRÉ, M. E.; VIGNOLO, G.; GARRO, O. Modeling lactic acid bacteria growth in vacuum- packaged cooked meat emulsion stored at three temperatures. Food Microbiology, v. 20, p. 561-566, 2003. CORRADINI, M.; PELEG, M. Estimating non-isothermal bacterial growth in foods from isothermal experimental data. Journal of Applied Microbiology, v. 99, p. 187-200, 2005. FRANÇOISE, L. Occurrence and role of lactic acid bacteria in seafood products. Food Microbiology, v. 27, p. 698-709, 2010. KOUTSOUMANIS, K. P.; STAMATIOU, A.; SKANDAMIS, P.; NYCHAS, G. J. Development of a microbial for the combined effect of temperature and pH on spoilage of ground meat and validation of the model under dynamic temperature conditions. Applied and Environmental Microbiology, v. 71, p. 124-134, 2006. LYHS, U.; BJORKROHT. Lactobacillus sakei curvatus is the prevailing lactic acid bacterium group in spoiled maatjes herring. Food Microbiology, v. 25, p. 529-533, 2008. LONGHI, D. A.; DALCANTON, F.; ARAGÃO, G. M. F.; CARCIOFI, B. A. M.; LAURINDO, J. B. Assessing the prediction ability of different mathematical models for the growth of Lactobacillus plantarum under non-isothermal conditions. Journal of Theoretical Biology, v. 335, p. 88-96, 2013. McMEEKIN, T. A.; OLLEY, J. N.; RATKOSWSKY, D. A.; ROSS, T. Predictive microbiology: towards the interface and beyond. International Journal of Food Microbiology, v. 73, p. 395-407, 2002. NYCHAS, G. J. E.; SKANDAMIS, P. N.; TASSOU, C. C.; KOUTSOUMANIS, K. P. Meat spoilage during distribution. Meat Science, v. 78, p. 77-89, 2008. PARK, S. W.; SOHN, K. H.; SHIN, J. H.; LEE, H. J. High hydrostatic pressure inactivation of Lactobacillus viridescens and its effects on ultrastructure of cells. International Journal of Food Science and Technology, v. 36, p. 775-781, 2001. PEXARA, E. S.; METAXPOULOS, J.; DROSINOS, E. H. Evaluation of shelf life of cured, cooked, sliced turkey fillets and cooked pork sausages – piroski – stored under vacuum and modified atmosphere at + 4 and + 10 °C. Meat Science, v. 62, p. 33-43, 2002. RATKOWSKY, D.; OLLEY, J.; MCMEEKIN, T.; BALL, A. Relationship between temperature and growth-rate of bacterial cultures. Journal of Bacteriology, v. 149, p. 1-5, 1982. ROSS, T. Indices for performance evaluation of predictive models in food microbiology. Journal of Applied Bacteriology, v. 81, p. 501-508, 1996. SPERBER, W. H.; DOYLE, M. P. Compendium of the microbiological spoilage of foods an beverages. London: Springer, 2009. SWINNEN, I. A. M.; BERNAERTS, K.; GYSEMANS, K.; VANIMPE, J. F. 5. REFERÊNCIAS BARANYI, J.; ROBERTS, T. A. A dynamic approach to predicting bacterial growth in food. International Jounal of Food Microbiology, v. 23, p. 277-294, 1994. CARR, F. J.; CHILL, D.; MAIDA, N. The lactic bacteria a literature survey. Critical Reviews in Microbiology, v. 28, n. 4, p. 281-370, 2002. 6 Área temática: Engenharia e Tecnologia de Alimentos 6 Área temática: Engenharia e Tecnologia de Alimentos 8 Quantifying microbial lag phenomena due to a sudden rise in temperature: a systematic macroscopic study. International Journal of Food Microbiology, v. 100, p. 85-96, 2005. VAN IMPE, J. F.; POSCHET, F.; GEERAERD, A. H.; VEREECKEN, K. M. Towards a novel class 7 7 of predictive microbial growth models. International Journal of Food Microbiology, v. 100, p. 97- 105, 2005. of predictive microbial growth models. International Journal of Food Microbiology, v. 100, p. 97- 105, 2005. of predictive microbial growth models. International Journal of Food Microbiology, v. 100, p. 97- 105, 2005. XANTHIAKOS, K.; SIMOS, D.; ANGELIDIS, A.; NYCHAS, G.; KOUTSOUMANIS, K. Dynamic modeling of Listeria monocytogenes growth in pasteurized milk Journal of Applied Microbiology, v. 100, p. 1289-1298, 2006. ZWIETERING, M. H.; WIT, J. C.; CUPPERS, H. G. A. M.; VAN’T RIET, K. Modeling of Bacterial Growth with Shifts in Temperature. Applied and Environmental Microbiology, v. 60, n. 1, p. 204-213, 1994. ZURERA-CONSANO, G.; GARCÍA-GIMENO, R. M.; RODÍGUEZ-PÉREZ, M. R.; HERVÁS- MARTÍNES, C. Validation an artificial neural network model of Leuconostoc mesenteroides in vacuum packaged sliced cooked meat products for shelf life estimation. Food Control, v. 17, p. 429- 438, 2006. Área temática: Engenharia e Tecnologia de Alimentos 8 8
https://openalex.org/W2883156727	https://www.matec-conferences.org/10.1051/matecconf/201817805005/pdf	English	null	An overview of possibilities for CAM machining	MATEC web of conferences	2,018	cc-by	3,514	1 Introduction Today’s CAM software solution have to provide tools in such a way that programmers have at their disposal all the instruments needed to get the job done. But CAM solutions have to encapsulate the same features for a variety of modern NC centers and it has to address each one in the same manner. Whether it is Mazak or Hurco NC centers, or Fanuc versus Sinumerik controllers CAM solutions must know their capabilities, acknowledge them and take advantage of their capabilities [1]. A modern CAM software solution knows about high speed machining versus prolonged tool life and is able to provide support to the programmer in sense of manufacturing strategies. Knowledge about software capabilities and the strategies that may be applied as well as the fact that it can be implemented onto existing machining equipment is a must for a senior programmer [2-3]. This paper aims to provide useful information about milling CAM operations and their machining optimization strategies and how software aids at their implementation. * Corresponding author: andrei.mihalache@yahoo.com An overview of possibilities for CAM machining Andrei Marius Mihalache* “Gheorghe Asachi” Technical University of Iasi, Department of Machine Manufacturing Technology, Boulevard Dimitrie Mangeron 59A, Iasi, Romania Andrei Marius Mihalache* “Gheorghe Asachi” Technical University of Iasi, Department of Machine Manufacturing Technology, Boulevard Dimitrie Mangeron 59A, Iasi, Romania Abstract. There is an entire array of options when you are planning a CAM machining strategy. Knowing your equipment possibilities and adjusting your software options to them has become mandatory in terms of cost and time efficiency. This paper aims to present the main options for most common software solutions used today in modern mechanical engineering which can be applied to the existing hardware manufacturing equipment found in enterprises or educational centers. © 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/). MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 https://doi.org/10.1051/matecconf/201817805005 2.2 Compound job 2.2 Compound job A machining sequence encapsulate all the machining processes as a single task or job. Jobs may be then introduced in an array thus the name of compound job list giving users the opportunity to structure them considering position, tool orientation or the machining step itself. The jobs are available inside a network making them visible or hidden to specific users according to the needs. Fig. 2. Batch of multiple jobs. They can be grouped and exported as a batch and may contain thousands of individual tasks that are available offline as a package. p j A machining sequence encapsulate all the machining processes as a single task or job. Jobs may be then introduced in an array thus the name of compound job list giving users the opportunity to structure them considering position, tool orientation or the machining step itself. The jobs are available inside a network making them visible or hidden to specific users according to the needs. Fig. 2. Batch of multiple jobs. They can be grouped and exported as a batch and may contain thousands of individual tasks that are available offline as a package. Fig. 2. Batch of multiple jobs. Fig. 2. Batch of multiple jobs. Jobs may be then introduced in an array thus the name of compound job list giving users the opportunity to structure them considering position, tool orientation or the machining step itself. The jobs are available inside a network making them visible or hidden to specific users according to the needs. Fig. 2. Batch of multiple jobs. They can be grouped and exported as a batch and may contain thousands of individual tasks that are available offline as a package. 2.1 Fast results 2.1 Fast results Fig. 1. Intuitive setup for faster results. This means that the system allows fast programming providing all the related tools in sequenced windows for a specific task with minimal errors. The software solution has automated functions dependent of each other that self-adjust on modification of various parameters. It provides a task oriented management system and all the way indications and descriptions of issues or errors in such a way that input data will be clear and within range. Fig. 1. Intuitive setup for faster results. There is a progress status displayed and any changes are automatically propagated throughout the system. There is also the possibility to copy the entire setup or just machining steps or tools or features and to easy adjust them to the required need. The system automatically recognizes erroneous entries and highlights them. Modern CAM software solutions allow multiple sequences of different projects thus making it possible for a project to be let’s say processed as other is being programmed [6]. On the other hand a management window allows multiple machining strategies to be part of the same job listed according to the programmer needs. It incorporates the CAD model with all relevant links available. 2.2 Compound job 2 General capabilities All modern CAM software solution have certain capabilities that form industry standards. They are about the possibility to provide fast and accurate results under user friendly settings, about encapsulation of different operations in batches and export them in such a manner that they would be available offline, the possibility for interchangeable settings and standard friendly formats as well as the possibility to program in a certain manner that would be recognizable by other software solutions in terms of parameter setup and the changes that are made upon them [4]. CAM software solutions have to understand the machining strategy that the programmer had in mind and to be able to output the right numerical code for the right equipment [5]. https://doi.org/10.1051/matecconf/201817805005 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 2.1 Fast results 2.3 Associative programming The software allows users to work on copies of the same job because often it is the case that just few parameters have to be adjusted for a new machining as the strategy stays the same. All the parameters are linked inside a reference job and any modification made to the original will be inherit by the parameters untouched in the copies that have been made. But copy related parameters may be unlinked and modified without affecting any other as well. Furthermore setups from a particular software solution may be exported with all relevant information including the CAD model that has been used in a general format that is readable by other CAM related solutions as well as for offline use [7]. 2 2 https://doi.org/10.1051/matecconf/201817805005 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 2.4 Parameter programming Programming in sequence allows the user to adjust each parameter for a flexible workflow. The user is aware at all-time about dependencies and how changes affect the overall process as variables are modified. This strategy also allows the software to implement changes about different setups or equipment as defining new zero points, accommodate additional clamping or adjusting tool positions [8]. Fig. 3. Parameter extended setup. It also allows definition of multiple zero points inside a compound job. 2.4 Parameter programming 2.4 Parameter programming Programming in sequence allows the user to adjust each parameter for a flexible workflow. The user is aware at all-time about dependencies and how changes affect the overall process as variables are modified. This strategy also allows the software to implement changes about different setups or equipment as defining new zero points, accommodate additional clamping or adjusting tool positions [8]. Fig. 3. Parameter extended setup. It also allows definition of multiple zero points inside a compound job. Fig. 3. Parameter extended setup. Fig. 3. Parameter extended setup. 3.1 Face milling This operation allows programming of large flat areas to be quickly machined. The tool moves one way or in zig-zag and multiple surfaces may be included in a single operation. It is very appropriate if the user has to program the numerical control code for parts that are mostly constituted of faces like squares or cubes which eventually have holes or other geometric features to be later machined. Fig. 4. Face milling setup. 3.2 Pocket milling The software has the means to process any type of pocket, open or closed, circular, square or other shape with various heights or depths. There is the possibility to define an outer starting point outside the machining area or if sequenced an intermediate step considers a ramping position or a helical movement to allow the tool to enter and continue the machining process depending on the type of tool and settings. Fig. 5. Pocket milling setup. The software also allows such movements to be preserved and saved as a machining cycle for future repetitive processes [9]. The strategy consists of computing the minimal number of rapid movements and the times between those or other strategies as Automatic feature recognition (see Figure 6), Complete machining of the bottom (see Figure 7) and Supports 2D controller cycles (see Figure 8). The software has the means to process any type of pocket, open or closed, circular, square or other shape with various heights or depths. There is the possibility to define an outer starting point outside the machining area or if sequenced an intermediate step considers a ramping position or a helical movement to allow the tool to enter and continue the machining process depending on the type of tool and settings. Fig. 5. Pocket milling setup. The software also allows such movements to be preserved and saved as a machining cycle for future repetitive processes [9]. The strategy consists of computing the minimal number of rapid movements and the times between those or other strategies as Automatic feature recognition (see Figure 6), Complete machining of the bottom (see Figure 7) and Supports 2D controller cycles (see Figure 8). Fig. 5. Pocket milling setup. The software has the means to process any type of pocket, open or closed, circular, square or other shape with various heights or depths. There is the possibility to define an outer starting point outside the machining area or if sequenced an intermediate step considers a ramping position or a helical movement to allow the tool to enter and continue the machining process depending on the type of tool and settings. The software has the means to process any type of pocket, open or closed, circular, square or other shape with various heights or depths. 3 CAM milling capabilities 3.1 Face milling This operation allows programming of larg flat areas to be quickly machined. The to moves one way or in zig-zag and multip surfaces may be included in a sing operation. It is very appropriate if the us has to program the numerical control cod for parts that are mostly constituted of fac like squares or cubes which eventually hav holes or other geometric features to be lat machined. Fig. 4. Face milling setup. 3.1 Face milling Fig. 4. Face milling setup. 3.1 Face milling Fig. 4. Face milling setup. 3.1 Face milling 3.2 Pocket milling There is the possibility to define an outer starting point outside the machining area or if sequenced an intermediate step considers a ramping position or a helical movement to allow the tool to enter and continue the machining process depending on the type of tool and settings. Fig. 5. Pocket milling setup. Fig. 5. Pocket milling setup. The software also allows such movements to be preserved and saved as a machining cycle for future repetitive processes [9]. The strategy consists of computing the minimal number of rapid movements and the times between those or other strategies as Automatic feature recognition (see Figure 6), Complete machining of the bottom (see Figure 7) and Supports 2D controller cycles (see Figure 8). The software also allows such movements to be preserved and saved as a machining cycle for future repetitive processes [9]. The strategy consists of computing the minimal number of rapid movements and the times between those or other strategies as Automatic feature recognition (see Figure 6), Complete machining of the bottom (see Figure 7) and Supports 2D controller cycles (see Figure 8). 3 3 https://doi.org/10.1051/matecconf/201817805005 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 IManE&E 2018 Fig. 6. Automatic feature recognition. Fig. 6. Automatic feature recognition. Fig. 7. Complete machining of the bottom. Fig. 8. Supports 2D controller cycles. Fig. 8. Supports 2D controller cycles. Fig. 7. Complete machining of the bottom. Fig. 6. Automatic feature recognition. Fig. 8. Supports 2D controller cycles. Fig. 7. Complete machining of the bottom. Fig. 7. Complete machining of the bottom. 3.3 Contour milling 3.3 Contour milling This operation is used when milling complex contours. The software takes into account G41/G42 tool paths compensation as the tool can start machining from the center path or follow the contour’s borderlines. It also detects areas that cannot be machined inside the same operation, bottle-necks, wrong retract moves or self-cuts within the containment zone. There are also aids in form of optimizations regarding the sorting of tool paths which are very helpful when dealing with multiple contours. Fig. 9. Contour milling tool path. Fig. 9. Contour milling tool path. Fig. 9. Contour milling tool path. This operation is used when milling complex contours. The software takes into account G41/G42 tool paths compensation as the tool can start machining from the center path or follow the contour’s borderlines. It also detects areas that cannot be machined inside the same operation, bottle-necks, wrong retract moves or self-cuts within the containment zone. There are also aids in form of optimizations regarding the sorting of tool paths which are very helpful when dealing with multiple contours. Fig. 9. Contour milling tool path. The software provides tips and identifies possible starting points taking into account overall tool moves and how to minimize them [10]. Also, the tool moves may be optimized in terms of Approach and Retract in such ways that we would have minimum distances or collision-free machining to ensure that infeed and transition movements are performed in the most suitable areas of the analyzed contours. contours. Fig. 10. Self-cut and bottleneck check- up. Fig. 11. Spiral machining down the bottom. Fig. 12. Fillet outside edges. Fig. 13. Z-infeed trimming against the model. Out of optimization functions the user has the possibility to choose from Self-cut and bottleneck check-up (see Figure 10), Spiral machining down the bottom (see Figure 11), Fillet outside edges (see Figure 12), Z-infeed trimming against the model (see Figure 13) and 2D multiple lateral infeed (see Figure 14). Fig. 14. 2D multiple lateral infeed. Fig. 11. Spiral machining down the bottom. Fig. 11. Spiral machining down the bottom. Fig. 13. Z-infeed trimming against the model. Fig. 10. Self-cut and bottleneck check- up. Fig. 10. Self-cut and bottleneck check- up. Fig. 13. Z-infeed trimming against the model. Fig. 13. Z-infeed trimming against the model. Fig. 12. Fillet outside edges. Fig. 11. Spiral machining down the bottom. Fig. 14. 2D multiple lateral infeed. Fig. 14. 2D multiple lateral infeed. 3.4 Rest machining 3.4 Rest machining It is not always possible to machine contours or pockets using the same tool as you did for large surfaces. This involves a more accurate approach thus a new strategy. It computes distinct tool paths for smaller tools. The software points out and highlights the areas that will be left unprocessed and proposes alternatives. Areas may not be only inside closed contours but the software finds them even if they are on different geometries. Fig. 15. Rest machining setup. Fig. 15. Rest machining setup. This strategy ensures a perfect finish of all areas by combining tool changes where they are necessary keeping in mind at all time a cost-time ratio [3]. As machining strategies we could mention Residual stock machined with a previous cycle or Tangential infeed for best contours. This strategy ensures a perfect finish of all areas by combining tool changes where they are necessary keeping in mind at all time a cost-time ratio [3]. As machining strategies we could mention Residual stock machined with a previous cycle or Tangential infeed for best contours Fig. 17. Tangential infeed for best contours. f f Fig. 16. Residual stock machined with a previous cycle. Fig. 17. Tangential infeed for best contours. f f Fig. 16. Residual stock machined with a previous cycle. F c f f Fig. 16. Residual stock machined with a previous cycle. Fig. 16. Residual stock machined with a previous cycle. Fig. 17. Tangential infeed for best contours. 3.3 Contour milling Out of optimization functions the user has the possibility to choose from Self-cut and bottleneck check-up (see Figure 10), Spiral machining down the bottom (see Figure 11), Fillet outside edges (see Figure 12), Z-infeed trimming against the model (see Figure 13) and 2D multiple lateral infeed (see Figure 14). Out of optimization functions the user has the possibility to choose from Self-cut and bottleneck check-up (see Figure 10), Spiral machining down the bottom (see Figure 11), Fillet outside edges (see Figure 12), Z-infeed trimming against the model (see Figure 13) and 2D multiple lateral infeed (see Figure 14). Fig. 14. 2D multiple lateral infeed. Fig. 14. 2D multiple lateral infeed. 4 https://doi.org/10.1051/matecconf/201817805005 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 3.5 Drilling The software offer possibilities for Centering, Simple drilling, Deep hole drilling, Deep drilling, Drilling with chip break, Reaming and Boring, Thread milling. Those are especially helpful when they are combined with so-called macro-milling. The machining can occur on its own or combined with other operations in a single job, compound jobs, subroutines or cycles [5]. They are available also for automatic recognition programming. From the optimization point of view the user may consider for Drilling the shortest path, X- parallel (see Figure 19) or Helical drilling with freely definable pitch (see Figure 20). Fig. 18. Identified drilling geometric features. Fig. 18. Identified drilling geometric features. Fig. 18. Identified drilling geometric features. Fig. 18. Identified drilling geometric features. 5 5 https://doi.org/10.1051/matecconf/201817805005 MATEC Web of Conferences 178, 05005 (2018) IManE&E 2018 The machining optimization strategy allows the user to define the pitch of the spiral. The other strategies are suitable as their names imply for deep holes or threads both exterior and interior. Fig. 19. Drilling on the shortest path. Fig. 20. Helical drilling. Fig. 19. Drilling on the shortest path. Fig. 20. Helical drilling. Fig. 19. Drilling on the shortest path. The machining optimization strategy allows the user to define the pitch of the spiral. The other strategies are suitable as their names imply for deep holes or threads both exterior and interior. Fig. 19. Drilling on the shortest path. Fig. 20. Helical drilling. Fig. 20. Helical drilling. 4 Conclusions In case of machining using CAM related optimization strategies there are various possibilities that carry different names on different software solutions but essentially do the same thing. For each operation the programmer is provided with several options that allows him to implement the desired policy whether is about prolonging tool life by using smaller feeds or is about cost-time efficiency by using more of the tool’s length deeper into the blank. When dealing with the same geometrical feature the CAM software solution allows the user to adjust the avoidance planes in such way that the tool does not have to withdraw to a safety position after each operation. Even more if the user has to deal with multiple machining operation patterns which would imply the use of several tool paths and inclinations, the software provides additional retraction positions in order to further reduce path lengths. The collision free politics is also present in case of changes between machined features and the machining planes. When detected, an automatic collision-free plane is created in order for the tool to be able to withdraw without any collision occurrence. Furthermore being able to program with available tools and make the best of it is something that defines a good programmer. There are many tweaks that can be applied in any machining strategy but in the end it all comes to knowledge, skills and available logistics. 1. V. Merticaru et al., Convergent use of advanced CAD/CAE/CAM capabilities for sustainable integrated engineering 12, 43-48 (2014) 1. V. Merticaru et al., Convergent use of advanced CAD/CAE/CAM capabilities for sustainable integrated engineering 12, 43-48 (2014) 2. A.S.M. Hoque, Computers & Industrial Engineering 66, 988–1003 (2013) 3. G. Musca, A. Mihalache, L. Tabacaru, IOP Conference Series-Materials Science and Engineering 161 (2016) 4. T. Dodoka, et al., Procedia Engineering 192, 113-118 (2017) 5. G. Musca, E. Musca, V. Merticaru, DAAAM Proceedings 8, 1485-1488 (2009) 6. M.I. Ripanu, et al., Applied Mechanics and Materials 371, 153-157 (2013) 7. P. Theodoroua, Omega 36, 107-121 (2008) 8. D. Luca, V. Schiopu, Journal of Optoelectronics and Advanced Materials 17, 997-1003 (2015) 9. G.M. Minquiz, Procedia CIRP 14, 581-586 (2014) 9. G.M. Minquiz, Procedia CIRP 14, 581-586 (2014) 10. G. Musca, L. Tabacaru, Performance in design with SolidEdge ST and technical data management in product design (translation from Romanian), (PIM, 2010) 6
https://openalex.org/W3160054294	https://boris.unibe.ch/156458/1/Kuehne_2021_EffectsOfPosedSmilingOnMemoryForHappyAndSadFacialExpressions_SciRep.pdf	English	null	Effects of posed smiling on memory for happy and sad facial expressions	Scientific reports	2,021	cc-by	10,499	Effects of posed smiling on memory for happy and sad facial expressions Maria Kuehne1,2, Tino Zaehle2,3 & Janek S. Lobmaier1 The perception and storage of facial emotional expressions constitutes an important human skill that is essential for our daily social interactions. While previous research revealed that facial feedback can influence the perception of facial emotional expressions, it is unclear whether facial feedback also plays a role in memory processes of facial emotional expressions. In the present study we investigated the impact of facial feedback on the performance in emotional visual working memory (WM). For this purpose, 37 participants underwent a classical facial feedback manipulation (FFM) (holding a pen with the teeth—inducing a smiling expression vs. holding a pen with the non-dominant hand—as a control condition) while they performed a WM task on varying intensities of happy or sad facial expressions. Results show that the smiling manipulation improved memory performance selectively for happy faces, especially for highly ambiguous facial expressions. Furthermore, we found that in addition to an overall negative bias specifically for happy faces (i.e. happy faces are remembered as more negative than they initially were), FFM induced a positivity bias when memorizing emotional facial information (i.e. faces were remembered as being more positive than they actually were). Finally, our data demonstrate that men were affected more by FFM: during induced smiling men showed a larger positive bias than women did. These data demonstrate that facial feedback not only influences our perception but also systematically alters our memory of facial emotional expressions. In human social interactions, facial expressions play an important role. Facial expressions transmit internal states such as motivations and feelings, which makes them an important source of non-verbal information. Various studies indicate that facial expressions are mimicked by eliciting facial muscular activity congruent with the presented facial expressions1,2. Generally, this mimicry process appears to be automatic and can occur without attention3. Theories of embodied simulation assume that the mimicked facial expression and the resulting feed- back from the facial muscles trigger a corresponding state in the observer’s motor, somatosensory, affective and reward system, helping to decode and understand the meaning of the perceived expression4. These mimicry processes seem to be implemented differently in men and women5. Early research has shown that women are emotionally more expressive6 and show more mimicry7 than men. www.nature.com/scientificreports www.nature.com/scientificreports Effects of posed smiling on memory for happy and sad facial expressions Facial feedback manipulation was implemented by different pen holding conditions equivalent to the study by Strack and colleagues16. While the results demonstrated that in particular the smiling condition differentially influenced the automatic processing of happy and sad facial expressions, the affected underlying cognitive process remains elusive. We assumed that the facial feedback manipulation influenced the encoding and retrieval of happy and sad facial expressions. Specifically, we interpreted these findings such that the smiling manipulation condition might have facilitated the encoding of happy while impeding the encoding of the sad faces. Therefore, the emotional valence of the happy face might have been stored more effectively than sad faces24.l h ppy gf y To date, only very few studies have looked at the influence of facial mimicry and the resulting facial feedback on the storage and retrieval of facial emotional expressions. One recent study by Pawling et al.25 demonstrated that the visual re-exposure to a facial expression reactivated the corresponding mimicry in a similar way as did the initial exposure. Interestingly, this emotional mimicry re-activation also occurred when the same face identity was displayed with a neutral expression during the re-exposure. These results are in accordance with the reactivation account of memory, indicating that the same brain regions are activated during retrieval and encoding (for review see Danker and Anderson26).h g ( ) The present study examined the role of facial feedback in memory processes of emotional facial expressions in a facial feedback manipulation study using an emotional working memory task. Facial feedback manipulation was administered following Strack et al.16. However, as previous studies have shown no or only minor effects of the holding-the-pen-between-the-lips-manipulation13,24 we restricted our manipulation to the smile-inducing condition, in which participants hold a pen with their teeth. We compared this manipulation to a neutral control condition (holding the pen with the non-dominant hand). Memory performance was investigated using a modi- fied emotional working memory (WM) paradigm with facial expressions, allowing us to separate overall WM accuracy from emotional biases27. The participants’ task was to encode, maintain and subsequently retrieve the valence as well as the intensity of happy and sad faces. In accordance with Mok and colleagues27 we predicted that the intensity of the expressions will affect memory, with better performance for less ambiguous emotional expres- sions. Further, we expected the teeth-manipulation to increase facial feedback of smiling, which in turn affects memory performance. Effects of posed smiling on memory for happy and sad facial expressions Additional analysis with ADS-K as well as with IPANAT is documented in supplementary material. Measure (n = 37 ) M(SD) Range Age 25 (3.42) 18–34 ADS-K 7.35 (3.92) 1–15 IPANAT 1 2.15PA (0.39) 1–3 1.82NA (0.51) 1–3 2 2.24PA (0.41) 2–3 1.68NA (0.42) 1–3 3 2.15PA (0.48) 1–3 1.77NA (0.38) 1–3 Measure (n = 37 ) M(SD) Range Age 25 (3.42) 18–34 ADS-K 7.35 (3.92) 1–15 IPANAT 1 2.15PA (0.39) 1–3 1.82NA (0.51) 1–3 2 2.24PA (0.41) 2–3 1.68NA (0.42) 1–3 3 2.15PA (0.48) 1–3 1.77NA (0.38) 1–3 Table 1. Sample characteristics. Age in years, ADS-K, IPANAT before (1), during (2) and after (3) the emotional WM task, separately for positive affect (PA) and negative affect (NA). Additional analysis with ADS-K as well as with IPANAT is documented in supplementary material. Table 1. Sample characteristics. Age in years, ADS-K, IPANAT before (1), during (2) and after (3) the emotional WM task, separately for positive affect (PA) and negative affect (NA). Additional analysis with ADS-K as well as with IPANAT is documented in supplementary material. and colleagues17–20, there is ample evidence that such facial feedback manipulations influence the conscious processing of emotional facial expressions13,21,22 and emotional body expressions23, as well as the automatic processing of unattended facial emotional expressions24. Recently, we investigated the impact of facial feedback on the automatic processing by electrophysiological measurements of the expression-related mismatch negativ- ity (eMMN). Facial feedback manipulation was implemented by different pen holding conditions equivalent to the study by Strack and colleagues16. While the results demonstrated that in particular the smiling condition differentially influenced the automatic processing of happy and sad facial expressions, the affected underlying cognitive process remains elusive. We assumed that the facial feedback manipulation influenced the encoding and retrieval of happy and sad facial expressions. Specifically, we interpreted these findings such that the smiling manipulation condition might have facilitated the encoding of happy while impeding the encoding of the sad faces. Therefore, the emotional valence of the happy face might have been stored more effectively than sad faces24.l and colleagues17–20, there is ample evidence that such facial feedback manipulations influence the conscious processing of emotional facial expressions13,21,22 and emotional body expressions23, as well as the automatic processing of unattended facial emotional expressions24. Recently, we investigated the impact of facial feedback on the automatic processing by electrophysiological measurements of the expression-related mismatch negativ- ity (eMMN). Effects of posed smiling on memory for happy and sad facial expressions Finally, following findings suggesting gender effects on recognition of emotional faces28,29 as well as on facial mimicry5–9, we assumed that memory performance might differ between women and men. Effects of posed smiling on memory for happy and sad facial expressions More recently, Niedenthal and colleagues showed that the duration of pacifier use in early childhood negatively impacts facial mimicry in boys but not in girls and additionally, that this pacifier use is correlated with emotional intelligence and perspective taking skills in men’s later life8. The authors interpret these findings such that the usage of a pacifier leads to a reduction of facial mimicry processes in the user as well as to reduced facial mimicry reactions towards the user. While girls are thought to compensate for these negative consequences, boys are thought to be at the mercy of these consequences9. q While some studies investigated facial mimicry using electromyographic measures2,10–12, others have experi- mentally manipulated the facial feedback processes to investigate its impact on the processing of emotional stimuli13–15. The classical facial feedback manipulation method was first introduced by Strack et al. in 198816. Here, participants are asked to hold a pen in their mouth, in different ways. The underlying principle behind this approach is that different pen holding conditions differentially activate facial muscles essential for smiling. In particular, when participants hold the pen with their teeth they activate the Musculus zygomaticus major and the Musculus risorius—both these muscles are activated while smiling. In contrast, when participants hold the pen with their lips they activate the Musculus orbicularis oris, the contraction of which is incompatible with smiling. Notwithstanding an ongoing intense debate about the replicability of the seminal study by Strack 1Department of Social Neuroscience and Social Psychology, Institute of Psychology, University of Bern, Bern, Switzerland. 2Department of Neurology, Otto-Von-Guericke-University Magdeburg, Leipziger Straße 44, 39120 Magdeburg, Germany. 3Center for Behavioral Brain Sciences, Magdeburg, Germany. email: maria.kuehne@med.ovgu.de Scientific Reports \| (2021) 11:10477 \| https://doi.org/10.1038/s41598-021-89828-7 www.nature.com/scientificreports/ Table 1. Sample characteristics. Age in years, ADS-K, IPANAT before (1), during (2) and after (3) the emotional WM task, separately for positive affect (PA) and negative affect (NA). Additional analysis with ADS-K as well as with IPANAT is documented in supplementary material. Measure (n = 37 ) M(SD) Range Age 25 (3.42) 18–34 ADS-K 7.35 (3.92) 1–15 IPANAT 1 2.15PA (0.39) 1–3 1.82NA (0.51) 1–3 2 2.24PA (0.41) 2–3 1.68NA (0.42) 1–3 3 2.15PA (0.48) 1–3 1.77NA (0.38) 1–3 Table 1. Sample characteristics. Age in years, ADS-K, IPANAT before (1), during (2) and after (3) the emotional WM task, separately for positive affect (PA) and negative affect (NA). Methods Participants. Participants. We investigated 37 healthy participants (19 women, 18 men, mean age 25 years ± 3.42). Sam- ple size was determined based on previous studies24,27,30. p p Participants had normal or corrected to normal vision and had no history of neurological or neuropsychi- atric diseases. After arriving at the laboratory, all participants provided written informed consent and filled in the short version of the Allgemeine Depressionsskala (ADS-K, self-report questionnaire measuring impairments due to depressive symptoms during the previous weeks31). Additionally, participants were asked to complete the Implicit Positive and Negative Affect Test (IPANAT, measuring implicit positive and negative affect as well as state variance32). They filled in the IPANAT three times, before, during and after the experiment. All sample characteristics are presented in Table 1. The study and its experimental procedures were conducted in accord- ance with the Declaration of Helsinki (1991; p. 1194) and were approved by the local Ethical Committee of the University of Magdeburg. Stimuli and procedure. At the beginning, participants read the instruction of the task and filled in the questionnaires. The experiment proper consisted of an emotional WM task (adapted from Mok et al.27). Six female and seven male characters, each with three different emotional expressions (neutral, happy and sad), were taken from the NimStim Set of Facial Expressions33 (Stimulus IDs: 01F, 02F, 03F, 05F, 07F, 09F, 20M, 21M, https://doi.org/10.1038/s41598-021-89828-7 Scientific Reports \| (2021) 11:10477 \| www.nature.com/scientificreports/ Figure 1. Procedure of the facial emotion WM task. On each trial, participants were asked to encode a target face with an emotional expression (sad or happy) of a certain intensity. After a delay, participants used a mouse to adjust the expression to match the emotion and intensity of the face in memory. (A) Trial example. Each trial began with a starting image, presented until participants press the right mouse button. The target image was displayed for 500 ms followed by a mask image of 100 ms. After a delay of 3000 ms the test image was shown and participants had to respond. After the response or after 11 s a fixation cross appeared for 800 ms before the next trial started. (B) Target Image. The target image was either a happy or a sad emotional face at one of 11 intensity steps (neutral, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% sad or happy). (C) Test Image. Methods Participants. The test image always started with a neutral face. By using the mouse wheel, participant could adjust the remembered emotion and the intensity. Scrolling the mouse wheel changed the intensity of the emotional face continuously in steps of 1%. By pressing the left mouse button, the participant made their final selection. Figure 1. Procedure of the facial emotion WM task. On each trial, participants were asked to encode a target face with an emotional expression (sad or happy) of a certain intensity. After a delay, participants used a mouse to adjust the expression to match the emotion and intensity of the face in memory. (A) Trial example. Each trial began with a starting image, presented until participants press the right mouse button. The target image was displayed for 500 ms followed by a mask image of 100 ms. After a delay of 3000 ms the test image was shown and participants had to respond. After the response or after 11 s a fixation cross appeared for 800 ms before the next trial started. (B) Target Image. The target image was either a happy or a sad emotional face at one of 11 intensity steps (neutral, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% sad or happy). (C) Test Image. The test image always started with a neutral face. By using the mouse wheel, participant could adjust the remembered emotion and the intensity. Scrolling the mouse wheel changed the intensity of the emotional face continuously in steps of 1%. By pressing the left mouse button, the participant made their final selection. 23M, 29M, 32M, 34M) and from the Karolinska face data-base34 (Stimulus ID: AM14). The stimuli were equally distributed to the different pen holding conditions (3male/female for hand and 3 male/female for teeth; 1 male, 29M, for practice). All stimuli were edited with GIMP software (Version 2.10.6). To avoid low-level visual influ- ence the hair region of each character was cut out by putting an eliptic shape around the head with grey back- ground. From this elliptic shape, a scrambled mask was created separately for each character by changing pixels into random colors thereby producing white noise (see Fig. 1a). To familiarize the participants with the emo- tional WM task, they performed a practice trial before starting the main task. Methods Participants. During the emotional WM task, participants had to encode, retrieve and maintain the emotion itself and the specific intensity of an emotional face while holding a pen either between their teeth or with the non-dominant hand. The two pen holding condi- tions alternated over 12 different blocks. Each block consisted of 21 trials (overall 126 trials for each pen holding condition). Each trial began with a starting screen lasting until participants pressed the right mouse button. Thereafter the target image appeared for 500 ms, followed by the mask for 100 ms. After a delay of 3000 ms, the test image was shown and participants gave their response (see below). After an interval of 800 ms, the next trial started (see Fig. 1a). The target image displayed a face with a specific intensity of either happy or sad emotion. For this purpose, morph sequences were created from neutral to happy and from neutral to sad expressions in 1-degree-steps from 0 to 100% using java psychomorph35 (version 6). For target images, intensities in 10% steps were used (0% happy/sad, 10% happy/sad, 20% happy/sad, 30% happy/sad, 40% happy/sad, 50% happy/sad, 60% happy/sad, 70% happy/sad, 80% happy/sad, 90% happy/sad, 100% happy/sad, see Fig. 1b). During the task, each character was presented with each intensity step as target image. The test image was always the neutral face of the character. By scrolling the mouse wheel back and forth, participants could adjust the emotion and the inten- sity of the emotion to match the memorised target face. All intensity levels from 0–100% were possible for the response selection (see Fig. 1c). The response time window was restricted to 11 s. There were 8 different versions of the task, varying the order of pen holding conditions (starting with hand or teeth), identity allocation to pen holding conditions and mouse wheel settings (scroll up: face becomes happier, scroll down: face becomes sadder or vice versa). The Versions were pseudorandomly assigned to the participants. Data analysis. To investigate the influence of facial feedback manipulation (FFM) on emotional memory we assessed the quality of WM representations for emotional facial expressions and the systematic affective biases in perceiving and interpreting these expressions27. Accordingly, we separately analyzed performance accu- racy (categorical judgment of a happy or sad face) and emotional bias (remembered emotional expression is https://doi.org/10.1038/s41598-021-89828-7 Scientific Reports \| (2021) 11:10477 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Accuracy of WM Performance. Methods Participants. (A) Percent correct responses for each emotional intensity of the target face during the hand condition for happy (solid grey) and sad (dashed grey) faces and during the teeth condition for happy (solid blue) and sad (dashed blue) faces. (B) Percent correct responses for highly (left) and less (right) ambiguous emotional faces. In comparison to less ambiguous faces, memory accuracy for highly ambiguous faces was significantly reduced. (C) Percent correct responses across all intensity levels for happy (left) and sad (right) faces during the hand (grey) and teeth (blue) FFM condition. Whereas FFM did not influence the memory accuracy to sad faces, FFM improved the accuracy for happy faces during the teeth condition. Happy faces were more often correctly remembered in the teeth compared to the hand condition. Error bars represent standard errors (SE). **p < .001, p < .05. Figure 2. Accuracy of WM Performance. (A) Percent correct responses for each emotional intensity of the target face during the hand condition for happy (solid grey) and sad (dashed grey) faces and during the teeth condition for happy (solid blue) and sad (dashed blue) faces. (B) Percent correct responses for highly (left) and less (right) ambiguous emotional faces. In comparison to less ambiguous faces, memory accuracy for highly ambiguous faces was significantly reduced. (C) Percent correct responses across all intensity levels for happy (left) and sad (right) faces during the hand (grey) and teeth (blue) FFM condition. Whereas FFM did not influence the memory accuracy to sad faces, FFM improved the accuracy for happy faces during the teeth condition. Happy faces were more often correctly remembered in the teeth compared to the hand condition. Error bars represent standard errors (SE). **p < .001, p < .05. more positive or more negative than the original) for the two pen holding conditions and the two facial emo- tions. Trials with neutral target faces were excluded from this analysis. To characterize the accuracy of WM performance, we assessed the percentage of correct responses. A response was considered correct when participant adjusted a face to the correct emotion type (e.g., reporting a happy face as happy and a sad face as sad). To analyze the effect of ambiguity, the intensity levels were median-split in two equal bins of high and low ambiguity and percent correct responses were computed for each target emotion intensity bin. Methods Participants. Mean percent correct responses were entered into a repeated measures (RM) -ANOVA with the within-participant factors FFM (hand vs. teeth), emotion (happy vs. sad) and ambiguity (high vs. low) and the between-participant factor gender (male vs. female). Additionally, an ANOVA including all intensity levels was performed.h p The emotional bias represents the signed percentage deviation of the test image from the target image, such that negative values imply that participants remembered the emotion as less positive/more negative than the target image originally was and positive values imply that they remembered it as more positive/less negative (see supplementary material for formulas). Consequently, an emotional bias of − 5% would indicate that a tar- get image is remember 5% less positive/more negative than it originally was. After calculating the percentage deviation, an outlier analysis was performed on individual level for each participant separately for the two pen holding conditions (hand, teeth) and the two emotion conditions (happy, sad). Values exceeding ± 2 standard deviations from the mean were excluded from further analysis. This resulted on average in 2.73 (± 1.03 SD) excluded trials for happy and 3.32 (± 1.01 SD) for sad faces during the hand and 2.65 (± 1.12 SD) excluded trials for happy and 3.05 (± 1.29 SD) for sad faces during the teeth condition (see supplementary material for more details). Data of the emotional bias were entered into a RM- ANOVA with the within-participant factors FFM (hand vs. teeth), emotion (happy vs. sad) and between-participant factor gender (male vs. female). If necessary, Greenhouse–Geisser adjustment was used to correct for violations of sphericity. All significant interactions were post-hoc examined by using paired t tests and Bonferroni family-wise error correction was applied. The statistical analysis was performed by using IBM SPSS (version 26). Results A subsequent step-down analysis by means of the factor ambiguity revealed a significant FFM × emotion interaction (F1,36 = 4.447, p = 0.042, η2 p = 0.110) for highly ambiguous emo- tional faces. This interaction is explained by a significant increase of correct responses in the teeth compared to the hand condition for happy (Mhand = 0.81, SDhand = 0.11, Mteeth = 0.86, SDteeth = 0.08, t(36) = − 2.665, p = 0.011, d = − 0.520) but not for sad faces (Mhand = 0.83, SDhand = 0.10, Mteeth = 0.81, SDteeth = 0.14, t(36) = 0.909, p = 0.369, d = 0.164,). In contrast, the RM-ANOVAs for the less ambiguous emotional faces revealed no significant effect of the factor FFM or its interactions (all ps > 0.8). The results were comparable when including all 10 levels of intensity, showing a significant main effect of the factor intensity level (F3.907,136.737 = 211.870, p < 0.001, η2 p = 0.858), a significant FFM × emotion interaction (F1,35 = 4.293, p = 0.046, η2 p = 0.109) and a marginally significant FFM × emotion × intensity level interaction (F3.779, 132.268 = 2.323, p = 0.063, η2 p = 0.062). Emotional bias. Figure 3 illustrates the results for the emotional bias. The RM-ANOVA revealed significant main effects of the factors FFM (F1,35 = 5.010, p = 0.032, η2 p = 0.125) and emotion (F1,35 = 7.288, p = 0.011, η2 p = 0.172) as well as a significant interaction between FFM and gender (F1,35 = 5.260, p = 0.028, η2 p = 0.131). The main effect of emotion results from a overall more negative bias for happy faces (M = − 4.39, SD = 6.88) compared to sad faces (M = 1.19, SD = 7.47, t(36) = − 2.738, p = 0.01, d = 0.778) (see Fig. 3A). Happy faces were remembered as less positive/more negative than their respective target images were. The main effect of FFM is shown in Fig. 3B. Independent of the emotion, the teeth condition reduced the negative bias, that is, faces were remembered to be more positive/less negative when participants held the pen with their teeth (M = − 0.91, SD = 4.54) compared to the hand condition (M = − 2.29, SD = 3.72, t(36) = − 2.057, p = 0.047, d = − 0.333). Results p To further examine the FFM × gender interaction post hoc comparisons between the hand and the teeth conditions were conducted separately for male and female participants. While in men the teeth condition sig- nificantly lowered the negative bias compared to the hand condition (Mhand = − 2.54, SDhand = 4.27, Mteeth = 0.22, SDteeth = 5.62, t(17) = − 2.473, p = 0.024, d = − 0.553) there was no influence of the FFM in women (Mhand = − 2.37, SDhand = 4.70, Mteeth = − 2.16, SDteeth = 3.81, t(18) = − 0.295, p = 0.771, d = − 0.049; see Fig. 3C). Results Accuracy of WM performance. Figure 2A illustrates the percentage of correct responses for each emo- tional intensity separately for both emotions and FFM conditions. The RM-ANOVA revealed a significant main effect of the factor ambiguity (F1,35 = 487,407, p < 0.001, η2 p = 0.933). As can be seen in Fig. 2B, memory accu- racy was reduced for more ambiguous faces (faces with low intensity levels, M = 0.83, SD = 0.05). Specifically, more ambiguous faces were more often incorrectly remembered as expressing the wrong emotion than faces with a more explicit emotion (high intensity levels, M = 0.98, SD = 0.02). The RM-ANOVA further revealed a https://doi.org/10.1038/s41598-021-89828-7 Scientific Reports \| (2021) 11:10477 \| www.nature.com/scientificreports/ Figure 3. Emotional bias in the working memory task. (A) Effect of emotion. Happy faces (green) were remembered as being less happy (i.e., more negative) for sad faces this negative bias was less pronounced. (B) Effect of FFM. Independent of the emotion, emotional faces were remembered as being more positive/less negative during teeth (blue) than during the hand (grey) condition. (C) Emotional bias for male (left side) and female (right side) participants for the hand (grey) and teeth (blue) FFM condition. Men remembered faces more positive during the teeth compared to the hand condition than did women. Error bars represent standard errors (SE). p < .05, p ≤ .01. Figure 3. Emotional bias in the working memory task. (A) Effect of emotion. Happy faces (green) were remembered as being less happy (i.e., more negative) for sad faces this negative bias was less pronounced. (B) Effect of FFM. Independent of the emotion, emotional faces were remembered as being more positive/less negative during teeth (blue) than during the hand (grey) condition. (C) Emotional bias for male (left side) and female (right side) participants for the hand (grey) and teeth (blue) FFM condition. Men remembered faces more positive during the teeth compared to the hand condition than did women. Error bars represent standard errors (SE). p < .05, **p ≤ .01. significant FFM × emotion interaction (F1,35 = 4.293, p = 0.046, η2 p = 0.109). Post-hoc comparisons showed that, compared to the hand condition, the teeth condition significantly increased the accuracy of happy faces only (Mhand = 0.90, SDhand = 0.06, Mteeth = 0.92, SDteeth = 0.04, t(36) = − 2.537, p = 0.016, d = − 0.392). Results There was no effect of FFM on correct responses to sad faces (Mhand = 0.90, SDhand = 0.06, Mteeth = 0.89, SDteeth = 0.08, t(36) = 0.808, p = 0.424, d = 0.141, see Fig. 2C). Finally the ANOVA revealed a significant FFM × emotion × ambiguity inter- action (F1,35 = 4.429, p = 0.043, η2 p 0.112). A subsequent step-down analysis by means of the factor ambiguity revealed a significant FFM × emotion interaction (F1,36 = 4.447, p = 0.042, η2 p = 0.110) for highly ambiguous emo- tional faces. This interaction is explained by a significant increase of correct responses in the teeth compared to the hand condition for happy (Mhand = 0.81, SDhand = 0.11, Mteeth = 0.86, SDteeth = 0.08, t(36) = − 2.665, p = 0.011, d = − 0.520) but not for sad faces (Mhand = 0.83, SDhand = 0.10, Mteeth = 0.81, SDteeth = 0.14, t(36) = 0.909, p = 0.369, d = 0.164,). In contrast, the RM-ANOVAs for the less ambiguous emotional faces revealed no significant effect of the factor FFM or its interactions (all ps > 0.8). The results were comparable when including all 10 levels of intensity, showing a significant main effect of the factor intensity level (F3.907,136.737 = 211.870, p < 0.001, η2 p = 0.858), a significant FFM × emotion interaction (F1,35 = 4.293, p = 0.046, η2 p = 0.109) and a marginally significant FFM × emotion × intensity level interaction (F3.779, 132.268 = 2.323, p = 0.063, η2 p = 0.062). significant FFM × emotion interaction (F1,35 = 4.293, p = 0.046, η2 p = 0.109). Post-hoc comparisons showed that, compared to the hand condition, the teeth condition significantly increased the accuracy of happy faces only (Mhand = 0.90, SDhand = 0.06, Mteeth = 0.92, SDteeth = 0.04, t(36) = − 2.537, p = 0.016, d = − 0.392). There was no effect of FFM on correct responses to sad faces (Mhand = 0.90, SDhand = 0.06, Mteeth = 0.89, SDteeth = 0.08, t(36) = 0.808, p = 0.424, d = 0.141, see Fig. 2C). Finally the ANOVA revealed a significant FFM × emotion × ambiguity inter- action (F1,35 = 4.429, p = 0.043, η2 p 0.112). Discussion However, as the IPANAT does not measure explicit mood but rather affective trait and state, it provides only indirect evidence for how the FFM might have influenced the mood of the participants. While there is some evidence suggesting that the FFM modifies mood, the three measurements taken in the present study do not permit unambiguous proof for the effects of facial feedback on emotional memory performance. Therefore, in future research, it would be interesting to systemati- cally measure the influence of facial feedback on mood changes.hfh Possibly, the observed effect of FFM is related to general mood modulation processes. The smiling manipula- tion may thus have activated the corresponding affective system in the participants and consequently resulted in a positive mood, which in turn helps to store congruent information in memory. Previous studies consistently demonstrated that FFM can systematically induce and modulate mood46–48. Further, some evidence suggests that mood itself can influence memory performance49,50. A mood-congruent memory effect is additionally supported by results demonstrating the tendency to better recall information that is congruent to the current mood in depressed and anxious participants38,51,52. In the present study, we assessed the influence of FFM on the ft yl g The FFM effect might also be attributable to changes in processing style. There is compelling evidence that happy mood triggers a global and automatic processing style while sad mood triggers more local and analytic processing53–55. Following this, the smiling manipulation might have caused a positive mood and consequently triggered a more global and automatic processing style in the participants. However, in the present task, which asked for memorizing facial emotional expressions at different intensity levels, a local, more analytical processing style might have been more favorable than a global automatic processing style, to allow for the processing of more subtle differences between the intensity levels. Thus, while the present FFM might have evoked mood changes, these mood changes may not fully explain the reduced bias observed during the smiling FFM.l g y y p g g Alternatively, the influence of FFM might also be explained on a more neural level. As mentioned above, the reactivation account of memory assumes that remembering a piece of information activates the same brain regions that were engaged during the encoding phase. Discussion Several studies have shown that facial muscle manipulation—and thus facial feedback manipulation—influences conscious as well as automatic processing of emotional faces, both on a behavioral and electrophysiological level. Yet, these studies did not ascertain whether the link between FFM and emotion processing are solely attribut- able to influences on a perceptual level or whether other cognitive processes are altered as well. To investigate the impact of FFM on memory processes for emotional faces we deployed a facial emotional working memory (WM) paradigm while participants held a pen either between their teeth or with the non-dominant hand. Holding a pen between the teeth leads to activating facial muscles used when smiling (smiling manipulation) and thus to an increased positive facial feedback whereas holding the pen in the non-dominant hand serves as control condition (no FFM). Overall, our data show that the smiling manipulation condition improved the memory performance selectively for happy faces, especially when highly ambiguous facial expression had to be remembered. Furthermore, we found that happy faces were remembered as being less happy (negative bias). For sad faces, we found no bias. Interestingly, the teeth manipulation (smiling feedback) resulted in less negative and more positive memory of emotional facial information. Finally, our data demonstrate that men were more strongly affected by the FFM than women; during smiling manipulation men showed a reduced negative bias. Scientific Reports \| (2021) 11:10477 \| https://doi.org/10.1038/s41598-021-89828-7 www.nature.com/scientificreports/ The data of the present study are in line with the results of previous reports using a comparable WM design27. As anticipated, the emotional faces with low intensity/high ambiguity were more often falsely remembered as the wrong emotion than faces with more clear expressions. This can be explained by the fact that facial expressions of low intensities are generally more difficult to recognize. Comparable recognition difficulties of low-intensity emotional expressions have also been reported by Montagne and colleagues in 200736. We found a pronounced influence of the FFM particularly for high ambiguous faces; this is in line with theories of embodied cognition which assume that facial mimicry and its resulting facial feedback contribute to facial emotion recognition, especially when the senders’ expressions is ambiguous37. p y p g Furthermore, our data revealed a stronger negative bias for happy faces than for sad faces. A negative memory bias was also reported by Mok and colleagues27, albeit selectively for fearful faces while there was no effect for happy faces. Discussion Negative memory bias is consistently reported in depressed and dysphoric participants with better memory performance for sad faces than for happy faces38–40. However, the present results cannot be explained by subtle depressive symptoms as indexed by statistical analysis of the ADS-K questionnaire (see supplementary material ADS-K analysis). Facial feedback manipulation influences memory. Importantly, our results indicate that FFM sys- tematically influences memory performance for facial emotional stimuli, at least in men. In contrast to the control manipulation, the smiling manipulation improved the memory performance selectively for happy faces and induced a positive bias independent of emotional quality. Numerous previous studies have demonstrated that facial mimicry and the resulting facial feedback are both important for the processing of emotional stimuli in general41 and for the perception of facial emotional expressions in particular13–15,22,42–44. It is thought that facial mimicry supports embodied simulation processes: the perception of an emotional expression results in an internal simulation of a relating affective state via the activation of corresponding motor, somatosensory, affective and reward systems. This in turn helps to understand and interpret the expression45.l p p To date, research assessing the influences of facial feedback on emotional memory is scarce. We can hence nly speculate about the processes underlying the link between facial muscle activation and memory:fh y p p y g y Possibly, the observed effect of FFM is related to general mood modulation processes. The smiling manipula- tion may thus have activated the corresponding affective system in the participants and consequently resulted in a positive mood, which in turn helps to store congruent information in memory. Previous studies consistently demonstrated that FFM can systematically induce and modulate mood46–48. Further, some evidence suggests that mood itself can influence memory performance49,50. A mood-congruent memory effect is additionally supported by results demonstrating the tendency to better recall information that is congruent to the current mood in depressed and anxious participants38,51,52. In the present study, we assessed the influence of FFM on the participants’ affect by asking them to complete the IPANAT32 before starting with the paradigm, midway after a smiling manipulation block, and at the end of the experiment (depending on the version of the paradigm either after a hand or after a teeth condition). Indeed, smiling significantly decreased participants’ negative affect while the positive affect remained unchanged (see supplementary material). www.nature.com/scientificreports/ Additionally, a sole influence of feedback manipulation on perceptual processes would have affected the target as well as the test image and both should have been perceived as more positive. As a consequence, such general perception biases should have cancelled each other out. Therefore, while it is possible that the undertaken FFM influenced the perception of the emotional faces, this could not fully explain the present results. Notwithstanding the female advantage in emotion recognition abilities, a gender difference in memorizing abilities for facial emotional expressions remains incompletely understood and should be topic of future studies. Limitations and further directions. There are limitations in this study that should be addressed in future research. First, because of the implementation of the FFM (holding a pen with the non-dominant hand vs. holding it with the teeth) the manipulation was maintained during the whole duration of the testing session, alternating between control and smiling blocks. For this reason, data do not allow for a detailed separation of the influence on the different stages of memory processing. We therefore cannot confidently assert whether the performed FFM influenced the storage, encoding, maintenance or the retrieval of emotional faces. Based on the present data, future studies should apply a more specific FFM, either during target or test image presentation. Second, in contrast to previous studies13,24,42 and to the seminal FFM study16, we did not assess the influence of a pen-between-lips condition in the present study. Since previous studies have shown only minor to no effects of this manipulation method13,24 and to ensure sufficient trial replications per condition and to prevent unneces- sary exhaustion we decided to exclude this manipulation condition. Using the pen between the teeth condition, we found that posing a smile affected the memory processes of emotional faces. We are hence convinced that the omission of the pen between the lips condition does not take away from our most important finding, that facial feedback from the smiling muscle (zygomaticus major) results in more positive memory to emotional facial expressions. It will have to be the aim of future research to specifically compare the effects between a frowning and a smiling manipulation condition.i g p A further limitation is related to the task specificity. In the present paradigm participants had to remember the facial expression and the intensity of this expression, allowing to investigate the influence of FFM on memory for emotional faces. www.nature.com/scientificreports/ communication process77. Thus, the FFM possibly primed the activation of those brain regions engaged during emotional memory processing and consequently facilitated the storage and retrieval of related information about facial expressions. With respect to future research, it would be interesting to shed further light on the activity of related brain regions during memorizing facial emotional expressions and the contribution of facial feedback to those processes. Gender difference. Our data show that men were more susceptible to the FFM. They remembered emo- tional expressions to be less negative/more positive in the teeth compared to the control manipulation condi- tion, thereby reducing the negative bias. An analogous gender effect has recently been shown by Wood and colleagues in 201978. There, the recognition of facial expressions and hand gestures was impaired after facial mobility restriction in men but not in women. Further evidence that male participants are more susceptible to FFMs comes from a study looking at pacifier use in childhood8. This study revealed a negative correlation between the duration of pacifier use and the amount of facial mimicry in boys but not in girls and that this effect seems to further impact social skills of men in later life. Especially skills that depend on the recognition of others’ emotion were affected. f Meanwhile, women generally outperform men during emotion perception tasks, with a more pronounced advantage for negative emotions (for review see Thompson and Voyer79). This advantage can be of biological as well as cultural origin—women as caregivers are more in demand of recognizing negative emotions79 and women as “emotion experts” profit of particular emotional stimulation in childhood80,81. Finally, there exists some evidence suggesting that women are more responsive towards emotional facial expressions in their own facial reactions1 and generally show more emotional expressions than men and tend to smile more82. Consequently, it might be that in the present study female participants reached a ceiling effect regarding the potential influence of FFM while male participants did not exploit their facial mimicry and expressivity to its full potential and could thus profit from the manipulation.fl i Since previous studies did not reveal gender differences in the influence of a smiling manipulation on emo- tion perception13,15 it is unlikely that our results rely on influences of perceptual processes only. www.nature.com/scientificreports/ We cannot exclude that the manipulation might have also influenced the visual memory for more static aspects of the target such as identity or gender. Accordingly, future research of this topic should consider control tasks where participants have to remember the facial identity of a perceived face. Finally, as in the most of previous studies using the FFM technique, the relatively small number of participants might have resulted in a rather low statistical power. Discussion One might speculate that the FFM in the present study primed the related brain regions which were activated during a smiling expression and—most importantly— which were also activated during the storage and the retrieval of related information like the memory of a smiling face. In the past, imaging studies provided insights into the brain regions that are involved in WM processes of emotional faces56–59. These studies found activation within frontal areas, especially within the dorsolateral prefrontal and orbitofrontal cortex (dlPFC, OFC), as well as within the superior temporal sulcus (STS) and the amygdala. The dlPFC plays a fundamental role within the WM network60–63. Both the OFC and the amygdala contain face-selective neurons64,65 and their connective activity is thought to be responsible for differentiating positive and neutral facial expressions from negative ones66. Further, amygdala activation is allegedly related to enhanced memory for emotional stimuli67–71. Finally, the STS is a well-known structure in processing change- able features of faces such as emotional expressions72,73. Previous research revealed that the activation of the amygdala, hippocampus (especially right) and STS are related to facial mimicry processes during the percep- tion of emotionally expressive faces43,44,74–77. It is thought that the activation of the right hippocampus displays the recruitment of memory contents for an improved understanding of the displayed facial expression76 while STS activation presents not only the sensory representation of the visual information but also an emotional Scientific Reports \| (2021) 11:10477 \| https://doi.org/10.1038/s41598-021-89828-7 www.nature.com/scientificreports/ Conclusionh The present study examined the influence of FFM on memory for emotional faces. For this purpose we applied classical FFM where holding a pen with the teeth induced a smiling expression and thus increasing positive facial feedback while holding it with the non-dominant hand served as control condition. Facial feedback of the participants was manipulated while they performed a visual WM paradigm where they had to remember the intensity of either a happy or sad facial emotional expression. Data show that the smiling manipulation condition improved memory performance selectively for happy faces, especially when highly ambiguous facial expressions had to be remembered. Furthermore, we found that FFM induced a general positive bias (and reduced the nega- tive memory bias) for the remembered emotional facial information. Finally, data demonstrated that men were more affected by the FFM than women. https://doi.org/10.1038/s41598-021-89828-7 Scientific Reports \| (2021) 11:10477 \| www.nature.com/scientificreports/ The influences of the smiling manipulation might be attributed to the priming of activation of a specific brain network engaged during memory processes for emotional faces. Consequently, this priming might facilitate the storage and retrieval of congruent information. Our data demonstrate that facial feedback not only influences perception but also systematically alters memory of the facial emotional expressions. This study constitutes a first step towards our understanding of the influence of facial feedback on memory of emotional facial expressions. Data availabilityh The conditions of our ethics approval do not permit public archiving of the data. Readers seeking access to the data should contact the corresponding author. 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LaFrance, M. & Hecht, M.A. Gender and smiling: A meta-analysis, in Studies in emotion and social interaction. Second series Gender and emotion: Social psychological perspectives (ed. Fischer A. H.) 118–142 (Cambridge University Press, 2000). Author contributions Author contributions M.K., T.Z. and J.S.L. designed the experiment. M.K. carried out the measurements, analyzed the data and wrote the manuscript. All authors reviewed and revised the manuscript and approved the final version. Funding g Open Access funding enabled and organized by Projekt DEAL. www.nature.com/scientificreports/ N., Fine-Goulden, M. R. & Dolan, R. J. fMRI-adaptation reveals dissociable neural representations of identity and expression in face perception. J. Neurophysiol. 92, 1830–1839 (2004).fh 73. Haxby, J. V., Hoffman, E. A. & Gobbini, M. I. The distributed human neural system for face perception. Trends Cogn. Sci. 4, 223–233 (2000). 4. Wild, B., Erb, M., Eyb, M., Bartels, M. & Grodd, W. Why are smiles contagious? An fMRI study of the interaction between percep tion of facial affect and facial movements. Psychiat. Res. Neuroimaging 123, 17–36 (2003). f ikowski, K. et al. Facial mimicry and the mirror neuron system: simultaneous acquisition of facial electromyography and func- ional magnetic resonance imaging. Front. Hum. Neurosci. 6 (2012).f f 75. Likowski, K. et al. Facial mimicry and the mirror neuron system: simultaneous acquisition of facial electromyography and func- tional magnetic resonance imaging. Front. Hum. Neurosci. 6 (2012).f 76. Schilbach, L., Eickhoff, S. B., Mojzisch, A. & Vogeley, K. What’s in a smile? Neural correlates of facial embodiment during social interaction. Soc. Neurosci. 3, 37–50 (2008).i ( ) 7. Lee, T.-W., Josephs, O., Dolan, R. J. & Critchley, H. D. Imitating expressions: emotion-specific neural substrates in facial mimicry Soc. Cogn. Aff. Neurosci. 1, 122–135 (2006). ( ) ee, T.-W., Josephs, O., Dolan, R. J. & Critchley, H. D. Imitating expressions: emotion-specific neural substrates in facial mimicry. oc. Cogn. Aff. Neurosci. 1, 122–135 (2006). https://doi.org/10.1038/s41598-021-89828-7 https://doi.org/10.1038/s41598-021-89828-7 Scientific Reports \| (2021) 11:10477 \| Competing interests h Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-89828-7. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-89828-7. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-89828-7. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-89828-7. Correspondence and requests for materials should be addressed to M.K. Correspondence and requests for materials should be addressed to M.K. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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https://openalex.org/W2337989073	https://link.springer.com/content/pdf/10.1007/s13205-016-0421-y.pdf	English	null	Purification and characterization of endo β-1,4-d-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse	3 biotech	2,016	cc-by	9,072	Puriﬁcation and characterization of endo b-1,4-D-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse Zabin K. Bagewadi1 • Sikandar I. Mulla1 • Harichandra Z. Ninnekar1 Received: 15 January 2016 / Accepted: 27 March 2016 / Published online: 12 April 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract An acidophilic-solvent-thermostable endo b- 1,4-D-glucanase produced from a potential Trichoderma harzianum strain HZN11 was puriﬁed to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with 33.12 fold puriﬁcation with speciﬁc activity of 66.25 U/mg and molecular mass of 55 kDa. The optimum temperature and pH were 60 C and 5.5 retaining 76 and 85 % of activity after 3 h, respectively. It showed stability between pH 4.5–6.0 and temperature between 50–70 C indicating thermostability. Endo b-1,4-D-glucanase was activated by Ca2? and Mg2? but inhibited by Hg2?, Pb2? and Cd2?. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Puriﬁed endo b-1,4-D-glucanase exhibited highest speciﬁcity towards carboxymethyl cellulose. Kinetic analysis showed the Km, Vmax and Ki (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of puriﬁed endo b-1,4-D- glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efﬁciency of puriﬁed endo b-1,4- D-glucanase mixed with cocktail was demonstrated over commercial enzyme. Optimized enzymatic hydrolysis of sweet sorghum and sugarcane bagasse released 5.2 g/g (36 h) and 6.8 g/g (48 h) of reducing sugars, respectively. Separate hydrolysis and fermentation of sweet sorghum bagasse yielded 4.3 g/L bioethanol (16 h) conﬁrmed by gas Electronic supplementary material The online version of this article (doi:10.1007/s13205-016-0421-y) contains supplementary material, which is available to authorized users. & Harichandra Z. Ninnekar hzninnekar@yahoo.com 1 Department of Biochemistry, Karnatak University, Dharwad 580 003, Karnataka, India Abstract An acidophilic-solvent-thermostable endo b- 1,4-D-glucanase produced from a potential Trichoderma harzianum strain HZN11 was puriﬁed to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with 33.12 fold puriﬁcation with speciﬁc activity of 66.25 U/mg and molecular mass of 55 kDa. The optimum temperature and pH were 60 C and 5.5 retaining 76 and 85 % of activity after 3 h, respectively. It showed stability between pH 4.5–6.0 and temperature between 50–70 C indicating thermostability. Endo b-1,4-D-glucanase was activated by Ca2? and Mg2? but inhibited by Hg2?, Pb2? and Cd2?. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Puriﬁed endo b-1,4-D-glucanase exhibited highest speciﬁcity towards carboxymethyl cellulose. 1 Department of Biochemistry, Karnatak University, Dharwad 580 003, Karnataka, India 3 Biotech (2016) 6:101 DOI 10.1007/s13205-016-0421-y ORIGINAL ARTICLE & Harichandra Z. Ninnekar hzninnekar@yahoo.com Puriﬁcation and characterization of endo b-1,4-D-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse Kinetic analysis showed the Km, Vmax and Ki (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of puriﬁed endo b-1,4-D- glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efﬁciency of puriﬁed endo b-1,4- D-glucanase mixed with cocktail was demonstrated over commercial enzyme. Optimized enzymatic hydrolysis of sweet sorghum and sugarcane bagasse released 5.2 g/g (36 h) and 6.8 g/g (48 h) of reducing sugars, respectively. Separate hydrolysis and fermentation of sweet sorghum bagasse yielded 4.3 g/L bioethanol (16 h) conﬁrmed by gas chromatography–mass spectrometry (GC–MS). Morpho- logical and structural changes were assessed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. Elemental analysis was carried out by SEM equipped with energy dispersive X-ray technique. These unique properties prove the potentiality of enzyme for biomass conversion to biofuel and other industrial applications. chromatography–mass spectrometry (GC–MS). Morpho- logical and structural changes were assessed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. Elemental analysis was carried out by SEM equipped with energy dispersive X-ray technique. These unique properties prove the potentiality of enzyme for biomass conversion to biofuel and other industrial applications. Keywords Endo b-1,4-D-glucanase Trichoderma harzianum strain HZN11 Puriﬁcation Characterization Enzymatic hydrolysis Bioethanol Keywords Endo b-1,4-D-glucanase Trichoderma harzianum strain HZN11 Puriﬁcation Characterization Enzymatic hydrolysis Bioethanol 123 Chemicals, substrate and culture All the chemicals and media components used were pro- cured from HiMedia, Sigma-Aldrich (USA) and Merck (USA). Sweet sorghum stalks were collected from University of agricultural sciences, Dharwad. Saccha- romyces cerevisiae NCIM 3594 was procured from National Collection of Industrial Microorganisms (NCIM). Introduction In the current scenario, the major concerns are towards the diminishing of fossil fuels which have forced the energy industries and researchers to develop alternatives to the existing fuels (Bentsen and Felby 2012). One of the attractive sustainable substitutes is the microbial produc- tion of bioethanol from lignocellulosic wastes as it is cost- effective and renewable (Ren et al. 2009). Plant biomass constitute of cellulose which is the major organic polysaccharide found in the biosphere (Bhat and Bhat 1997) and is renewable. Biodegradation of plant based biomass requires cellulose and hemicellulose saccharifying enzymes. For example, cellulases participate in sacchari- ﬁcation of biomass for bioethanol production (Dhillon et al. 2011), by mainly acting on b-1,4-glycosidic bonds of cel- lulose. Cellulolytic enzymes have been classiﬁed as: endoglucanase (endo-1,4-D-glucanase, EG), cellobiohy- drolase (exo-1,4-D-glucanase, CBH) and glucosidase (1,4- D-glucosidase, BG) (Saha 2004), which have been shown to act synergistically for effective degradation (Lynd et al. Electronic supplementary material The online version of this article (doi:10.1007/s13205-016-0421-y) contains supplementary material, which is available to authorized users. & Harichandra Z. Ninnekar hzninnekar@yahoo.com 1 Department of Biochemistry, Karnatak University, Dharwad 580 003, Karnataka, India 123 3 Biotech (2016) 6:101 101 Page 2 of 10 101 Page 2 of 10 2002) whereas xylanases (1,4-b-D-xylanohydrolase) hydrolyze xylan, a major component of hemicellulose (Zhang et al. 2011). The fungal endoglucanases ﬁnds its applications in biomass bioconversions, pulp and paper, textile, detergents, starch processing, grain alcohol fer- mentation, brewery, wine making, extraction of fruit and vegetable juices (Karmakar and Ray 2011; Kuhad et al. 2011). These applications certainly require endoglucanases with industrial attributes like thermostability, stability at varying pH, substrate speciﬁcities (Bhat 2000), solvent tolerant, detergent compatibility, chemical stability, etc. Solid state fermentation (SSF) for cellulase production is an advantageous process as it reduces the capital invest- ment with easy operating conditions (Pandey et al. 1999). Cellulose sacchariﬁcation can be carried out by separate hydrolysis and fermentation (SHF) process with an ease of optimizing the enzymatic hydrolysis conditions (Zhu et al. 2012) for ethanol production. Ethanol quantiﬁca- tion can be achieved by employing methods like GC– MS. Desirable for better speciﬁcity, few mass spectro- metric (MS) methods for ethanol analysis have been reported (Tiscione et al. 2011). Insights of molecular level changes and functional groups in the lignocellu- losic material at various fermentation steps could be studied by employing FTIR (Adapa et al. 2011; Sim et al. Puriﬁcation of endo b-1,4-D-glucanase and molecular mass determination Endo b-1,4-D-glucanase produced from sweet sorghum bagasse by Trichoderma harzianum strain HZN11 was subjected for puriﬁcation. The cell debris was removed by vacuum ﬁltration (Millipore India Ltd.) and the enzyme protein was subjected to fractionation by (NH4)2SO4 (70 % w/v). The precipitate was centrifuged at 8000 rpm for Enzyme assay and protein determination Endo b-1,4-D-glucanase hydrolyzes CMC to produce free carboxymethyl glucose units. Endo b-1,4-D-glucanase activity was estimated using CMC as substrate under standard conditions according to Standard International Union of Pure and Applied Chemistry (IUPAC) method described by Ghose (1987). The reducing sugars released from the reaction were determined according to Miller (1959) by dinitrosalicylic acid (DNS) method. In the above assay, one unit (U) of enzyme was deﬁned as the amount of enzyme that released 1 lmol of the glucose per minute under standard assay conditions (30 min incubation at 50 C with 50 mM acetate buffer pH 6.0). The concen- trations of soluble proteins were estimated according to Lowry et al. (1951) using bovine serum albumin (BSA) as the standard. Keeping in view the industrial applications of the endo b-1,4-D-glucanase, this study was carried out to purify and characterize a novel endo b-1,4-D-glucanase from Tricho- derma harzianum strain HZN11. Enzymatic hydrolysis and ethanol fermentation was successfully achieved. Further, sweet sorghum bagasse was molecularly characterized with techniques like FTIR, SEM and SEM/EDX. Introduction 2012), morphological changes by SEM and substrate elemental analysis by higher throughput techniques like SEM equipped with EDX technique. sequencing (data was not shown). The nucleotide sequence of the strain was deposited to NCBI (National Center for Biotechnology Information) GenBank with accession number KP050786. The newly isolated Trichoderma har- zianum strain HZN11 is maintained at the Department of Biochemistry, Karnatak University, Dharwad on potato dextrose agar (PDA) enriched with carboxymethyl cellu- lose (CMC) at 4 C. Endo b-1,4-D-glucanase was produced by Trichoderma harzianum strain HZN11 in SSF using alkali pretreated sweet sorghum bagasse as substrate. SSF was carried out in 250 mL Erlenmeyer ﬂasks containing 10 g of pretreated substrate in Mandels–Weber medium containing (g/L) urea 0.3; ammonium sulfate 1.4; KH2PO4 0.3; CaCl2 0.3; MgSO4.7H2O 0.3; protease peptone 1.0; lactose 10; and (mg/L) FeSO4.7H2O 5.0; MnSO4.7H2O 1.6; ZnSO4.7H2O 1.4; CoCl2 2; Tween-80 0.1 %; and pH 6 with 70 % moisture content. Sterilized ﬂasks were inocu- lated with 4 mL spore suspension and incubated at 35 C under static condition for 7 days. The crude enzyme was extracted with 50 mM sodium acetate buffer, pH 6 with 1:2 solid to liquid ratio under shaking (150 rpm) at 35 C for 30 min, followed by ﬁltration through muslin cloth. The ﬁltrate was centrifuged at 8000 rpm for 20 min at 4 C. The clear supernatant was used as crude enzyme for puriﬁcation. 123 Fungal strain and production of endo b-1,4-D-glucanase Trichoderma harzianum strain HZN11 previously isolated from forest soil was identiﬁed based on 18S rDNA 123 Page 3 of 10 101 3 Biotech (2016) 6:101 15 min at 4 C. Enzyme was resuspended in a minimum volume of 50 mM sodium acetate buffer (pH 6.0) and dialyzed against the same buffer for 6 h at 4 C and lyo- philized. Total proteins and endo b-1,4-D-glucanase activ- ity of partially puriﬁed fraction were determined before and after dialysis. Endo b-1,4-D-glucanase was further puriﬁed by column chromatography. The lyophilized enzyme was dissolved in 50 mM sodium acetate buffer (pH 6.0). The enzyme protein was loaded onto DEAE-Sepharose column (40 9 2 cm) pre-equilibrated with 50 mM sodium acetate buffer (pH 6.0). The ﬂow rate was maintained at 1 mL/min and eluted with gradient of 0.1–1 M NaCl. The pooled fractions were dialyzed and lyophilized. The fractions were re-dissolved in buffer and loaded onto the Sephadex G-100 column (30 9 2 cm) pre-equilibrated and eluted with 50 mM sodium acetate buffer (pH 6.0) at a ﬂow rate of 0.5 mL/min. The fractions were analyzed for proteins by A280 method and endo b-1,4-D-glucanase activities were determined. The resulting concentrated active endo b-1,4- D-glucanase fractions were pooled and used for further characterization (Bakare et al. 2005). The apparent molecular weight of the puriﬁed endo b-1,4-D-glucanase was determined by sodium dedocylsulphate polyacry- lamide gel electrophoresis (SDS-PAGE) with protein molecular weight ladder [lysozyme (14.3 kDa), b-lac- toglobulin (20 kDa), carbonic anhydrase (29 kDa), oval- bumin (43 kDa), bovine serum albumin (66 kDa) and phosphorylase B (97.4 kDa) run along with sample], according to the method described by Laemmli (1970). Protein bands were visualized by staining with coomassie brilliant blue R-250. bromosuccinimide, dimethyl sulfoxide (DMSO), iodoac- etamide, p-chloromercuribenzoate (p-CMB) and 1,10- phenanthroline, at various concentrations (1–10 mM). Relative activities of endo b-1,4-D-glucanase in the pres- ence of various detergents like sodium dodecyl sulfate (SDS), sodium tetraborate, and commercial detergents (Tide, Ariel and Surf Excel), surfactants like tween-20, tween-40, tween-80 and triton X-100 and oxidizing agents like sodium perborate, sodium hypochlorite and hydrogen peroxide at varying concentrations (0.1–1 %) were deter- mined. Endo b-1,4-D-glucanase stability in the presence of various organic solvents like glycerol, ethanol, methanol, acetone, formic acid, propanol, petroleum ether, iso- propanol, benzene, cyclohexane, hexane, butanol and toluene at different concentrations (10–30 %) were evalu- ated (control was 100 %). Enzymatic hydrolysis and ethanol fermentation Enzymatic hydrolysis of untreated and alkali pretreated sweet sorghum bagasse and sugarcane bagasse was studied using puriﬁed endo b-1,4-D-glucanase (53 U/g) mixed with crude multi-enzyme cocktail (exoglucanase 15 U/g, ﬁlter paper activity (FP) 15 U/g, cellobiase 18 U/g, xylanase 1740 U/g and b-glucosidase 13 U/g) produced by Tricho- derma harzianum strain HZN11 from sweet sorghum bagasse and commercial cellulase from Trichoderma sps. (9 FP U/mL) individually. Reaction constituted of 2 % substrate in 50 mM sodium acetate buffer pH 5.5, 0.1 % tween-40 and ﬁlter sterilized enzyme in a volume of 30 mL incubated at 40 C at 150 rpm. The reaction was fortiﬁed with 0.005 % sodium azide. Samples were withdrawn, centrifuged at 8000 rpm for 15 min and the clear super- natants were analyzed for reducing sugars according to the method described by Miller (1959). Controls were kept for each reaction with heat-inactivated enzyme. Parameters such as hydrolysis time (12–72 h) and temperature (40–60 C) for enzymatic hydrolysis of alkali pretreated sweet sorghum bagasse and sugarcane bagasse was opti- mized. SHF experiments were designed in which Fungal strain and production of endo b-1,4-D-glucanase Substrate speciﬁcity of endo b- 1,4-D-glucanase was tested against variety of substrates like 1 % microcystalline cellulose, CMC, chitin, cellobiose, starch, ﬁlter paper, PNP-a-galactopyranoside, PNP-glu- copyranoside, PNP-cellobioside, brichwood xylan and oat spelt xylan. Endo b-1,4-D-glucanase kinetics with CMC concentration range of 2.5–30 mg/mL was studied and kinetic parameters Km and Vmax were determined by linear transformations of the Michaelis–Menten model to Line- weaver–Burk. Inhibition of endo b-1,4-D-glucanase with cellobiose was studied at 5 mM and 10 mM and inhibition constant Ki were determined by Lineweaver–Burk plot. The storage stability of the puriﬁed endo b-1,4-D-glucanase was monitored for 60 days. 123 Analytical methods The SHF aliquot samples were ﬁltered through 0.2 mm membrane ﬁlters for the analysis of ethanol by GC–MS (Shi- madzuQP2010 Plus) equipped with an autosampler. GC–MS accelerates ethanol analysis with its simultaneous separation and identiﬁcation. GC–MS was equipped with quadruple mass ﬁlter Rtx-5MS capillary column (30 m 9 0.25 mm), scan interval 0.5 s and mass range 40–500 m/z. The column tem- perature was maintained at 50 C for 1 min, and then ramped with 20 C increase per min to a ﬁnal temperature at 280 C for 14.5 min and the injector temperature was kept at 250 C. Helium was used ascarrier gasin the gas chromatographer with 1 lL injection volume. The MS was operated at electron ion- ization energy of 70 eV. The sample was run for 20 min. Absolute ethanol was used as standard. The puriﬁed endo b-1,4-D-glucanase showed a single pro- tein band on SDS-PAGE with molecular weight *55 kDa (Fig. 1) and was found to be a monomeric protein from native gel (data not shown). There are reports of varying molecular mass of endo b-1,4-D-glucanase from different organisms, 29 kDa from Aspergillus niger AT-3 (Dutt and Kumar 2014) and 62 kDa from P. betulinus (Valaskova and Baldrian 2006). Results and discussion hydrolyzates of sweet sorghum bagasse were collected and centrifuged at 8000 rpm for 15 min. The supernatant con- taining reducing sugars was transferred to serum bottles for fermentation process with pH maintained to 7 by 1 N NaOH. Glucose fermenting yeast, Saccharomyces cere- visiae NCIM 3594 was inoculated and incubated at 30 C for 72 h under shaking at 120 rpm. Aliquots were with- drawn at different time intervals for the estimation of ethanol, reducing sugars and biomass. Puriﬁcation of endo b-1,4-D-glucanase and molecular mass determination The endo b-1,4-D-glucanase produced by Trichoderma harzianum strain HZN11 was puriﬁed to homogeneity by (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 chromatography. The enzyme was puriﬁed to 33.12 fold with speciﬁc activity of 66.25 U/mg protein as shown in Table 1. The elution proﬁles of DEAE-Sepharose and Sephadex G-100 chromatography are shown in Fig. S1 (Supplementary Information, SI) which did not show any multiple isoforms of enzyme. Characterization of puriﬁed endo b-1,4-D-glucanase Optimum pH of the puriﬁed endo b-1,4-D-glucanase was determined by incubating the enzyme at different pH ranging from pH 3–11 using 0.1 M buffers of sodium citrate (pH 3–4.5), sodium acetate (pH 5–6), sodium phosphate (pH 6.5–7.5), Tris–HCl (pH 8.0–9.5), and gly- cine–NaOH (pH 10.0–11.0). The stability of pH was determined at optimum range of pH 5–6 for 3 h and the relative activity was calculated. Optimum temperature was evaluated in range of 20–85 C and the thermo stability of endo b-1,4-D-glucanase was assessed between optimum temperature ranges of 50–65 C for 3 h at pH 5.5 by measuring the relative activity. Effect of various metal ions like Co21, Zn21, Ca21, Mg21, K1, Na1, Cu21, Hg21, Fe21, Pb21, Ni21, Mn21 and Cd21 was determined at different concentrations (1–10 mM). Endo b-1,4-D-glu- canase relative activity was evaluated in the presence of various additives like dithiothreitol (DTT), b-mercap- toethanol, ethylene diamine tetra acetic acid (EDTA), urea, phenyl methyl sulphonyl ﬂuoride (PMSF), N- 3 Biotech (2016) 6:101 101 Page 4 of 10 Effect of metal ions, additives, detergents, surfactants, oxidizing agents and organic solvents Similar results have been reported for cellulases from Bacillus subtilis YJ1 (Yin et al. 2010) and Bacillus vallismortis RG- 07 (Gaur and Tiwari 2015). Fig. 2 Determination of kinetic parameters Km and Vmax by Line- weaver–Burk plot (a) and inhibition kinetics by cellobiose on puriﬁed endo b-1,4-D-glucanase (b) in Fig. S2A (SI). Fungal cellulases have found to be active in acidic pH. The charge distribution on substrate and enzyme is greatly affected by variation in pH intern affecting the mechanism of substrate binding and catalysis (Shah and Madamwar 2005). Enzyme stability at its opti- mum pH is a required for efﬁcient catalysis. 81, 85 and 77 % of activity was retained at pH 5, 5.5 and 6, respec- tively, after 3 h (Fig. S2B, SI), thereby anticipating good pH stability. Similarly, effect of temperature was evaluated in the range between 20–85 C. Endo b-1,4-D-glucanase from Trichoderma harzianum strain HZN11 was found to be highly stable between 50–70 C with optimum activity at 60 C indicated in Fig. S2C (SI). The relative activity was recorded to be 80, 77, 76 and 66 % at 50, 55, 60 and 65 C, respectively, after 3 h (Fig. S2D, SI). The results reveal an acidic pH stable and thermostable endo b-1,4-D- glucanase from Trichoderma harzianum strain HZN11 which could be employed for enzymatic hydrolysis of lignocellulosic biomass. Thermal stability of an enzyme is an attractive property for signiﬁcant industrial applications (Asgher and Iqbal 2011). Many investigators reported optimum reaction temperature of 50–75 C. Our results were similar to other endoglucanases from Trichoderma viride HG 623 with temperature and pH of 55 C with pH 5 Endo b-1,4-D-glucanase showed good stability in the presence of detergents retaining 90, 85, 68, 71 and 76 % activity with 1 % of SDS, sodium tetraborate, tide, ariel and surf excel, respectively. Surfactants like tween-20, tween-40, tween-80 and triton X-100 retained greater than 80 % of the activity at 1 %. Endo b-1,4-D-glucanase retained 64, 73 and 40 % of activity with 1 % oxidizing agents like sodium perborate, sodium hypochlorite and hydrogen peroxide, respectively (Table S2, SI). Detergents may cause alterations in the structural and conformational characteristics of enzymes (Bajaj et al. 2009). The deter- gent stability of cellulase is an important attribute to be an effective additive in commercial detergents. Effect of metal ions, additives, detergents, surfactants, oxidizing agents and organic solvents The effect of various metal ions on endo b-1,4-D-glucanase activity at different concentrations (1–10 mM) is shown in Table S1 (SI). The metal ions such as Ca2?, Mg2?, Mn2?, Fe2? and Co2? activated the enzyme at 1 mM concentra- tion whereas Hg2?, Pb2?, Zn2? and Cd2? inhibited the enzyme. Similar results have been reported for endo b-1,4- D-glucanase from Fusarium oxysporum JRE1 (Dar et al. 2013) and Penicillium pinophilum MS20 (Pol et al. 2012). Additives like DTT, b-mercaptoethanol, EDTA and urea activated the enzyme and PMSF, N-bromosuccinimide, DMSO, iodoacetamide, p-CMB and 1, 10-phenathroline inhibited the enzyme as shown in Table S1 (SI). Endo b- 1,4-D-glucanase inhibition in the presence of iodoac- etamide and p-CMB indicate the binding to the –SH groups. Activation by DTT and b-mercaptoethanol indicate the presence of thiol groups at the active sites and these chemicals cause the reduction of disulphide bonds and reactivate the enzyme (Singh et al. 1990). Inhibition by N- bromosuccinimide suggests the presence of tryptophan residues in the active sites (Kaur et al. 2015). Similar results have been reported for cellulases from Bacillus subtilis YJ1 (Yin et al. 2010) and Bacillus vallismortis RG- 07 (Gaur and Tiwari 2015). The effect of various metal ions on endo b-1,4-D-glucanase activity at different concentrations (1–10 mM) is shown in Table S1 (SI). The metal ions such as Ca2?, Mg2?, Mn2?, Fe2? and Co2? activated the enzyme at 1 mM concentra- tion whereas Hg2?, Pb2?, Zn2? and Cd2? inhibited the enzyme. Similar results have been reported for endo b-1,4- D-glucanase from Fusarium oxysporum JRE1 (Dar et al. 2013) and Penicillium pinophilum MS20 (Pol et al. 2012). (B) -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1/V U/ml 1/[CMC] mg/ml 0mM 5mM 10mM Ki (B) Additives like DTT, b-mercaptoethanol, EDTA and urea activated the enzyme and PMSF, N-bromosuccinimide, DMSO, iodoacetamide, p-CMB and 1, 10-phenathroline inhibited the enzyme as shown in Table S1 (SI). Endo b- 1,4-D-glucanase inhibition in the presence of iodoac- etamide and p-CMB indicate the binding to the –SH groups. Activation by DTT and b-mercaptoethanol indicate the presence of thiol groups at the active sites and these chemicals cause the reduction of disulphide bonds and reactivate the enzyme (Singh et al. 1990). Inhibition by N- bromosuccinimide suggests the presence of tryptophan residues in the active sites (Kaur et al. 2015). Effect of pH and temperature Cellulase from Fusarium oxysporum JRE1 strain exhibited maximum activity at pH 5.6 retain- ing 75 % activity between pH 4.0–7.0 and 70 % activity around 25–37 C (Dar et al. 2013). (A) Effect of pH and temperature The puriﬁed endo b-1,4-D-glucanase was remarkably active in the pH range of 4–6.5 with optimum pH at 5.5 as shown The morphology and physical property changes of untreated, alkali pretreated and Trichoderma harzianum strain HZN11 hydrolyzed sweet sorghum bagasse samples were analyzed by SEM (JOEL-JSM 5600, JAPAN). The lyophilized samples were mounted on aluminum stubs, and sputter-coated with a gold layer. The SEM images were taken at different magniﬁcations. SEM equipped with EDX technique was used for elemental analysis of the substrate. Fig. 1 SDS-PAGE with lane a: puriﬁed endo b-1,4-D-glucanase and lane b: Molecular weight markers FTIR (Perkin Elmer, FTIR1760) was used to investigate the structural changes in untreated, alkali pretreated and Trichoderma harzianum strain HZN11 hydrolyzed sweet sorghum bagasse samples. Samples were mixed with KBr (potassium bromide) and prepared pellets. The spectra of samples were obtained using 32 scans with the spectra resolution measured as 4 cm-1 with a scanning range of 500–4000 cm-1. Fig. 1 SDS-PAGE with lane a: puriﬁed endo b-1,4-D-glucanase and lane b: Molecular weight markers of endo b-1,4-D-glucanase from Trichoderma harzianum strain HZN11 Table 1 Puriﬁcation summary of endo b-1,4-D-glucanase from Trichoderma harzianum strain HZN11 Puriﬁcation steps Total volume (mL) Total protein (mg) Total activity (U) Speciﬁc activity (U/mg) Yield (%) Fold puriﬁcation Crude extract 200 1800 3600 2 100 1 Ammonium sulfate 25 325 1150 3.53 32 1.765 Fractionation (70 %) DEAE-Sepharose 8 32.8 456 13.9 12.7 6.95 Sephadex G-100 4 3.2 212 66.25 5.9 33.12 1 3 Table 1 Puriﬁcation summary of endo b-1,4-D-glucanase from Trichoderma harzianum strain HZN11 3 Biotech (2016) 6:101 Page 5 of 10 101 (A) (B) -0.01 0 0.01 0.02 0.03 0.04 0.05 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1/V (U/ml) 1/[S] (mg/ml) 1/Vmax -1/Km -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1/V U/ml 1/[CMC] mg/ml 0mM 5mM 10mM Ki Fig. 2 Determination of kinetic parameters Km and Vmax by Line- weaver–Burk plot (a) and inhibition kinetics by cellobiose on puriﬁed endo b-1,4-D-glucanase (b) (A) -0.01 0 0.01 0.02 0.03 0.04 0.05 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1/V (U/ml) 1/[S] (mg/ml) 1/Vmax -1/Km and stable for 1 h from 35 to 55 C between 3.0–7.5 pH (Huang et al. 2013). 123 Effect of metal ions, additives, detergents, surfactants, oxidizing agents and organic solvents A cold-active endoglucanase from Aspergillus terreus strain AKM-F3 was found to be resistant to triton X-100 (Maharana and Ray 2015). Appreciable stability of cellulase from Bacillus vallismortis RG-07 for non-ionic surfactants and detergent at 1 % and oxidizing agents at 0.1 % has been reported (Gaur and Tiwari 2015). The enzyme showed more than 75 % of relative activity in most of the organic solvents like glycerol, ethanol, 123 123 3 Biotech (2016) 6:101 101 Page 6 of 10 101 sorghum bagasse and sugarcane bagasse were used to test the ability of the puriﬁed endo b-1,4-D-glucanase with other crude hydrolytic enzyme cocktail and commercial cellulase for the production of fermentable sugar by enzymatic hydrolysis. Alkali pretreated sweet sorghum and sugarcane bagasse released higher amounts of reducing sugars as compared to untreated. Maximum reducing sug- ars of 3.7 and 5.2 g/g were produced from sweet sorghum and sugarcane bagasse, respectively, at 48 h when treated with puriﬁed endo b-1,4-D-glucanase mixture of cocktail, in comparison to commercial cellulase which produced 2.4 and 4.3 g/g of reducing sugars with sweet sorghum and sugarcane bagasse, respectively, at 48 h represented in Fig. 3a. Hence, the efﬁcient bioconversion of lignocellu- losic biomass necessarily requires the synergetic action of cellulolytic enzymes, depolymerizing and debranching hemicellulolytic enzymes. Enzymatic hydrolysis of the alkali pretreated sweet sorghum and sugarcane bagasse into glucose was optimized. Optimization of hydrolysis time and temperature revealed high amounts of sugars 5.2 g/g at 36 h and 6.8 g/g at 48 h from alkali pretreated sweet sor- ghum and sugarcane bagasse, respectively, at 50 C indi- cated in Fig. 3b. Therefore, these process parameters play an essential role in hydrolysis of lignocelluloses to get efﬁcient yield of reducing sugars. Moreover, short pro- cessing times are a key parameter to an economically viable industrial process. Formulation of cellulase enzymes is challenging for hydrolysis process but will not only lower the enzyme loadings but also reduce the capital cost in a cellulosic bioethanol production. In agreement to our results, crude enzyme cocktail from T. harzianum KUC1716 and S. commune KUC9397 could replace the commercial enzymes with approx 80 % hydrolysis yield (Lee et al. 2015). A similar study reported by Patel et al. (2015) on hydrolysis of pretreated maize stover suggests that enzyme cocktail with commercial cellulase results in better sugar release. Vimala et al. Substrate speciﬁcity and enzyme kinetics Puriﬁed endo b-1,4-D-glucanase exhibited highest substrate speciﬁcity with CMC (Table S4, SI). It also showed slight activity with ﬁlter paper and microcrystalline cellulose. In agreement with our ﬁndings, endoglucanse from P. betulinus, Bacillus subtilis YJ1 and Daldinia eschscholzii also showed highest substrate speciﬁcity for CMC (Karn- chanatat et al. 2008; Valaskova and Baldrian 2006; Yin et al. 2010). Endoglucanase reported from Aspergillus nidulans showed activity with pNP-b-D-lactopyranoside and pNP-cellobioside substrates, and hence belonged to GH7 family (Kaur et al. 2015). Furthermore, endo b-1,4-D-glucanase activity increased with increasing substrate concentration up to 22.5 mg/mL, and then saturation was observed due to the saturation of enzyme active sites. Kinetics of enzyme revealed the Km and Vmax of 2.5 mg/mL and 83.75 U/mg, respectively, for CMC shown in Fig. 2a. Lower Km value indicates strong afﬁnity for the substrate CMC. A Km and Vmax of 19.39 g/L and 0.0948 mM/L/min, respectively, was reported by T. harzianum IOC-3844 (de Castro et al. 2010). Endoglu- canase from Penicillium pinophilum MS20 was active towards CMC with Km of 4.8 mg/mL and Vmax of 78.5 U/mg (Pol et al. 2012). Inhibition studies showed cel- lobiose to be competitive inhibitor of endo b-1,4-D-glu- canase in the presence of CMC substrate with a Ki of 0.066 M shown in Fig. 2b. Similar observations were also reported for endoglucanase from Daldinia eschscholzii (Karnchanatat et al. 2008). About 87 % of activity was retained by puriﬁed endo b-1,4-D-glucanase when com- pared to crude enzyme with 30 % activity after 60 days of storage stability (Fig. S3, SI). Effect of metal ions, additives, detergents, surfactants, oxidizing agents and organic solvents Moreover, short pro- cessing times are a key parameter to an economically viable industrial process. Formulation of cellulase enzymes is challenging for hydrolysis process but will not only lower the enzyme loadings but also reduce the capital cost in a cellulosic bioethanol production. In agreement to our results, crude enzyme cocktail from T. harzianum KUC1716 and S. commune KUC9397 could replace the commercial enzymes with approx 80 % hydrolysis yield (Lee et al. 2015). A similar study reported by Patel et al. (2015) on hydrolysis of pretreated maize stover suggests that enzyme cocktail with commercial cellulase results in better sugar release. Vimala et al. (2011) studied the effect of different pretreatment strategies for enzymatic hydrol- ysis of sorghum straw and found that alkali deligniﬁed treatment released higher sugars. To minimize the process cost is of great importance for an economical cellulose- based ethanol production. Even if the new enzyme mixes are said to have improved efﬁciency, the enzyme cost is still contributing to a big part of the total production cost. The present study indicated that the puriﬁed endo b-1,4-D- glucanase with cocktail obtained from Trichoderma har- zianum strain HZN11 enhanced the efﬁciency of biomass hydrolysis. In SHF, ethanol produced was qualitatively detected by l l i f h l d d f 123 Effect of metal ions, additives, detergents, surfactants, oxidizing agents and organic solvents (2011) studied the effect of different pretreatment strategies for enzymatic hydrol- ysis of sorghum straw and found that alkali deligniﬁed treatment released higher sugars. To minimize the process cost is of great importance for an economical cellulose- based ethanol production. Even if the new enzyme mixes are said to have improved efﬁciency, the enzyme cost is still contributing to a big part of the total production cost. The present study indicated that the puriﬁed endo b-1,4-D- glucanase with cocktail obtained from Trichoderma har- zianum strain HZN11 enhanced the efﬁciency of biomass hydrolysis. methanol, acetone, propanol, petroleum ether, isopropanol, benzene, cyclohexane, hexane, butanol and toluene even at 30 % concentration (Table S3, SI). Solvent tolerant enzymes are needed for industrial applications. The enzyme stability in organic solvents may be attributed to its ability to form numerous hydrogen bonds with water, leading to structural ﬂexibility and conformational mobility (Klibanov 2001). In agreement to our results, cold-active endoglucanase from Aspergillus terreus strain AKM-F3 and cellulase from Bacillus vallismortis RG-07 was also found to be stable in most organic solvents (Gaur and Tiwari 2015; Maharana and Ray 2015). sorghum bagasse and sugarcane bagasse were used to test the ability of the puriﬁed endo b-1,4-D-glucanase with other crude hydrolytic enzyme cocktail and commercial cellulase for the production of fermentable sugar by enzymatic hydrolysis. Alkali pretreated sweet sorghum and sugarcane bagasse released higher amounts of reducing sugars as compared to untreated. Maximum reducing sug- ars of 3.7 and 5.2 g/g were produced from sweet sorghum and sugarcane bagasse, respectively, at 48 h when treated with puriﬁed endo b-1,4-D-glucanase mixture of cocktail, in comparison to commercial cellulase which produced 2.4 and 4.3 g/g of reducing sugars with sweet sorghum and sugarcane bagasse, respectively, at 48 h represented in Fig. 3a. Hence, the efﬁcient bioconversion of lignocellu- losic biomass necessarily requires the synergetic action of cellulolytic enzymes, depolymerizing and debranching hemicellulolytic enzymes. Enzymatic hydrolysis of the alkali pretreated sweet sorghum and sugarcane bagasse into glucose was optimized. Optimization of hydrolysis time and temperature revealed high amounts of sugars 5.2 g/g at 36 h and 6.8 g/g at 48 h from alkali pretreated sweet sor- ghum and sugarcane bagasse, respectively, at 50 C indi- cated in Fig. 3b. Therefore, these process parameters play an essential role in hydrolysis of lignocelluloses to get efﬁcient yield of reducing sugars. Enzymatic hydrolysis and ethanol fermentation In SHF, ethanol produced was qualitatively detected by GC–MS. Spectral analysis for ethanol produced from sweet sorghum bagasse after 24 h by Saccharomyces cerevisiae NCIM 3594 showed two peaks in Fig. S4A (SI). Peak 1 showing a retention time of 1.585 with a molecular mass of In the process of production of cellulosic bioethanol, pre- treatment and enzymatic hydrolysis is the crucial steps for sacchariﬁcation and the reducing sugars could be fer- mented to ethanol. Untreated and alkali pretreated sweet 123 (A) (B) 0 1 2 3 4 5 0 20 40 60 80 Reducing sugars (g/g) Time (h) Alkali pretreated with enzyme cocktail Crude with enzyme cocktail Alkali pretreated with Commercial enzyme Crude with Commercial enzyme 0 2 4 6 8 0 20 40 60 80 Reducing sugar (g/g) Time (h) Sweet sorghum with enzyme cocktail at 50°C Sugarcane with enzyme cocktail at 50°C Sweet sorghum with enzyme cocktail at 60°C Sugarcane with enzyme cocktail at 60°C (C) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 5 6 0 10 20 30 40 Cell Biomass (g/L) Reducing sugars (g/g) or Production of ethanol (g/L) Time (h) Initial glucose conc g/g Ethanol g/L Cell Biomass g/L Fig. 3 Enzymatic hydrolysis of untreated and pretreated sweet sorghum bagasse with puriﬁed endo b-1,4-D-glucanase mixed cocktail and commercial enzyme at 40 C (a), optimization of temperature and time for enzymatic hydrolysis of alkali pretreated sweet sorghum and sugarcane bagasse with puriﬁed endo b-1,4-D-glucanase mixed cocktail (b), and production of ethanol from sweet sorghum bagasse hydrolyzate fermented by Saccharomyces cerevisiae NCIM 3594 (c). Enzymatic hydrolysis and ethanol fermentation 2011) whereas peak 2 showing a retention time of 1.643 with a mass of 46 indicating the presence of ethanol by comparison with standard ethanol peak in Fig. S4B (SI). Tiscione et al. (2011) also obtained a similar mass spectrum for ethanol by GC–MS indicating this to be an excellent method for ethanol detection by GC-FID and simultaneous conﬁrma- tion by MS. In SHF, the fermentation of sweet sorghum hydrolyzate by Saccharomyces cerevisiae NCIM 3594 produced ethanol (4.3 g/L) at 16 h indicating the utilization of reducing sugars. Reducing sugar concentration in hydrolyzate decrease from 5.2 to 0.38 g/L within 24 h of fermentation with a cell biomass of 4.4 g/L indicated in Fig. 3c. Mukhopadhyay and Chatterjee (2010) reported 4.5 g/L ethanol with pretreated water hyacinth after 72 h of enzymatic hydrolysis in SHF. Sorghum juice and sorghum press cake fermentation produced 9.2 and 5.6 % (w/v) ethanol (Mamma et al. 1995). Interesting one-pot bioe- thanol production process with A. cellulolyticus C-1 and S. Cerevisiae co-cultures using Solka-Floc were reported (Park et al. 2012). End product inhibition by glucose is a serious problem in SHF. The main advantage is the pos- sibly to separately optimize the process steps. Hence, we report the successful production of bioethanol by SHF of sweet sorghum bagasse with good ethanol concentration. (B) 0 2 4 6 8 0 20 40 60 80 Reducing sugar (g/g) Time (h) Sweet sorghum with enzyme cocktail at 50°C Sugarcane with enzyme cocktail at 50°C Sweet sorghum with enzyme cocktail at 60°C Sugarcane with enzyme cocktail at 60°C (B) 8 Sweet sorghum with enzyme cocktail at 50°C Sugarcane with enzyme cocktail at 50°C Sweet sorghum with enzyme cocktail at 60°C Sugarcane with enzyme cocktail at 60°C (C) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 5 6 0 10 20 30 40 Cell Biomass (g/L) Reducing sugars (g/g) or Production of ethanol (g/L) Time (h) Initial glucose conc g/g Ethanol g/L Cell Biomass g/L Enzymatic hydrolysis and ethanol fermentation Data values represent average of triplicates and error bars represent standard deviation 3 Biotech (2016) 6:101 Page 7 of 10 101 3 Biotech (2016) 6:101 101 (A) (B) 0 1 2 3 4 5 0 20 40 60 80 Reducing sugars (g/g) Time (h) Alkali pretreated with enzyme cocktail Crude with enzyme cocktail Alkali pretreated with Commercial enzyme Crude with Commercial enzyme 0 2 4 6 8 0 20 40 60 80 Reducing sugar (g/g) Time (h) Sweet sorghum with enzyme cocktail at 50°C Sugarcane with enzyme cocktail at 50°C Sweet sorghum with enzyme cocktail at 60°C Sugarcane with enzyme cocktail at 60°C (A) 0 1 2 3 4 5 0 20 40 60 80 Reducing sugars (g/g) Time (h) Alkali pretreated with enzyme cocktail Crude with enzyme cocktail Alkali pretreated with Commercial enzyme Crude with Commercial enzyme 58 indicating the presence of acetone according to the data published previously (Tiscione et al. 2011) whereas peak 2 showing a retention time of 1.643 with a mass of 46 indicating the presence of ethanol by comparison with standard ethanol peak in Fig. S4B (SI). Tiscione et al. (2011) also obtained a similar mass spectrum for ethanol by GC–MS indicating this to be an excellent method for ethanol detection by GC-FID and simultaneous conﬁrma- tion by MS. In SHF, the fermentation of sweet sorghum hydrolyzate by Saccharomyces cerevisiae NCIM 3594 produced ethanol (4.3 g/L) at 16 h indicating the utilization of reducing sugars. Reducing sugar concentration in hydrolyzate decrease from 5.2 to 0.38 g/L within 24 h of fermentation with a cell biomass of 4.4 g/L indicated in Fig. 3c. Mukhopadhyay and Chatterjee (2010) reported 4.5 g/L ethanol with pretreated water hyacinth after 72 h of enzymatic hydrolysis in SHF. Sorghum juice and sorghum press cake fermentation produced 9.2 and 5.6 % (w/v) ethanol (Mamma et al. 1995). Interesting one-pot bioe- thanol production process with A. cellulolyticus C-1 and S. Cerevisiae co-cultures using Solka-Floc were reported (Park et al. 2012). End product inhibition by glucose is a serious problem in SHF. The main advantage is the pos- sibly to separately optimize the process steps. Hence, we report the successful production of bioethanol by SHF of sweet sorghum bagasse with good ethanol concentration. 58 indicating the presence of acetone according to the data published previously (Tiscione et al. SEM and elemental analysis (C) Scanning electron microscopy studies depict the plant tis- sue’s morphological and structural changes occurring during pretreatment and hydrolysis. Untreated sweet sor- ghum bagasse showed a unique structure of the ﬁbers. No pores occurred in large amount as the entire structure was closed indicating to be recalcitrant observed in Fig. 4a. It also reveals intact plant cell wall with vascular bundles and a highly ﬁbrillar structure.SEM image of alkali pretreated sweet sorghum bagasse depicts the alteration in ﬁbrillar structure analyzed in Fig. 4b. Pretreatment usually dislo- cates the bonding among cellulose, hemicelluloses and lignin by dissolving hemicelluloses, but major microﬁbrous cellulose structures remain unaltered and some lignin-car- bohydrate complexes may be packed on the surface of the cellulose ﬁbers. During pretreatment, hydrocarbons are removed and initiates development of cracks on the lig- nocellulosic ﬁber, further increasing the porosity which exposure the cellulosic portion for efﬁcient bioconversion. SEM image of sweet sorghum bagasse hydrolyzed by Trichoderma harzianum strain HZN11 showed intact cells on the substrate particles clearly observed in Fig. 4c. Fig. 3 Enzymatic hydrolysis of untreated and pretreated sweet sorghum bagasse with puriﬁed endo b-1,4-D-glucanase mixed cocktail and commercial enzyme at 40 C (a), optimization of temperature and time for enzymatic hydrolysis of alkali pretreated sweet sorghum and sugarcane bagasse with puriﬁed endo b-1,4-D-glucanase mixed cocktail (b), and production of ethanol from sweet sorghum bagasse hydrolyzate fermented by Saccharomyces cerevisiae NCIM 3594 (c). Data values represent average of triplicates and error bars represent standard deviation 123 3 Biotech (2016) 6:101 101 Page 8 of 10 Fig. 4 SEM analysis of untreated sweet sorghum bagasse (a), alkali pretreated sweet sorghum bagasse (b) and pretreated sweet sorghum bagasse hydrolyzed by Trichoderma harzianum strain HZN11(c) evidenced indicating sacchariﬁcation process by SEM analysis (Irfan et al. 2011). A smooth surfaced compact structure and intact morphology of untreated and signiﬁ- cant increase in porosity of pretreated sorghum stem was visualized by SEM (Nikzad et al. 2014). The elemental analysis of biomass was carried out by SEM equipped with EDX technique. Usually raw biomass has high amounts of potassium (K) and chorine (Cl) as they remain in ionic form and are not metabolized by the plant (Lehmann et al. 2011). The SEM/EDX analyses are shown in Fig. S5A (SI) for untreated, Fig. S5B (SI) for alkali pretreated and Fig. S5C (SI) for Trichoderma harzianum strain HZN11hydrolyzed sweet sorghum bagasse. SEM and elemental analysis The untreated bagasse was composed of carbon (65.47 %) which was gradually reduced to 60.15 % in pretreatment and further reduced to 58.32 % after hydrolysis indicating the utilization of carbon by the organism for enzyme pro- duction (Table S5, SI). The nitrogen content in pretreated bagasse may be exposed due to deligniﬁcation. The pres- ence of sodium may be attributed to alkali pretreatment process. Gradual decrease in the content of minerals and metals in the hydrolyzed bagasse may be due to its uti- lization by the organism. The increase in nitrogen content as compared to the raw biomass may be predicted due to the stability of nitrogen containing compounds such as heterocyclic aromatic compounds (Cantrell et al. 2012). Fuel characteristics of biomass were studied by this tech- nique. Hence, we report an interesting study on elemental analysis of sweet sorghum bagasse, thereby understanding the insights of nutritional changes and its utilization by Trichoderma harzianum strain HZN11during biomass hydrolysis. 123 FTIR analysis A C–O or C–O–C stretching around 1161 cm-1 was observed predict- ing cellulose and hemicellulose structure and peaks around 1100–1050 cm-1 were attributed to b(1-3) polysaccharide in pretreated and hydrolyzed bagasse. A peak found around 898 cm-1 was established for cellulose which increases after pretreatment. FTIR spectra illustrated the deligniﬁcation activities during pretreatment and further degradation of bagasse by enzymatic hydrolysis by Trichoderma harzianum strain HZN11. Previous reports on FTIR analysis of ligno- cellulosic biomass also conclude similar observations (Adapa et al. 2011; Qian et al. 2013). Similar structural changes have been studied in undecayed and decayed lime wood with T. viride (Popescu et al. 2010). The effective utilization of sweet sorghum bagasse for enzyme production is clearly evidenced in our study. wavelength in pretreated and hydrolyzed bagasse signiﬁes deligniﬁcationeffect. Intreatedandhydrolyzedbagasse peaks around 1315 cm-1 are correlated to absorption by C–H and C–O stretching of acetyl group in hemicelluloses. C–O stretching of aryl ethers, and phenolics of lignin-derived compounds and C–O stretching of pyranone rings and gua- iacyl monomers around 1250 cm-1 and presence of aliphatic amines C–N stretch around 1244 cm-1 in untreated bagasse were observed. Glycosidic linkage was predicted in untreated and pretreated bagasse around 1200–1150 cm-1. A C–O or C–O–C stretching around 1161 cm-1 was observed predict- ing cellulose and hemicellulose structure and peaks around 1100–1050 cm-1 were attributed to b(1-3) polysaccharide in pretreated and hydrolyzed bagasse. A peak found around 898 cm-1 was established for cellulose which increases after pretreatment. FTIR spectra illustrated the deligniﬁcation activities during pretreatment and further degradation of bagasse by enzymatic hydrolysis by Trichoderma harzianum strain HZN11. Previous reports on FTIR analysis of ligno- cellulosic biomass also conclude similar observations (Adapa et al. 2011; Qian et al. 2013). Similar structural changes have been studied in undecayed and decayed lime wood with T. viride (Popescu et al. 2010). The effective utilization of sweet sorghum bagasse for enzyme production is clearly evidenced in our study. 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. References Adapa PK, Tabil LG, Schoenau GJ, Canam T, Dumonceaux T (2011) Quantitative analysis of lignocellulosic components of non- treated and steam exploded barley, canola, oat and wheat straw using Fourier transform infrared spectroscopy. 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https://openalex.org/W2232911174	https://hal.inria.fr/hal-01463654/file/978-3-642-39377-8_33_Chapter.pdf	English	null	Using Bloom’s Taxonomy for Information Security Education	IFIP advances in information and communication technology	2,013	cc-by	3,862	To cite this version: Johan Van Niekerk, Rossouw Von Solms. Using Bloom’s Taxonomy for Information Security Educa- tion. 8th World Conference on Information Security Education (WISE), Jul 2009, Bento Gonçalves, Brazil. pp.280-287, 10.1007/978-3-642-39377-8_33. hal-01463654 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01463654 https://inria.hal.science/hal-01463654v1 Submitted on 9 Feb 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Using Bloom’s Taxonomy for Information Security Education Johan Van Niekerk1 and Rossouw Von Solms2 Institute for ICT Advancement, Nelson Mandela Metropolitan University johan.vanniekerk@nmmu.ac.za, rossouw.vonsolms@nmmu.ac.za Institute for ICT Advancement, Nelson Mandela Metropolitan University johan.vanniekerk@nmmu.ac.za, rossouw.vonsolms@nmmu.ac.za Abstract. The importance of educating organizational end users about their roles and responsibilities towards information security is widely ac- knowledged. However, many current user education programs have been created by security professionals who do not necessarily have an educa- tional background. This paper show how the use of learning taxonomies, speciﬁcally Bloom’s taxonomy, can improve such educational programs. It is the authors belief that proper use of this taxonomy will assist in ensuring the level of education is correct for the intended target audience. 1 Introduction The primary aim of corporate information security education is to ensure that each and every employee is instilled with the requisite knowledge and/or skills to perform his or her function in a secure way [1]. Most current information security educational programs are constructed by information security special- ists who do not necessarily have a strong educational background. Studies have shown that the vast majority of current awareness approaches lacks theoretical grounding [2, pp. 33-56]. The nature of security educational or awareness issues are often not understood, which could lead to programs and guidelines that are ineﬀective in practice [3]. This paper shows how the use of Bloom’s revised tax- onomy [4], as a pedagogical framework, can assist the creators of information security educational programs in deﬁning more pedagogically sound learning objectives for the humans involved in information security processes. The work in this paper is based on qualitative research methods. This paper should thus be seen as ”an inquiry process of understanding based on distinct methodological traditions of inquiry that explore a social or human problem” [5, p. 15]. Since education, as a ﬁeld of study, is normally seen as a ”human science” it was deemed ﬁtting to also ”borrow” the research paradigm used in this paper from the humanities. The research presented here does not attempt to deﬁne new knowledge, but rather to show how an existing taxonomy, Bloom’s taxonomy, could be used to improve information security educational programs. This paper is an expansion on ideas previously published by the authors in [6]. It is the authors’ belief that the use of Bloom’s taxonomy could improve the understanding of the pedagogical, or learning, objectives that should be considered in any educational program, amongst information security specialists. The rest of this paper will brieﬂy examine this taxonomy, before discussing its possible use in information security education. The rest of this paper will brieﬂy examine this taxonomy, before discussing its possible use in information security education. 2 Bloom’s taxonomy of the cognitive domain Bloom’s taxonomy is possibly one of the best known and most widely used models of human cognitive processes. Bloom’s model was originally developed in the 1950’s and remained in use more or less unchanged until fairly recently [7, p. 249]. A revised version of the taxonomy was published in 2001 [4]. This revised taxonomy has become accepted as more appropriate in terms of current educational thinking [7, pp. 249-260]. Both versions of Bloom’s taxonomy consist of six levels which increases in complexity as the learner moves up through these levels. Figure 1 shows both versions of this taxonomy. Knowledge Comprehension Evaluation Synthesis Analyses Application Complexity Levels of Bloom’s Taxonomy (original version (1956)) Remember Understand Create Evaluate Analyze Apply Complexity Levels of Bloom’s Taxonomy (revised version (2001)) Fig. 1. Blooms Taxonomy, Original and Revised (Adapted from Sousa (2006) pp. 249- 250) Complexity Fig. 1. Blooms Taxonomy, Original and Revised (Adapted from Sousa (2006) pp. 249- 250) There are two main diﬀerences between the original and the revised ver- sions of the taxonomy. Firstly, the revised version uses descriptive verbs for each level that more accurately describes the intended meaning of each level. Sec- ondly, the revised version has swapped the last two levels of the original version around. This was done because recent studies have suggested that generating, planning, and producing an original ”product” demands more complex thinking than making judgements based on accepted criteria [7, p. 250]. The hierarchy of complexity in the revised taxonomy is also less rigid than in the original in that it recognizes that an individual may move among the levels during extended cog- nitive processes. This paper will focus on the revised version of the taxonomy. Wherever this paper mentions Bloom’s taxonomy, it should be assumed that the revised version is intended, unless otherwise stated. The following is a brief explanation of each of the six levels of this revised taxonomy [7, pp. 250-252]: – Remember: Remember refers to the rote recall and recognition of previously learned facts. This level represents the lowest level of learning in the cognitive domain because there is no presumption that the learner understands what is being recalled. – Understand: This level describes the ability to ”make sense” of the material. In this case the learning goes beyond rote recall. If a learner understands material it becomes available to that learner for future use in problem solving and decision making. 2 Bloom’s taxonomy of the cognitive domain Subject speciﬁc skills, algorithms, techniques, and methods as well as knowledge of criteria for determining when to use appropriate procedures. skills, algorithms, techniques, and methods as well as knowledge of criteria for determining when to use appropriate procedures. – Meta-Cognitive Knowledge - An awareness and knowledge of one’s own cog- nition. I.e. Strategic knowledge, Self-knowledge, knowledge about cognitive tasks, including contextual and conditional knowledge. – Meta-Cognitive Knowledge - An awareness and knowledge of one’s own cog- nition. I.e. Strategic knowledge, Self-knowledge, knowledge about cognitive tasks, including contextual and conditional knowledge. Activities at these six levels of the cognitive domain are usually combined with the one or more of the four types of knowledge in a collection of statements outlining the learning objectives of an educational program. Usually a learning objective statement will be used to create a set of learning activities. Learning activities are activities which help learners to attain the learning objectives. A Learning activity consist of a verb that relates to an activity at one of the levels of the cognitive domain, and a noun providing additional insight into the relationship of the speciﬁc learning objective to a category of knowledge [4, pp. 93-109]. The use of a taxonomy often assist educators in gaining better understanding of learning objectives, and activities. However, it is not always clear how this increased understanding can help the educators. [4, pp. 6-10] identiﬁes the following four ”organizing questions” as the most important areas in which a taxonomy like Bloom’s can assist educators: – The Learning Question: What is the most important for learners to learn in the limited time available – The Instruction Question: How does one plan and deliver instruction that will result in high levels of learning for large numbers of learners – The Assessment Question: How does one select or design assessment in- struments and procedures to provide accurate information about how well students are learning – The Alignment Question: How does one ensure that objectives, instruction, and assessment are consistent with each other. In most cases, the correct usage of a taxonomy table, like the one given in Table 2, which combines elements from both the cognitive and knowledge dimensions, will allow educators to answer these question to some extent. 2 Bloom’s taxonomy of the cognitive domain – Apply: The third level builds on the second one by adding the ability to use learned materials in new situations with a minimum of direction. This includes the application of rules, concepts, methods and theories to solve problems within the given domain. This level combines the activation of procedural memory and convergent thinking to correctly select and apply knowledge to a completely new task. Practice is essential in order to achieve this level of learning. – Analyze: This is the ability to break up complex concepts into simpler com- ponent parts in order to better understand its structure. Analysis skills in- cludes the ability to recognize underlying parts of a complex system and examining the relationships between these parts and the whole. This stage is considered more complex than the third because the learner has to be aware of the thought process in use and must understand both the content and the structure of material. – Evaluate: Evaluation deals with the ability to judge the value of something based on speciﬁed criteria and standards. These criteria and/or standards might be determined by the learner or might be given to the learner. This is a high level of cognition because it requires elements from several other levels to be used in conjunction with conscious judgement based on deﬁnite criteria. To attain this level a learner needs to consolidate their thinking and should also be more receptive to alternative points of view. – Create: This is the highest level in the taxonomy and refers to the ability to put various parts together in order to formulate an idea or plan that is new to the learner. This level stresses creativity and the ability to form new patterns or structures by using divergent thinking processes. In addition to these levels of the cognitive domain [4] also places major emphasis on the use of the following categorization of the knowledge dimension [4, pp. 45-62]: – Factual Knowledge - The most basic elements the learner must know in order to be familiar with a discipline. I.e. Terminology or speciﬁc details and elements. – Conceptual Knowledge - The interrelationships among the basic elements of larger structures that enable these elements to function together. I.e. Clas- siﬁcation, categories, principles, theories, models, etc. – Procedural Knowledge - How to do something, methods of inquiry, how to use skills, apply algorithms, techniques and methods. I.e. 3 Bloom’s Taxonomy for Information Security Education Learning taxonomies assist the educationalist to describe and categorize the stages in cognitive, aﬀective and other dimensions, in which an individual oper- ates as part of the learning process. In simpler terms one could say that learning taxonomies help us to ”understand about understanding” [8]. It is this level of meta-cognition that is often missing in information security education. Accord- ing to Siponen awareness and educational campaigns can be broadly described by two categories, namely framework and content [3]. The framework category contains issues that can be approached in a structural and quantitative manner. These issues constitute the more explicit knowledge. The second category, how- ever, includes more tacit knowledge of an interdisciplinary nature. Shortcomings in this second area usually invalidate awareness frameworks [3]. How to really motivate users to adhere to security guidelines, for example, is an issue that would form part of this content category. Level Verb Sample Activities Create design Write a new policy item to prevent users from putting sensitive information on mobile devices. (A6) Evaluate critique Critique these two passwords and explain why you would recommend one over the other in terms of the security it provides.(A5) Analyze analyze Which of the following security incidents involving stolen passwords are more likely in our company?(A4) Apply execute Use the appropriate application to change your password for the ﬁnancial sub-system. (A3) Understand discuss Why should non alpha-numeric characters be used in a password? (A2) Remember deﬁne What is the deﬁnition of access control? (A1) Table 1. Abbreviated example of Learning Activities based on Bloom’s Taxonomy fo Information Security, adapted from Anderson et al., 2001 Level Verb Sample Activities Create design Write a new policy item to prevent users from putting sensitive information on mobile devices. (A6) Evaluate critique Critique these two passwords and explain why you would recommend one over the other in terms of the security it provides.(A5) Analyze analyze Which of the following security incidents involving stolen passwords are more likely in our company?(A4) Apply execute Use the appropriate application to change your password for the ﬁnancial sub-system. (A3) Understand discuss Why should non alpha-numeric characters be used in a password? (A2) Remember deﬁne What is the deﬁnition of access control? (A1) Table 1. 3 Bloom’s Taxonomy for Information Security Education Abbreviated example of Learning Activities based on Bloom’s Taxonomy for Information Security, adapted from Anderson et al., 2001 In order to ensure successful learning amongst all employees, it is extremely important to fully understand the educational needs of individual employees. Managers often attempt to address the security education needs of employees without adequately studying and understanding the underlying factors that con- tribute to those needs [9, pp. 27-36]. It has been argued before that educational material should ideally be tailored to the learning needs and learning styles of in- dividual learners [10][11, p. 19]. One could also argue that awareness campaigns that have not been tailored to the speciﬁc needs of an individual, or the needs of a speciﬁc target audience, will be ineﬀective. It is in the understanding of these needs, that a learning taxonomy can play an important enabling role. Information security specialists should use a taxonomy, like Bloom’s taxon- omy, before compiling the content category of the educational campaign. The use of such a taxonomy could help to understand the learning needs of the target audience better. It could also reduce the tendency to focus only on the frame- work category of these campaigns. For example, simply teaching an individual what a password is, would lie on the remember, and possibly understand level(s) of Bloom’s taxonomy. However, the necessary information to understand why their own passwords is also important and should also be properly constructed and guarded might lie as high as the evaluate level of the taxonomy. An infor- mation security specialist might think that teaching the users what a password is, is enough, but research have shown that understanding why is essential to obtaining buy-in from employees. It is this level of understanding that acts as a motivating factor and thus enables behaviour change [3][10][9, pp. 78-79]. The use of an educational taxonomy in the construction of information se- curity educational programs requires that both the content and the assessment criteria for this program is evaluated against the taxonomy in order to ensure that learning takes place at the correct level of the cognitive domain. The ref- erence point for any educational program should be a set of clearly articulated ”performance objectives” that have been developed based on an assessment of the target audience’s needs and requirements [9, p. 96]. 3 Bloom’s Taxonomy for Information Security Education Correct usage of an edu- cational taxonomy not only helps to articulate such performance objectives but, more importantly, helps the educator to correctly gauge the needs and require- ments of the audience. An example of how Bloom’s revised taxonomy could be used in an informa- tion security context is supplied in Table 1. This example contains learning activ- ities for a learning objective (LO1) that can be brieﬂy expressed as: ”Learners should be able to understand, construct and use passwords in the correct con- text”. This example is not intended to be a deﬁnitive work, but rather to serve, with taxonomy table Table 2, towards clarifying the use of Bloom’s taxonomy in an information security context. The The Cognitive Process Dimension Knowledge Dimension Remember Understand Apply Analyze Evaluate Create Factual Knowledge A1 A6 Conceptual Test1A Test1B Knowledge A2 A4 A6 Procedural LO1 Knowledge A3 A6 Meta- Cognitive Knowledge A5 Table 2. Example Taxonomy Table adapted from Anderson et al., 2001 The The Cognitive Process Dimension Knowledge Dimension Remember Understand Apply Analyze Evaluate Create Factual Knowledge A1 A6 Conceptual Test1A Test1B Knowledge A2 A4 A6 Procedural LO1 Knowledge A3 A6 Meta- Cognitive Knowledge A5 Table 2. Example Taxonomy Table adapted from Anderson et al., 2001 It was mentioned earlier that answering the four ”organizing questions” is one of the most diﬃcult things for creators of educational matter to do. The following sub-section will brieﬂy explain how the taxonomy table, Table 2 could be used to assist in answering these question for the learning activities, as shown in Table 1. 3.1 Answering the four ”Organizing Questions” Each learning activity in Table 1 consist of a verb that relates to one of the cognitive domain levels in Bloom’s Taxonomy [4, pp 67-68]. Each activity also has a noun relating to knowledge that could be categorized as one of the four categories of knowledge. By marking the appropriate spaces in the taxonomy table for each activity, the educator can derive a lot of useful information about the ”coverage” provided by the activities. As an example, the activity marked A1 Lies at the remember level of the cognitive domain and since it deals with basic subject terminology it deals with the ”factual” category of knowledge. This is reﬂected by its positioning in Table 2. Each of the other activities, A2 to A6, as shown in Table 1 has also been appropriately placed in Table 2. A complete information security educational program will obviously include many more activities, which would result in many more entries in the taxonomy table. Such a table do not always have to deal with an entire program, but could, like the given example, focus on a single learning objective, or even on a few related objectives. By examining the taxonomy table the educator can easily identify areas of knowledge, or levels of the cognitive domain, that has not been covered by the learning activities. Similarly, areas where multiple activities covers the same levels of cognition and categories of knowledge can be identiﬁed. This can assist in answering the so-called ”learning question”, i.e. ”are most important activities receiving the larger share of the available resources?”. In order to design activities that will result in maximum learning, thus answering the ”learning question”, one can look for activities that involves more than just one type of knowledge. For example, in order to create a new policy item (Activity A6), the learner will need to know; basic terminology (factual knowledge), how items relate to each other (conceptual knowledge), and which steps to follow to create a policy (procedural knowledge). To answer the ”assessment question” the educator could choose to focus on the learning objective itself, and thus, in the example given, only use assessment methods that require the learner to apply procedural knowledge. Or the assessor might decide to focus on one or more learning activities and thus have a wider range of assessment coverage. 3.1 Answering the four ”Organizing Questions” By noting assessment activities on the same taxonomy table, the educator can ensure that the chosen assessments correspond directly to what he/she intends to assess. For example, that learners must understand the concept of a password (Test1A) and must be able to analyze the relative strength of a given password ( Test1B). The table will also, at a glance, show which areas are not being assessed. Finally, given a complete taxonomy table, the ”alignment question” should be relatively easy to answer. In the given example, a clear ”disconnect” between the assessment and the learning objective itself exist. Instead of focusing on the application, or use, of passwords the assessments focus on the concept of what a password is, and how to determine its relative strength. Similarly, other ”miss-alignments” can be identiﬁed with the help of this taxonomy table. 4 Conclusion This paper suggested that information security educational programs would be more eﬀective if they adhered to pedagogical principles. It was speciﬁcally sug- gested that an educational taxonomy, like Bloom’s taxonomy should be used to accurately deﬁne the security education needs of organizational users. Through the use of such a taxonomy certain common weaknesses in current security aware- ness and educational programs might be addressed. An example of how Bloom’s taxonomy might be applied to a learning ob- jective in an information security educational program was provided. The paper used this brief example, to show how a taxonomy table based on this example, could assist educators in addressing the four ”organizing questions” faced by educators. The primary weakness of this paper is the lack of empirical evidence to support the suggested use of Bloom’s taxonomy. Due to space limitations, the examples are also by necessity, very brief. Future research in this regard should focus on addressing the lack of empirical evidence, and on expanding the examples to be more comprehensive. It has been argued before that secu- rity practitioners who engage in research or activities that relate to the human sciences should not re-invent the wheel, but should rather ”borrow” from the humanities when appropriate. This paper is one such an attempt, to ”borrow” from the humanities. References [1] Van Niekerk, J., Von Solms, R.: An holistic framework for the fostering of an information security sub-culture in organizations. Information Security South Africa (ISSA), Johannesburg, South Africa (2005) [2] Puhakainen, P.: A design theory for information security awareness. PhD thesis, Acta Universitatis Ouluensis A 463, The University of Oulu (2006) y ( ) [3] Siponen, M.: A conceptual foundation for organizational information security awareness. Information Management & Computer Security 8(1) (2000) 31–41 [4] Anderson, L., Krathwohl, D., Airasian, P., Cruikshank, K., Mayer, R., Pintrich, P., Raths, J., Wittrock, M.: A Taxonomy for Learning, Teaching, and Assessing: A Revision of Bloom’s Taxonomy of Educational Objectives, Complete Edition. Longman (2001) ( ) [5] Creswell, J.W.: Qualitative Inquiry and Research Design: Choosing among Five Traditions. Thousand Oaks, CA: Sage, 1998. Thousand Oaks, CA: Sage (1998) ( ) [6] Van Niekerk, J., Von Solms, R.: Bloom’s taxonomy for information security ed- ucation. Information Security South Africa (ISSA), Johannesburg, South Africa (2008) ( ) [7] Sousa, D.A.: How the brain learns. 3rd edn. Corwin Press (2006) [ ] [8] Fuller, U., Johnson, C.G., Ahoniemi, T., Cukierman, D., Hern´an-Losada, I., Jack- ova, J., Lahtinen, E., Lewis, T.L., Thompson, D.M., Riedesel, C., Thompson, E.: Developing a computer science-speciﬁc learning taxonomy. SIGCSE Bull. 39(4) (2007) 152–170 ( ) [9] Roper, C., Grau, J., Fischer, L.: Security Education, Awareness and Training: From Theory to Practice. Elsevier Butterworth Heinemann (2005) [10] Van Niekerk, J., Von Solms, R.: Corporate information security education: Is out- comes based education the solution? 10th IFIP WG11.1 Annual Working Confer- ence on Information Security Management, World Computer Congress (WCC), Toulouse, France (2004) [11] National Institute of Standards and Technology: NIST 800-16: Information Tech- nology Security Training Requirements: A Role- and Performance-Based Model. NIST Special Publication 800-16, National Institute of Standards and Technology. (1998)
https://openalex.org/W2913623747	https://www.frontiersin.org/articles/10.3389/fpsyg.2019.00168/pdf	English	null	Sex Differences in Body Ownership in Adults With Autism Spectrum Disorder	Frontiers in psychology	2,019	cc-by	8,716	Sex Differences in Body Ownership in Adults With Autism Spectrum Disorder Silvia Guerra1, Andrea Spoto1, Umberto Castiello1* and Valentina Parma2,3,4* A strong male prevalence has been observed in autism spectrum disorder (ASD) since its deﬁnition, but the behavioral manifestations of sex disparity have yet to be clariﬁed. Here, we investigate sex differences in the perception of the Numbness Illusion (NI), a procedure based on a tactile conﬂict, in adults with ASD and with typical development. We aim to assess if women and men with ASD perceive NI-dependent body ownership differently and whether sex differences emerge in individuals with typical development. To elicit the NI, participants pressed their right-hand palm against the confederate’s hand and stroked with the thumb and the index ﬁnger of their left hand the joined index ﬁngers in a synchronous or asynchronous way. Results reveal that women with ASD present a reversed and atypical pattern for the NI compared to men with ASD and a group of matched controls. In particular, women with ASD report a stronger illusion than men with ASD, that is more evident in the asynchronous conditions. In the asynchronous condition, women in the ASD group report stronger NI as compared to women and men in the Control group, whereas men with ASD only to men in the Control group. In the typical sample, the NI emerges only in the synchronous condition and no sex difference is observed. We discuss our results in terms of potential advantage of women in sociality and sensory information processing that might lead women with ASD to use different modalities to solve the illusion compared to men with ASD. In sum, these outcomes describe sex differences in individuals with ASD in the domain of illusory perception. This may be used in the future to support the characterization of the female phenotype of autism. Edited by: Mariska Esther Kret, Leiden University, Netherlands Reviewed by: Francois Quesque, INSERM U1028 Centre de Recherche en Neurosciences de Lyon, France Laura Anne Harrison, University of Southern California, United States Christiana Butera, University of Southern California, United States, in collaboration with reviewer LH *Correspondence: Umberto Castiello umberto.castiello@unipd.it Valentina Parma valentina.c.parma@gmail.com Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Keywords: autism spectrum disorders, body ownership, female phenotype, numbness illusion, sex differences Received: 11 September 2018 Accepted: 17 January 2019 Published: 04 February 2019 Keywords: autism spectrum disorders, body ownership, female phenotype, numbness illusion, sex differences ORIGINAL RESEARCH published: 04 February 2019 doi: 10.3389/fpsyg.2019.00168 Edited by: Mariska Esther Kret, Leiden University, Netherlands Citation: The RHI is an experimental paradigm that modulates the sense of body ownership by presenting incongruent sensory stimulations (i.e., looking at a rubber hand being stroked, while perceiving one’s unseen hand to be similarly touched), which generate a multisensory conﬂict that is solved by relocating the sense of feeling touched on one’s hand on the visible rubber hand. Several variants of RHI were developed to investigate the sense of body ownership: the virtual body illusion (Slater et al., 2008), the presentation of multiple hands (Folegatti et al., 2012) and the numbness illusion (NI; Dieguez et al., 2009; Martuzzi et al., 2015). j g In this connection, several studies based on clinical observations have suggested that high-functioning girls and women with ASD show diﬀerent and less severe social and communication impairments compared to boys and men with ASD (Rivet and Matson, 2011; Werling and Geschwind, 2013a). In particular, girls and women with ASD tend to have better expressive behavior (e.g., sharing interest and/or reciprocal conversation; Lai et al., 2011; Head et al., 2014), less impaired social and communication skills (e.g., desire to interact with other individuals and/or better linguistic ﬂuency; Carter et al., 2007) and diﬀerent repetitive and stereotyped interest and/or activities (e.g., women’s interest tend to involve other people or animals rather than objects; Hiller et al., 2014; Lai et al., 2015) compared to boys and men with ASD. These greater social and communication abilities attributed as a feature of the female phenotype of ASD may help them to cope with social situations, masking some of the symptoms recognized as core symptoms of the male phenotype of ASD and causing misdiagnoses or late identiﬁcation of ASD in girls and women (Wing, 1981; Attwood, 2007; Dworzynski et al., 2012; Hiller et al., 2016). The latter is an experimental paradigm that allows for the manipulation of the experience of the body-ownership of ﬁngers (Dieguez et al., 2009). In this paradigm, two individuals (i.e., the participant and a confederate) press the palm of their hands against each other. Then, both the participant and the confederate stroke with the thumb and the index ﬁnger of their respective free hand two joint index ﬁngers in a synchronous (i.e., the two index ﬁngers are stroked at the same time in up and down movement) or in an asynchronous way (i.e., one ﬁnger is stroked a time). Citation: In particular, in presence of intellectual disability, male and female individuals with ASD meet the diagnostic criteria in a similar way and the ratio of ASD diagnoses is 1F:1M; however, at high IQ scores, female individuals with ASD are underrepresented (4M:1F; Van Wijngaarden-Cremers et al., 2014). In this view, it has been supposed that high IQ scores may represent a confounding factor that leads to a missed diagnosis or a misdiagnosis in girls and women with ASD (Van Wijngaarden-Cremers et al., 2014). recognizing other people’s thoughts, perspectives and mental states [i.e., theory of mind (ToM)] are a trait frequently found in individuals with ASD (Baron-Cohen et al., 1985). Developing functional motor, social and communication skills (Gallagher, 2000) and eﬃciently “walking in someone’s shoes” require the acquisition of the ability to diﬀerentiate the self from others and to compare the two entities. Such distinction can be achieved by developing a coherent sense of “bodily self,” which involves two distinct and interdependent aspects: agency and body ownership. Agency refers to the experience of generating and controlling actions and the events caused by them in the environment (Gallagher, 2000; David et al., 2008). Body ownership refers to “the feeling that my body belongs to me” (as in Stone et al., 2018), and to the fact that my body is diﬀerent from other people’s bodies or external objects. The sense of body ownership, which origins from the integration of diﬀerent sensory information (i.e., proprioceptive, tactile and visual stimuli) is present not only when we act, but also during passive movements (Van den Bos and Jeannerod, 2002) and it can be perturbed by the induction of illusions. Successful perturbation of body ownership has been achieved by presenting incongruent sensory stimulation able to shift the belonging of one body part to either external objects (e.g., a rubber object shaped like a human hand, Botvinick and Cohen, 1998; Ehrsson, 2007) or to another person’s body part (e.g., someone else’s ﬁnger, Dieguez et al., 2009; Martuzzi et al., 2015). The multisensory foundations of body ownership and its underpinnings have been usually investigated by means of the rubber hand illusion (RHI; Botvinick and Cohen, 1998). Citation: Guerra S, Spoto A, Castiello U and Parma V (2019) Sex Differences in Body Ownership in Adults With Autism Spectrum Disorder. Front. Psychol. 10:168. doi: 10.3389/fpsyg.2019.00168 Guerra S, Spoto A, Castiello U and Parma V (2019) Sex Differences in Body Ownership in Adults With Autism Spectrum Disorder. Front. Psychol. 10:168. doi: 10.3389/fpsyg.2019.00168 Autism spectrum disorder (ASD) is deﬁned as a heterogeneous disorder characterized by impairments in social interactions, communication, and repetitive and stereotyped behaviors, which is more commonly diagnosed in male than in female individuals (4M: 1F; Werling and Geschwind, 2013a,b; Halladay et al., 2015). The male prevalence in ASD is known from the origin of the disorder. Indeed, both Kanner (1943) and Asperger (1944) reported that the children with February 2019 \| Volume 10 \| Article 168 Frontiers in Psychology \| www.frontiersin.org 1 Sex Differences in Body Ownership in ASD Guerra et al. autism that they examined were exclusively boys. Consequently, the majority of the research on ASD has chieﬂy focused on male participants. As a consequence, the female phenotype of ASD is still poorly understood and research results in that area are highly inconsistent (Head et al., 2014). To date, these accounts have been used to explain the mechanisms underlying sex disparity in ASD with some studies suggesting that the female phenotype of ASD may be the result of innate characteristics that protect girls and women from ASD and make them less vulnerable to develop the core symptoms of the disorder (female protective eﬀect or FPE; Robinson et al., 2013; Werling and Geschwind, 2013a). Other studies have advanced that female and male individuals are equally predisposed to develop ASD at the genetic level, but female individuals may have some factors - at the cognitive or/and neurobiological level – enabling them to better compensate for this risk during the lifespan (e.g., Skuse, 2007). Moreover, it has been proposed that sex diﬀerences in the development of the cognitive proﬁle may lead to diﬀerent manifestations of ASD in women and men (Carter et al., 2007). From the studies that have addressed sex disparity in ASD, it becomes evident that intellectual abilities play a role in facilitating the diagnosis of ASD in female individuals. Frontiers in Psychology \| www.frontiersin.org Participants The sample for the two experiments (also previously included in Guerra et al., 2017) consisted of 108 participants. Sample size for the group∗sex interaction∗conditions was estimated by means of the G∗Power 3.1 software (Faul et al., 2009) to have a power ≥95%, even in the case of a medium-small eﬀect size (0.22). In Experiment 1, 39 adults with high-functioning ASD were enrolled (ASD; 29 M and 10 F; mean age ± 24.72; age range 19–31 years). In Experiment 2, 69 age- and FSIQ-matched control adults with typical development were recruited (43 M and 26 F; mean age ± 23.64; age range 19–33 years). Individuals with ASD were age-, gender- and full scale IQ-matched with the individuals with typical development (see Table 1). Full scale IQ was measured via the Wechsler Adult Intelligence Scale – Fourth edition (WAIS-IV; Wechsler, 2008; Italian language adaptation: To delineate whether the ASD diagnosis modulates the expression of body ownership and whether sex diﬀerences in the NI are evident irrespective of the ASD diagnosis, we additionally tested a Control sample of typical individuals. Indeed, evidence has suggested that sex inﬂuences many aspects of typical development (Kimura, 1992; Baron-Cohen et al., 2005). In particular, men score higher in spatial abilities (e.g., mental rotation tasks, map reading tasks; Kimura, 1999), while women exhibit better-than-male performance in social sensitivity, emotional recognition and verbal ﬂuency tasks. Thus, TABLE 1 \| Sample description. Citation: In brief, stroking the ﬁngers synchronously generates in the participants the sensation of owning the confederate’s ﬁnger as if it were his/her own ﬁnger. This illusion only emerges when the stroking occurs simultaneously. When the stimulation is asynchronous or performed by another person, the illusion is not perceived (or its illusory eﬀects are reduced). Such illusory experience has been replicated in individuals with typical development (Dieguez et al., 2009; Martuzzi et al., 2015) and we have recently demonstrated its presence in adults with ASD The above-mentioned evidence is in line with the extreme male brain theory (EMB; Baron-Cohen, 2002), which posits that the underlying sex disparity in ASD might be the ‘hyper- masculinization’ of some behaviors. In other words, the ASD proﬁle may represent an extreme form of the typical male proﬁle, which is characterized by enhanced systemizing and reduced empathizing skills. Deﬁcits in empathy, in understanding and February 2019 \| Volume 10 \| Article 168 2 Sex Differences in Body Ownership in ASD Guerra et al. (Guerra et al., 2017). However, whether body ownership illusory experiences are comparable among women and men with ASD is still unknown. considering the potential female advantage in the social domain and given that the development of adaptive social functioning requires an eﬃcient sense of body ownership, we would expect a stronger disruption of the sense of body ownership during synchronous stroking in women with typical development compared to men. Here, we test whether women and men with ASD experience the NI in a similar way. Considering that women with ASD reportedly show less impairment in social information processing (e.g., Werling and Geschwind, 2013a), we foresee that the NI experience would be more eﬃcient in women with ASD as compared to their male counterparts. In other words, we expect women with ASD to be more subjected to the NI, in virtue of a greater disruption of the sense of body ownership. If this were true, outcomes may point out for the ﬁrst time to the existence of sex diﬀerences in the domain of sensory and illusory experiences in ASD and they may contribute to further support the characterization of the female phenotype of ASD. Participants The stroking was performed synchronously (i.e., the joint index ﬁngers were stroked at the same time) or asynchronously (i.e., the joint index ﬁngers were stroked alternatively) by the participant. In this posture, the participant stroked the dorsal side of the distal phalanges of the joined index ﬁngers with the thumb and the index ﬁnger of the other hand, either in a synchronous or in an asynchronous way. Speciﬁcally, in synchronous conditions both the index ﬁnger and the thumb of the participant’s free hand started from the ﬁrst phalanx and moved toward the third phalanx of the index ﬁnger of the receiver in a repetitive up- to-down movement. Instead, in asynchronous conditions the index ﬁnger of the agent started from the ﬁrst phalanx, whereas the thumb started from the third phalanx of the index ﬁnger of the receiver and they moved in opposite directions stroking one ﬁnger at a time, alternatively. Before the beginning of the experimental phase, participants were trained to achieve a consistent stroking frequency and pressure. The frequency in stroking (i.e., 10 strokes in 10 s; 1 Hz) was constantly monitored by a co-experimenter by means of a timer to ensure that it was comparable across participants. The experimental design was a 2 × 2 factorial design. The factor Synchrony – how the joint index ﬁngers were stroked – had two levels, namely synchronous (i.e., ﬁngers stroked simultaneously) or asynchronous (i.e., ﬁngers alternatively stroked). The factor Agent – who performed the stroking of the joint index ﬁngers –had two levels, namely self (i.e., participant) and other (i.e., the experimenter). Given that the NI emerges only when the stimulation is self-administered and it primarily depends on the synchrony of the stimulation (i.e., Dieguez et al., 2009; Martuzzi et al., 2015; Guerra et al., 2017), the ‘Other’ condition has not been considered in this study (see Guerra et al., 2017 for an account on such condition in individuals with ASD). Indeed, the stroking performed by other people is not eﬀective in inducing changes in the experience of the NI, irrespective of the type of synchrony of the stimulation. By removing this condition, we were able to gain power to evaluate the sex eﬀects on the NI. This led to two experimental conditions, namely self-synchronous and self-asynchronous. Each condition was repeated four times in a pseudo-randomized order for a total of 8 trials. Each trial lasted 10 s. Data Analysis All statistical analyses were carried out with the R software (R package version 3.3.9; R Core Team, 2013) and, more speciﬁcally, by means of the lme function (nlme package version 3.1-131) to perform linear mixed eﬀect models. For each participant, the mean of the responses across all conditions were computed to produce an individual index of the illusion experienced by the participant during the task (as in Dieguez et al., 2009 and Martuzzi et al., 2015). At ﬁrst, the data from the ASD group were analyzed by means of ﬁtting a linear mixed-eﬀect model with Participants Participants with ASD received a formal diagnosis from an expert, licensed clinical psychologist based on the Diagnostic and Statistical Manual of Mental Disorder – 5 (DSM-5) and the clinical evaluation was supported by meeting criteria on at least the Autism Diagnosis Observation Schedule (ADOS; Lord et al., 2000) or the Autism Diagnostic Interview – Revised (ADI-R; Lord et al., 1994; see Table 1), either on both. Participants with typical development had no history of ASD and they did not have any ﬁrst or second- degree relatives with a diagnosis of ASD. Participants with ASD were recruited via the local Pediatric and Developmental Neuropsychiatric Clinics, while volunteers were recruited on campus at the University of Padova (Italy). The project was approved by the local ethical committee and the experimental procedures were in accordance with the Declaration of Helsinki (Williams, 2008). All participants signed a written informed consent prior to the beginning of their experimental session. Participants At the end of each trial, participants rated the strength of the illusion experienced during the task by means of a questionnaire composed by 5 questions presented on 5-point Likert scale (Dieguez et al., 2009; Martuzzi et al., 2015; Table 2). The scale ranged from 1 (completely disagree) to 5 (completely agree). In line with previous studies (e.g., Dieguez et al., 2009; Martuzzi et al., 2015), we considered scores higher than 3 indicating that a signiﬁcant illusory experience was reported. Questions were repeated in a pseudo-randomized order across all trials, to reduce contextual inﬂuences on responses. FIGURE 1 \| Procedure to induce the NI: the participant pressed the palm of his/her right hand against the left palm of the experimenter. In this posture, the participant stroked with the index and thumb of his/her free hand the joined index ﬁngers (participant + experimenter). The stroking was performed synchronously (i.e., the joint index ﬁngers were stroked at the same time) or asynchronously (i.e., the joint index ﬁngers were stroked alternatively) by the participant. Orsini and Pezzuti, 2013) or via the Wechsler Abbreviated Scale of Intelligence (WASI; Wechsler, 1999). Participants with ASD received a formal diagnosis from an expert, licensed clinical psychologist based on the Diagnostic and Statistical Manual of Mental Disorder – 5 (DSM-5) and the clinical evaluation was supported by meeting criteria on at least the Autism Diagnosis Observation Schedule (ADOS; Lord et al., 2000) or the Autism Diagnostic Interview – Revised (ADI-R; Lord et al., 1994; see Table 1), either on both. Participants with typical development had no history of ASD and they did not have any ﬁrst or second- degree relatives with a diagnosis of ASD. Participants with ASD were recruited via the local Pediatric and Developmental Neuropsychiatric Clinics, while volunteers were recruited on campus at the University of Padova (Italy). The project was approved by the local ethical committee and the experimental procedures were in accordance with the Declaration of Helsinki (Williams, 2008). All participants signed a written informed consent prior to the beginning of their experimental session. Orsini and Pezzuti, 2013) or via the Wechsler Abbreviated Scale of Intelligence (WASI; Wechsler, 1999). Participants ASD Group Males Females test p-value N 74% (n = 29) 26% (n = 10) χ2(1) = 9.26 < 0.01∗ AGE 24.93 ( ± 3.33; range 19–31) 24.1 ( ± 3.60; range 19–30) t37 = −0.66 0.509 Full scale IQ 113.33 ( ± 11.15; range 89–129) 114.14 ( ± 12.29; range 98–128) t37 = 0.16 0.879 ADOS (total) 10.6 ( ± 5.41; range 3–23) 10.9 ( ± 4.61; range 6–22) t37 = 0.16 0.871 ADI-R (total) 39.79 ( ±14.33; range 20–67) 35.1 ( ± 5.67; range 27–44) t37 = −1.00 0.323 Control Group Males Females test p-value N 62% (n = 43) 38% (n = 26) χ2(1) = 4.19 < 0.05∗ AGE 24.14 ( ± 3.38; range 19–33) 22.73 ( ± 2.47; range 19–28) t67 = −1.78 0.079 Full scale IQ 107.25 ( ± 12.28; range 92–121) 112.67 ( ± 4.50; range 108–117) t67 = 0.72 0.487 ASD Group Control Group test p-value N F 9% (n = 10) 24% (n = 26) χ2(1) = 7.11 < 0.01∗ M 27% (n = 29) 40% (n = 43) χ2(1) = 2.72 0.099 total 36% (n = 39) 64% (n = 69) χ2(1) = 1.62 0.202 AGE F 24.1 ( ± 3.60) 22.73 ( ± 2.47) t34 = 1.25 0.220 M 24.93 ( ± 3.33) 24.14 ( ± 3.38) t70 = 0.98 0.330 total 24.72 ( ± 3.37) 23.64 ( ± 3.10) t106 = 1.68 0.095 Full scale IQ F 114.14 ( ± 12.29) 112.67 ( ± 4.50) t34 = 0.19 0.849 M 113.33 ( ± 11.15) 107.25 ( ± 12.28) t70 = 1.30 0.202 total 113.5 ( ± 11.21) 108.7 ( ± 10.77) t106 = 1.23 0.226 ASD, autism spectrum disorder; IQ, intelligence quotient; ADOS, autism diagnosis observation schedules; ADI–R, autism diagnostic interview-revised. Age, IQ, ADOS and ADI-R refer to the mean, while standard deviation and range are given in parentheses. F, females; M, males; ∗p < 0.05. Frontiers in Psychology \| www.frontiersin.org February 2019 \| Volume 10 \| Article 168 3 Sex Differences in Body Ownership in ASD Guerra et al. FIGURE 1 \| Procedure to induce the NI: the participant pressed the palm of his/her right hand against the left palm of the experimenter. In this posture, the participant stroked with the index and thumb of his/her free hand the joined index ﬁngers (participant + experimenter). Procedure The procedures were the same as in Guerra et al., 2017. Each participant and the experimenter sat facing each other. At the beginning of each trial, the participant was asked to press the palm of his/her right hand against the experimenter’s left-hand palm, which was lifted in the air (Figure 1). February 2019 \| Volume 10 \| Article 168 Frontiers in Psychology \| www.frontiersin.org 4 Sex Differences in Body Ownership in ASD Guerra et al. TABLE 2 \| Numbness illusion self-report. During the stroking of the ﬁngers. . . 1. The felt sensation was strange 2. I felt a sensation of numbness 3. It seemed like my own stroked ﬁnger became wider in size 4. It seemed like the experimenter’s ﬁnger became my own ﬁnger 5. It seemed like I felt only the big ﬁnger was being touched Completely disagree Disagree Neutral Agree Completely agree 1 2 3 4 5 p = 0.007). Furthermore, women with ASD reported a greater disruption of ﬁnger’s ownership when the stroking was asynchronous than men with ASD both in the asynchronous (t37 = 2.885; p = 0.031) and the synchronous conditions (t37 = 3.108; p = 0.018). No main eﬀect of Synchrony emerged [F(1,37) = 0.97; p = 0.331; partial-η2 = 0.042; Figure 2]. Sex Affects the NI Experience in Individuals With ASD, but Not in the Control Group To test whether this sex diﬀerence is characteristic of the ASD experience of the NI or it is also experienced by individuals with typical development, we ﬁtted a linear mixed- eﬀect model including the variable Group as a between factor. Results indicated a signiﬁcant eﬀect of Sex [F(1,104) = 4.79; p = 0.031; partial-η2 = 0.050]. Pairwise comparisons showed that women experienced the illusion more than men of both groups (t104 = 2.34; p = 0.021). More speciﬁcally, results indicated that women with ASD perceived a stronger illusion compared to women (t104 = 3.75; p = 0.007) and men (t104 = 4.34; p < 0.001) in the Control group, when the stroking was asynchronous. Furthermore, when women with ASD performed the stroking synchronously, results showed that the illusion was diﬀerently compared to men (t104 = 3.54; p = 0.014) in the Control group in the asynchronous condition. The same pattern was also observed in the comparison between men in the ASD and the Control group with respect to the asynchronous conditions (t104 = 3.27; p = 0.031). However, diﬀerently from what we found in the ASD group (see paragraph above), no signiﬁcant sex diﬀerences Synchrony (synchronous and asynchronous) as within factor, Sex (women and men) as between factors. Then, data from the two groups (ASD and Control) were analyzed by means of ﬁtting a linear mixed-eﬀect model with Synchrony (synchronous and asynchronous) as within factor, while Sex (women and men) and Group (ASD and Control) as between factors. When signiﬁcant interactions were retrieved, we conducted pairwise comparisons. The signiﬁcance level was set at p < 0.05. RESULTS Women With ASD Experience the NI More Strongly Than Men With ASD The analysis revealed that the NI was perceived diﬀerently by men and women with ASD [Sex: F(1,37) = 8.22; p = 0.007; partial-η2 = 0.182]. Indeed, the NI was perceived more clearly by women with ASD compared to men with ASD (t37 = 2.867; FIGURE 2 \| Strength of the NI for women (F) and men (M) of ASD group. Error bars indicate the standard error of the mean (SEM). ∗= p < 0.05; The horizontal line with intercept 3 refers to the level at which the illusion was experienced by participants. FIGURE 2 \| Strength of the NI for women (F) and men (M) of ASD group. Error bars indicate the standard error of the mean (SEM). ∗= p < 0.05; The horizontal line with intercept 3 refers to the level at which the illusion was experienced by participants. February 2019 \| Volume 10 \| Article 168 Frontiers in Psychology \| www.frontiersin.org 5 Sex Differences in Body Ownership in ASD Guerra et al. were reported in the subjective experience of the illusion in the Control group [Group∗Sex: F(1,104) = 0.684; p = 0.410; partial-η2 = 0.007]. This suggests that women and men with typical development experience the NI in a similar manner (synchronous: t104 = 1.89; p = 0.555; asynchronous: t104 = 0.51; p = 0.999). These results were ascribable to the signiﬁcant eﬀect of Synchrony [F(1,104) = 30.91; p < 0.0001; partial-η2 = 0.104]. Indeed, the strength of the NI changed depending on the type of stroking (synchronous or asynchronous; Figure 3). This pattern holds true both for women (t104 = −5.85; p < 0.0001) and men (t104 = −5.01; p = 0.0001) with typical development and in the comparison between them (t104 = −4.67; p = 0.0002), but it is not evident in the ASD group (Figure 4). Indeed, the type of illusion perceived was diﬀerent between the ASD and the Control group [Group: F(1,104) = 6.61; p = 0.012; partial-η2 = 0.075]. More speciﬁcally, the synchronous self-stroking produces the illusory eﬀect of the NI in individuals of both the ASD and the Control groups. Such eﬀect is not evident in Controls when the movement was performed asynchronously [Group∗Synchrony: F(1,104) = 26.77; p < 0.0001; partial-η2 = 0.204]. RESULTS Average scores to each item separately per group, per gender and per condition are reported in Table 3, while a frequency table with respect of participants’ self-report responses are given in Supplementary Table S1. inﬂuence of sex diﬀerences in sensory experiences in ASD has only been marginally addressed. This study aimed to explore whether the experience of sensory-induced body ownership – measured behaviorally by means of the NI - diﬀers between women and men with ASD. Our ﬁndings showed a clear sex diﬀerence in the strength of the NI experienced by individuals with ASD. Despite both women and men with ASD reported to experience the disruption of the body ownership over their own ﬁnger in the synchronous and in the asynchronous conditions, women reported to experience the illusion signiﬁcantly more strongly than men. To evaluate whether such sex diﬀerence in the experience of the NI is speciﬁc to ASD or it is a more general phenomenon, we also tested the eﬀect of sex on the NI in a group of women and men with typical development. Comparing the performance of individuals with ASD with that of a group of matched controls, it emerges that women with ASD were more susceptible to the NI than women and men with typical development, especially when considering the asynchronous condition. This result acquires even more relevance when considering that no sex diﬀerences appeared when analyzing the Control group alone, showing that the NI manifests in a similar manner in both typically developing women and men. When focusing on the performance of men in both groups it is evident that both men in the ASD and Control groups were less susceptible to the NI compared to the women in both groups. DISCUSSION Two explanations, one focused on social skills and the other on sensory abilities, can be advanced to interpret these data. First, this ﬁnding may be interpreted as a reﬂection of the ability of women with ASD to better deal with socialization and empathy (e.g., Werling and Geschwind, 2013a). Indeed, the sense Researchers and clinicians are devoting more and more eﬀort to understanding whether and how the diﬀerences between the female and male phenotypes of ASD can emerge. So far, the FIGURE 3 \| Strength of the NI for women (F) and men (M) in the ASD and Control groups in the synchronous and asynchronous conditions. ∗= p < 0.05; Error bars refer to the standard error of the mean (SEM). The horizontal line with intercept 3 refers to the level of which the illusion was experienced by participants. FIGURE 3 \| Strength of the NI for women (F) and men (M) in the ASD and Control groups in the synchronous and asynchronous conditions. ∗= p < 0.05; Error bars refer to the standard error of the mean (SEM). The horizontal line with intercept 3 refers to the level of which the illusion was experienced by participants. February 2019 \| Volume 10 \| Article 168 Frontiers in Psychology \| www.frontiersin.org 6 Sex Differences in Body Ownership in ASD Guerra et al. FIGURE 4 \| Strength of the NI for women (F) and men (M) in the Control group in the synchronous and asynchronous conditions. ∗= p < 0.05; ns = lack of statistical signiﬁcance. Error bars refer to the standard error of the mean (SEM). The horizontal line with intercept 3 refers to the level of which the illusion was experienced by participants. FIGURE 4 \| Strength of the NI for women (F) and men (M) in the Control group in the synchronous and asynchronous conditions. ∗= p < 0.05; ns = lack of statistical signiﬁcance. Error bars refer to the standard error of the mean (SEM). The horizontal line with intercept 3 refers to the level of which the illusion was experienced by participants. et al., 2012) who exhibited reduced empathetic skills were also those who were less susceptible to the RHI. Although not tested directly, we may speculate that the women with ASD in the present sample may present better empathic skills than their male counterparts. DISCUSSION A second explanation, not in contrast with the previous one, may suggest that the increased susceptibility to the NI in women is the result of the ability of women to diﬀerentially focus on the sensory input received. Women seem to be more focused on the sensory information to solve the mismatch produced by the tactile conﬂict in the NI, whereas men rely less on such sensory information. This idea is in line with the evidence showing that girls with ASD score higher in the subscales of “Touch Response and Use” in the Tokyo version of the Childhood Autism Rating Scale (CARS) scale (Kumazaki et al., 2015) and that women with ASD report more sensory-motor symptoms than men with ASD (Moseley et al., 2018). Furthermore, increased sensory issues (e.g., noise hypersensitivity, unusual sensory interest, . . .) were reported more frequently in females with ASD than in males with ASD (Gould and Ashton-Smith, 2011; Lai et al., 2011). When directly comparing women with ASD and with typical development, self-reports suggest that they are both more sensitive to sensory stimulation than men (Tavassoli et al., 2014). This sensory perspective also ﬁts with the evidence of a stronger NI’s experience in women in the synchronous (but not in the asynchronous) condition as compared to men. Indeed, results showed that the NI emerged in both groups when the stroking was synchronous, while, when the movement was performed asynchronously, of body ownership has been deemed crucial in the development of adaptive social skills, particularly imitation and empathy (Gallese, 2003). The literature exploring the link between body ownership and empathy reveals that both participants with ASD (Cascio et al., 2012) and typical development (Farmer TABLE 3 \| Self-report’s scores. DISCUSSION ASD group Control group Males Females Males Females Synchronous Item 1 3.38 ± 0.57 3.63 ± 0.44 2.99 ± 0.97 3.34 ± 0.94 Item 2 3.08 ± 0.68 4.03 ± 0.36 2.60 ± 1.10 2.88 ± 0.99 Item 3 3.07 ± 0.79 3.60 ± 0.49 3.02 ± 1.06 3.14 ± 0.97 Item 4 3.26 ± 0.51 2.85 ± 1 3.38 ± 0.94 3.73 ± 0.77 Item 5 2.97 ± 0.62 3.20 ± 1.21 3.64 ± 0.88 3.97 ± 0.73 3.15 ± 0.38 3.46 ± 0.44 3.13 ± 0.70 3.41 ± 0.62 Asynchronous Item 1 3.36 ± 0.71 3.43 ± 0.59 2.66 ± 0.93 2.83 ± 0.85 Item 2 3.15 ± 0.63 3.90 ± 0.70 2.34 ± 0.96 2.60 ± 0.95 Item 3 3.10 ± 0.81 3.93 ± 0.58 2.71 ± 1.02 2.47 ± 0.82 Item 4 3.27 ± 0.69 3.33 ± 0.81 2.88 ± 0.77 2.99 ± 0.96 Item 5 3.04 ± 0.76 3.58 ± 1.03 2.95 ± 0.91 3.05 ± 0.93 3.18 ± 0.45 3.63 ± 0.42 2.71 ± 0.72 2.79 ± 0.65 Average scores for each questionnaire’s item across all condition of both ASD and Control groups. Values refer to the mean and standard deviation of participants’ ratings to the self-reports’ questions in both synchronous and asynchronous conditions, respectively. See Table 2 for the description of each item. Values in bold type refer to the mean and standard deviation of the strength of illusion experienced by participants in different conditions. February 2019 \| Volume 10 \| Article 168 Frontiers in Psychology \| www.frontiersin.org 7 Sex Differences in Body Ownership in ASD Guerra et al. Furthermore, studies on unusual tactile sensitivity in autism reported no diﬀerences in the domain of tactile perception across diﬀerent tactile stimuli (e.g., detection of light touch, discrimination of the roughness of diﬀerent sandpapers,. . .) when high-functioning individuals with ASD were compared with individuals with typical development (O’Riordan and Passetti, 2006; Cascio et al., 2008). However, these ﬁndings reﬂect a non-social aspect of sensory suggestibility and may not be impaired in ASD. In lack of other evidence directly linking socially relevant sensory suggestibility to the tactile domain, we turn to the evidence gathered from eye-witnesses. In this case, individuals with ASD are reported to be “no more or less suggestible than their typical counterparts” when directly asked to report about their experiences (Maras and Bowler, 2014). DISCUSSION Second, if sensory suggestibility in the tactile domain is key to the perception of the NI, we should expect that the variability in sensory suggestibility would also be reﬂected at the level of the Control group. However, this is not the case, since a diﬀerence between the synchronous and asynchronous conditions is reported in the Control group, as expected when also looking at other illusory paradigms (Stone et al., 2018), but not in ASD. only the participants in the ASD group experienced the illusion. This seems to suggest that the process diﬀerentiating between self and other in individuals with ASD is impaired, in that an excessive focalization on one’s own self can alter the perception of the self-other boundary (Noel et al., 2017). An interesting observation, that was not part of our initial set of hypotheses, is related to the range of the responses given by the ASD group. Both women and men with ASD were highly reliable in providing the same rating of the strength of the NI. In other words, at the group level, the responses are locked around a limited range of options, as the inspection of the error bars suggests. This ﬁnding might be taken as evidence that the participants with ASD had diﬃculties in the understanding of the questions posed in the self-report. However, all participants with ASD presented a full-scale IQ comparable to that of controls, and this seems to be suﬃcient reason to believe that the instructions were understood and complied to the same extent as in the Control group. A more likely explanation for the reduced range of responses in the ASD sample can be found when interpreting this outcome in the context of the aberrant precision theory (Bolis et al., 2017). Such theory posits that individuals with ASD use abnormal strategies (i.e., perceptual hypersensitivity, hyper-attention to details,. . .) to make perceptual inferences. Such strategies, rather than maximizing the conﬁdence in the sensory evidence estimated based on a priori beliefs (i.e., reducing the prediction error), tend to produce sub-optimal inferences about the nature of the sensory information. In other words, and compatibly with the neural instantiation of Bayesian inference from which this principle is extracted (e.g., Friston, 2005; Bastos et al., 2012), aberrant precision strategies emerge when the sensory bottom-up input and the top-down predictions about a stimulus are mismatching (i.e., the prediction error). DISCUSSION In the context of the NI, the expectation of the participant is to feel their own index ﬁnger pressed against the hand of the experimenter as part of their own body. However, the sensory inputs (visual and tactile) produce an experience compatible with the reduction of body ownership for such ﬁnger (self) and attributing the ownership of that ﬁnger to the experimenter (other). Put in these words, it appears evident how the precision ascribed to the sensory evidence retrieved is imbalanced with respect to the a priori belief hold about the experience. In line with the call from the ASD and the scientiﬁc community for research into the female autistic phenotype (Halladay et al., 2015; Lai et al., 2015), the present study contributes to uncover of sex diﬀerences in adults with ASD in the ﬁeld of the perception of body ownership by means of the NI, a novel procedure to better understand sensory and social issues in individuals with ASD. As for most innovative studies, some limitations in data interpretation exist and future studies will be needed to address them. First, although proportional to what found in the ASD population, the sample of women with ASD included in the present study is rather limited. Therefore, to conﬁrm the present set of results we call for the replication of this study in a larger sample of individuals with ASD. Second, to better characterize the female phenotype of ASD a developmental perspective is needed. Indeed, testing our hypotheses from childhood to adulthood will allow to understand more deeply how sex diﬀerences in body ownership emerge over development. Third, to conﬁrm the speciﬁcity of these results to ASD, it would be important to investigate sex diﬀerences in the NI in a group of individuals with non-ASD atypical development. Fourth, the administration of standardized self- reports on sensory perception (e.g., the sensory perception quotient; Tavassoli et al., 2014) and empathy (e.g., empathy quotient; Baron-Cohen and Wheelwright, 2004) can be used to probe the link between empathizing skills and the sense of body ownership during the NI. Indeed, testing the possible relationships between sex diﬀerences in the NI experience and individual empathic competences in typical and atypical populations might contribute to better understanding the processing underlying the behavioral sex disparity in ASD. Frontiers in Psychology \| www.frontiersin.org REFERENCES disorders? J. Am. 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AUTHOR CONTRIBUTIONS VP, UC, and SG conceived and designed the study. SG contributed to testing and data acquisition. AS, SG, and VP analyzed and interpreted the data. VP and SG drafted the manuscript. VP, UC, AS, and SG reviewed and edited the manuscript. All authors approved the ﬁnal version of the manuscript for submission. VP, UC, and SG conceived and designed the study. SG contributed to testing and data acquisition. AS, SG, and VP analyzed and interpreted the data. VP and SG drafted the manuscript. VP, UC, AS, and SG reviewed and edited the manuscript. All authors approved the ﬁnal version of the manuscript for submission. VP, UC, and SG conceived and designed the study. SG contributed to testing and data acquisition. AS, SG, and VP ACKNOWLEDGMENTS We are indebted to our participants for having given us their time and best eﬀort to complete the tasks. We are indebted to our participants for having given us their time and best eﬀort to complete the tasks. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2019.00168/full#supplementary-material FUNDING To summarize, this is the ﬁrst study exploring how women and men with ASD are aﬀected by a sensory-induced illusion on the sense of body ownership. These results, discussed in the context of social and sensory issues typical of women with ASD, pave the way for the investigation of how sensory experiences can help deﬁne the female phenotype of ASD. This work was supported by the Strategic Project (No. 2010XPMFW4) to UC. DISCUSSION Fifth, to further test whether the results hereby presented are confounded by sensory suggestibility, we suggest the inclusion of the sensory suggestibility scale (Gheorghiu et al., 1995) in future investigations. p Despite the attention to details, including sensory ones in ASD (Martínez-Sanchis, 2014), one might also consider the lack of a signiﬁcant diﬀerence between the synchronous and asynchronous conditions in ASD as the reﬂection of the lack in the perception of the NI and, instead, the evidence of greater sensory suggestibility in ASD. Although our data are not directly able to test this issue, we contend that this may be unlikely for at least two reasons. First, sensory suggestibility does not seem to be impaired in ASD. 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https://openalex.org/W1564804666	https://journal.fi/scripta/article/download/67228/27526	English	null	Dance as Aggressiveness	Scripta Instituti Donneriani Aboensis	1,996	cc-by	7,402	Dance as Aggressiveness The woman who founded Tenho-kötai-jingii-kyö, Kitamura Sayo (1900- 1967), publicly announced in July 1945 that the world was coming to an end and that she had been chosen by the absolute deity Tensho Kotai Jingu to be the savior of the world. People began to gather to her banner, a relig- ious organization was formed, and legal incorporation of the group as a religious juridical person took place in January 1947. Teaching that regret, desire, hatred, love and other emotional antipathies were the cause of all misfortune, the founder urged people to free themselves of such restraints by praying earnestly until they attained a state in which the self was com- pletely forgotten. Since the members of the group perform a ritual dance and fall into an ecstatic condition at the group meetings, the movement is called the Dancing Religion (Matsuno 1972: 230). Kitamura Sayo, or Ögamisama, was born in the South-West of Japan, in the Yamaguchi prefecture. She was a farmer's wife, worked hard in the Fields and never got enough sleep. In July 1942 a barn on the Kitamura family was burnt down and Sayo saw herself as the cause of the accident. Serving the kami of the ancestors had for her been the first thing in life, so she was deeply disturbed by the fire. She blamed herself for having de- stroyed the ancestor's property and disturbing the neighbours. After pray- ing and praying for forgiveness, she still suffered torments of conscience day after day. She went to a well-known healer in a nearby village, who gave her an explanation (the fire was caused by a jealous arsonist) and some advice for the future. In her diary two days later, and thirteen days later, we can read, "Got up at 2.00 a. m. and took cold bath of penance and prayed... each time I take a cold bath of penance, I feel as if My body and soul have been purified and that I have come nearer to God. My mind is filled only with the eagerness of praying to purify Myself and I have no worldly cares" (Tensho-kotai-jingu-kyo 1954: 15). In March 1944 the healer living nearby had a divine message for Kita- mura Sayo: "All the Angels of Heaven and Earth will descend upon You and You will become a living God on earth." (Tensho-kotai-jingu-kyo 1954: 16). TINA HAMRIN TINA HAMRIN Dance as Aggressiveness Dance as Aggressiveness Dance as Aggressiveness After that she began to receive inspiration from an "almighty kami" 13 176 TINA HAMRIN and she became the mouthpiece of the deity who possessed her. What the entity inside commanded her to say always flabbergasted people and was often taken as a sign of madness or insanity. She could not act in accor- dance with her own intentions but had involuntarily to obey the dictates of the one in her body. Otherwise she got headaches, stomach pains and suf- fered attacks of diarrhea. and she became the mouthpiece of the deity who possessed her. What the entity inside commanded her to say always flabbergasted people and was often taken as a sign of madness or insanity. She could not act in accor- dance with her own intentions but had involuntarily to obey the dictates of the one in her body. Otherwise she got headaches, stomach pains and suf- fered attacks of diarrhea. According to Kitamura Sayo's life history, what started with animal spirit possession, which is quite common in the area, became something else when the snake spirit transformed itself into two beings, that of Kotai- jin (a kanit connected to the Ise shrines) and Amaterasu (the Sun goddess, here as Tenshö, meaning heavenly) who united in the body of the selected woman as "The Almighty God of Heaven". In the middle of autumn 1944 she was led by the divinity inside her to a mountain on her property at 02.00 a.m. Kitamura Sayo rode her bicycle to the foot of the mountain, about two miles from her house, and climbed the hill. At the top she commenced a prayer that was given to her as the only prayer in accordance with God's will: NA-MYO-HO-REN-GE-KYO.1 "Her prayer `1■Ta-myo-ho-ren-ge-kyo', sometimes drawn out and some- times brief, was chanted with natural rhythm and reverberated among the surrounding mountains. When she finished praying, She lay on the ground, face upward, and watched the dawn breaking. After a pause, She resumed Her prayer. Once celestial lotus flowers in full bloom showered down from Heaven as the reddish sun ascended amongst the blossoms. Angels in full costume were dancing before Her... this is a dance of Angels, the One in Her body said. While She was watching the dance in admiration, She was in a celestial bliss and felt as if She had also become a dancing Angel" (Tensho-kotai-jingu-kyo 1954: 29-30). 1 Before 1964 Tenshö-kötai-jingi1-kyö used the same kanji as the Nichiren sect to indi- cate namu-myöhö-renge-kyö, but ceased doing so as a consequence of a lawsuit initiated by the Nishiren Shö shi. Inside the Dancing Religion the formula has been interpreted as "A woman with a little name has contacted the law of Heaven binding it into a teaching". Nowadays the members are instructed that the words are untranslatable, reflecting in their sounds a transforming influence of evil or negative unredeemed spirits. Kerner 1979: 317. Dance as Aggressiveness In July 1945 Kitamura Sayo gave her first public sermon and her relig- ious group was called Odori-shrhyö, the Dancing Religion. The dance was something given to the foundress as a way to restore peace. "You have been appointed to perform a Salvation Dance when the present world is on the verge of collapse", the divinity inside Kitamura Sayo told her (Tensho- kotai-jingu-kyo 1954: 46). 177 DANCE AS AGGRESSIVENESS To her followers Ögamisama said, "I will establish true peace on this earth... World peace advocated by politicians is nothing but a camouflage of national interest based on selfishness... Peace can be brought only by prac- ticing God's teaching" (Tensho-kotai-jingu-kyo 1960: 1-2). The foundress of the Dancing Religion danced with the Almighty God that struck in Hiroshima in 1945, allied with the enemy. "The other name for MacArthur is the `Messenger of Divinity'. He was sent to Japan as an agent of the Divine Wind... The members of the Diet were high-handed criminals, and the Cabinet Ministers were even worse. Who put them in jail? Nobody but MacArthur—Is this not the work of a good Divine Wind?" (Tensho-kotai-jingu-kyo 1954: 60-61). When the Japanese emperor denied being divine in his New Year speech, on the first ofJanuary 1946, the first year of God's Kingdom began, accord- ing to Kitamura Sayo. The sun-goddess Amaterasu was a part of her and the heavenly god would now expand the spiritual world instead of the Japanese territory. As Kitamura Sayo established the foundation of God's Kingdom within Japan, she followed her Almighty God's command and designated Hawai'i as "the Bridge to the World". Hawai'i The situation among Japanese Americans in post-war Hawai'i was that issei (the immigrants) were enemy, but nisei (second generation) were American citizens. "This often meant changed parent-child roles and the loss of parental authority over children... [since] Issei accepted from the beginning the role of their children as American citizens" (Kimura 1992: 226). In Hawai'i, after the war, it was very important for the American Japa- nese to be Americans only — to identify with the other, the hostile one that used to be a friend. Issei, the immigrants, were enemy aliens, and therefore enemies in American eyes, and since public use of the Japanese language was forbidden, it was the interpretations, made by their children, of regu- lations concerning enemy aliens that issei had to depend on. In contrast to traditional customs, some had to substitute I-neither-saw-nor-heard for helpfulness. The "arrest of almost all the Issei leaders and the closing of all the Japanese instituitions stopped the functioning of their society, making the Issei the most desolate and isolated element of the island population. The freezing of Japanese assets, including bank accounts, and the loss of 178 TINA HAMRIN livelihood due to wartime regulations added to the atmosphere of insecu- rity" (Kimura 1992: 225). The immigrants tried to eliminate everything Japanese. Cultural objects, as well as letters, books, photographs and Japanese flags were burnt. Issei and their children made great efforts to be identified with the United States; together with American flags they placed portraits of George Washington, Abraham Lincoln, and President Roosevelt in their homes. "The Issei chose these American figures as legitimate representations of America with the symbolic value of protection" (Kimura 1992: 225). After the war, Kitamura Sayo arrived in Hawai'i to teach those who would become her followers what otherness was. She gave sermons and talked about the Almighty God who expanded his kingdom thanks to her, the Great Goddess, in command of lost human beings who would dance for world peace. Kitamura Sayo identified with the enemy when Japan was invaded. To escape the anxiety stemming from the presence of a threatening authority, the so-called Caucasians, many Japanese in Hawai'i did the same thing. Sometimes the need for strength results in identification with the authori- tarian for the purpose of neutralizing the threatening presence (cf. Moloney 1954: 116). DANCE AS AGGRESSIVENESS 179 conduct themselves as loyal Americans and never let their fathers' situa- tion affect the duties they have to "their native land". Many of them excuse themselves for being internees, afraid that their children will feel humili- ated. On the path to assimilation the private aspects of ethnicity can be turned upside down, as in this case. That is because those aspects remain just as long as the group wants them. Under the pressure from an over- arching majority some ethnic content might be lost or altered, but it should not be seen as some blind turning away from the roots. Nisei desired change therefore the process of Americanisation came from within spon- sored by the ethnic group itself. "The general implication here is that 'de- ethnicisation' occurs because of the formidable attraction of mainstream life" (Edwards 1989: 180). What comes from the inside because of lust for something new, and what comes from necessity for this other, is retrospectively always hard to tell. The extreme expression of the combination of Japanese morality and loy- ality to America was found in the combat teams composed of second gen- eration Japanese in Hawai'i who volunteered during World War II. "Local Japanese attained the unprecedented ethnic glory through their heroic commitment to the American cause; their ethnic pride reached a peak paradoxically when the whole Japanese community was suddenly `de- Japanized' and the authority of Issei was downgraded to a nonentity" (Lebra 1972: 10). Ethnicity was not the only basis for Japanese group for- mation; nationality was also important. Nationality involves a wider range than ethnicity and when Kitamura Sayo arrived, she told the people who gathered around her that Japan was divine and the real war began when she became the mouthpiece of the sun-goddess Amaterasu; therefore Japan did not really lose the war. Kitamura Sayo alluded to the former expansion of the Japanese empire when she preached about the expansion of God's kingdom on earth. Her sermons were usually nothing but scoldings. The converts were chartised. Kitamura Sayo was often frantic with anger and accused her followers of not polishing their souls enough. In an ecstatic way she screamed about political leaders and priests, and she blamed the men in high positions for being "maggots". The voice of God inside her made her shout. Hawai'i On October 12, 1952, Ögamisama made a trip around the island of Oahu and danced in ecstasy in public places, like parks and at religious sights (Tensho-kotai-jingu-kyo 1954: 180). The dance, muga-no-mai, surrendering and conquering, was used to proselytize, symbolizing the union of man and God. This dance was very offensive among the male converts after the war; it was performed in a way that reminded the spectators of military marches. According to members of the Dancing Religion who were con- verted when Ögamisama made her first missionary tour, the more people that surrounded them when they were dancing in a public park, the more strongly the converts became convinced they were powerful servants of God. In compliance with the teaching of the Dancing Religion, to prosetyl- ize spectators is to spread peace in the world, and this was extremely im- portant after the war. In Hawai'i, the Japanese as a group went down in the social scale and there was a huge change within the group itself, when it comes to status. What usually happens in societies in dangerous situations is that the authority of elders over juniors is stressed. This was inverted in Hawai'i after the Pearl Harbor attack. It is interesting to read the letters sent by issei that were put in internment camps. They tell their wives to look upon themselves as mothers of American citizens, and they instruct their sons to TINA HAMRIN predisposition. "That which society defines, recognizes and problematizes as aggression may well be a set of functional activities dressed up as a cul- tural drama" (Rogers et al. 1995: 166). predisposition. "That which society defines, recognizes and problematizes as aggression may well be a set of functional activities dressed up as a cul- tural drama" (Rogers et al. 1995: 166). The Situation at the Time for the Founding of the Dancing Religion The Situation at the Time for the Founding of the Dancing Religion Kitamura Sayo, with bomber-planes around her in Japan, danced with an- gels for world peace. She left her ordinary self to escape the terrible world and to be with God. It was the God of the winners who once gave birth to Buddha, Jesus, and then to Ögamisama. Through dancing a dance born in a wish to escape reality she was one with God's liberators, the Americans. She sang: Oh, bring on the bombs — bring on the bombs - May they exterminate the maggots of this rotten world, And burn all their lairs to ashes, whereupon Let there appear God's New Kingdom. (Tensho-kotai-jingu-kyo 1954: 35). The voice inside told her she should burn down the Imperial Palace, only leaving one wing. It would not be disrespectful, since the emperor had for- saken the nation. According to the one inside, Japan was founded upon the union of three things: the national domain, Tenshö-kötai-jingri-kyö, and the emperor as the guardian of the nation. The voice said, "I have descended into Your body, and therefore equip yourself with the necessary knowledge to meet the situation. Even if the Emperor and Empress took cold baths of penance for three years, I would never descend into such defiled bodies as theirs... perform a Salvation Dance... they have failed to govern their coun- try properly" (Tensho-kotai-jingu-kyo 1954: 46). After MacArthur's victory many male converts danced for peace in a very intense military marchlike way. "Dance is... an active creation of meanings, that is, social action de- pendent upon social relationships at the time" (Brinson 1988: 211). The Japanese nation as a social institution fostered violence in a variety of ways during the war. Institutions can produce a weakened sense of per- sonal responsibility for aggressive actions, and they can also dehumanize the victims of aggression. DANCE AS AGGRESSIVENESS "I think, that we are concerned here with a widespread use of spirit- possession, by means of which women and other depressed categories exert mystical pressures on their superiors in circumstances of deprivation and frustration when few other sanctions are available to them" (Lewis 1989: 39). To Ögamisama aggressiveness was a drama set up by her, the dancing goddess, as a way to win converts. It became a taken-for-granted human 180 TINA HAMRIN DANCE AS AGGRESSIVENESS 513). When Kitamura Sayo gave her sermons, both in Japan and in Ha- wai'i, she put herself aside and acted as an aggressive doomster. Her act showed physical arousal and in many ways her performance was a case of deindividuation. 513). When Kitamura Sayo gave her sermons, both in Japan and in Ha- wai'i, she put herself aside and acted as an aggressive doomster. Her act showed physical arousal and in many ways her performance was a case of deindividuation. The moral code all Japanese share is one inhibition against expressing aggression and thereby harming others. As children they are taught not to be aggressive and offensive. But punishing someone for aggression can have a paradoxical effect. Watching other people being aggressive may lead to the disinhibition of aggression. Kitamura Sayo, maltreated by her mother-in-law, who functioned as an arbitrator in the marriage between Sayo and her husband, might in some ways have acted as a punished child. When you punish a child to inhibit the behaviour for which the child was punished, the punishment can have another effect, it might provide a model of aggression that encourages aggression. Kitamura Sayo's mother- in-law used arbitrary power, she disciplined Sayo without explaining the reasons for that discipline. The old lady taught her daughter-in-law "that acting in a hostile way was an appropriate way to get what one wants (through modeling)" (cf. Sabini 1992: 506). During all the years with her really mean tyrant Sayo got more and more aggressive. But "aggression needs to be understood within the manifold of sets of meanings whereby aggression is constituted... not as an action or a state but as a set of inter- woven textual identities, actions and descriptions" (Rogers et al. 1995: 169). Kitamura Sayo was a frustrated woman, though with well-developed self-defence mechanisms, and became more and more offensive instead of defensive. It is that the dance became something like a cathartic activity I find interesting. In the middle of the night Sayo rode her bicycle Long dis- tances, upset and physiologically aroused she prayed and danced. Aroused she "used up" her aggression through intense dance, a process of catharsis. But did the opportunities to discharge her aggressions actually reduce it? According to several research results it seems to work the other way (see Buss 1966; Loew 1967) In her sermons Kitamura Sayo is usually more ag- gressive after the dance than before. TINA HAMRIN As an example, Japanese officers degraded the victims of South-East Asia through making them seem as if they were less than human. Features of a situation that lead to a disinhibition of aggres- sion through deindividuation can be anonymity, loss of sense of responsibil- ity, physiological arousal and intense sensory stimulation (Sabini 1992: 181 DANCE AS AGGRESSIVENESS DANCE AS AGGRESSIVENESS The dance can be seen as a safe outlet for aggressive energy, as cathartic activity it releases the plug and allows aggressive energy to be released, but she is as mad, or even more, after than before. Of course the word "aggression" means several very different things, and I use the same word for different forms, but it is all about aggression as a combination of influences. "We need also to address the problem that ag- 182 TINA HAMRIN gression is simply not accessible to empirical investigation other than as a set of socially negotiated meanings" (Rogers et al. 1995: 170). gression is simply not accessible to empirical investigation other than as a set of socially negotiated meanings" (Rogers et al. 1995: 170). Kitamura Sayo acted aggressively and found she was reinforced in some way, by getting what she wanted. She was therefore more likely to act in that way again. She gained power and social status through her aggres- siveness. During war many people are rewarded for acting aggressively, and other people learn through vicarious reinforcement to imitate those that succeed. However, for anger to be converted to aggression, certain cues must be available in the environment. It seems like cues acts as triggers to aggression and aggression plus frustration creates anger, then anger to- gether with the cue to act out cause aggressive behaviour (Rogers et al. 1995: 170). The leader of the Dancing Religion pestered all other religious move- ments and molested their leaders. According to the dancing goddess, those who did not follow her teaching, Mioshie, would fall into hell. The salvation of the world should be carried out at all costs, and Ögamisama "mobilized" her followers to attain world peace (Ogamisama's Sermon 1986: 6). Kitamura Sayo worked hard and was always aroused when she shouted in front of people during her sermons. It seems that she could not relieve her arousal through the dance, she often became frustrated with what she saw and with the people she confronted, a frustration that enhanced ag- gression. She had very high blood pressure, usually around 230-240. One interpretation of the enhancement of aggression by extraneous arousal gives drive a direct role. According to this interpretation, extraneous arousal `energizes' behaviour; it makes one do whatever one happens to be doing more intensely. Kitamura Sayo was a doomwatcher and her fire-and- brimstone sermons were intensified through her dance. DANCE AS AGGRESSIVENESS Aggression and the transfer of arousal are important elements when the dance is seen as in- strumental offensive power. Because of the socio-cultural background in the specific war/post-war situation there was a militancy among the male converts in the peace dance. The members took a militant stand toward the outside society and Ögamisama told them to "defend vigilantly and to expand aggressively the Kingdom of God" (Lebra 1967: 69). Since one of the major factors to be taken into account when interpreting the dance "is the range and type of human movement, expression and communication used by the people from whom the particular dance emerges, for this determines, in part, the meaning of the dance" (Hodgens 1988: 65), we have to look at the break- down in Japanese society in the 1930s, from Taisho democracy to trium- phant militarism. 183 DANCE AS AGGRESSIVENESS Ögamisama acted like a military, in a very unfeminine way, after she had been possessed by Tenshö-kötai-jing5-kyö. She reflected Japan. Ögamisama made a political association with emperorship and thereby she made an accomodation to the world. The paradoxical militarism in the peace-dance was nurtured by male issei in Hawai'i. When they were stud- ied in a park, it was noticed that they were waving their arms downward left and right, as in a march, and when it came to the melodies that were sung, "some are reminiscent of the songs taught in Japanese grade schools, of military marching songs" (Lebra 1967: 116). The dance performed by some of the members of the Dancing Religion was a way to manifest Japanese features important at that time, a kind of imperialism. Historically the United States had provided demonstrations of imperialist methods, and as a means of self-preservation Japan identi- fied herself with her enemy. The history of the United States and Japan in Hawai'i demonstrates the Japanese imitation of American methods. After Perry effectively opened the doors of Japan, the actions of the American Marines were often copied by the Japanese. The emigration from Japan to Hawai'i, initially permitted by the Hawaiian king Kalakaua, grew enormous and justified an interest in the Hawaiian Islands. The manner in which the Japanese emissary aproached Hawai'i was patterned after Perry's "visit" to Japan. DANCE AS AGGRESSIVENESS The dignitaries came in a ship of war, and in the same fashion as Perry had made his demands to Japan, they asked for the ichi bun (chief/headman) In 1898 the United States negotiated with the Hawaiians over the treaty of annexation. They reassured the Japanese of- ficials that they had no such plans as a transfer of the islands to the United States. The Japanese were deceived and remembered this. On the 7' of December, 1941, the Japanese emissaries in Washington talked of peace and called the idea of war ridiculous (Moloney 1954: 124). Through out history it seems that the rulers of Japan have often identi- fied with the enemy, and then the enemy has not been the other but the self. The dance of Ögamisama was to some extent that of Christian angels. The Other was in form the God of the other, and the issei members tried to leave their selves for The Other (in muga-no-mai ) and the other (in the acculturation process). Ögamisama told her followers to stand up and dance, and tried to explain that "I danced a similar dance in 1945 by the order of God in My body. The dance you just saw is an Angels' dance which you have seen in pictures or heard about in stories. If a person enters a state of non-ego, God will guide him to dance. Soon everyone of you will be able to perform this celestial dance (Tensho-kotai-jingu-kyo 1954: 68). Communion was a gathering of members who wished to get rid of the "me" 184 TINA HAMRIN through the "ego-less" dance. But it was also a spiritual march towards the Kingdom of God, and the dance was a piece of a strenuous life, a struggle in itself, striving for harmony through a release of tensions, aiming at Utopia and a society in which they all, sick and poor and filled with guilt, would be happy and healthy, free from frustration and anger. The dance was a war against the Jack of peace. g p For issei with hazy, undefined identities and a relaxation of discipline inside the families, to submerge in a group in a way that gave a new iden- tity, partly through deindividuation, seemed attractive. DANCE AS AGGRESSIVENESS DANCE AS AGGRESSIVENESS had been taught the traditional customs issei had tried to keep, they wanted to be "totally American". Thanks to the internalisation, they had a base as American citizens, also mind-wise, and they felt more secure than their parents. Later on they could learn from issei the traditions the par- ents secretly kept. To issei the dance was a dance of tenshi, influenced by Christian angels, performed by a Japanese Goddess. It was a dance of sur- render and victory, it was harmonizing, a goal in itself. To nisei it became a means for a new goal: to spread God's kingdom on earth. They chose con- version instead of compliance: something more indirect, since they realized how a minority can influence a majority. With the dance they got in touch with Americans and tried to convince them that their views were valid. It has been said that the consistency of the argument is the most important factor, and who in post-war Hawai'i did, and does, not want peace? (cf. Hayes 1993: 49-50). Ögamisama's dance is supposed to be spontaneous and a dance of joy, but I have seen many men marching around like soldiers, aggressively, while they are listening to the taped sermons of the foundress. She shouts and seems to enjoy freaking out. "Having cried out vehemently in this way, Ögamisama closed the grand public sermon by saying, is an im- portant place from which, as a foothold, the Divine Doctrine is to be intro- duced to the world. Therefore, no error or misunderstanding of the doctrine will be tolerated" (Tensho-kotai-jingu-kyo 1954: 179). The peace dance is shaped in a form of blind discipline, demanding unquestioning obedience. Out in the parks, among tourists and so-called Caucasians, aggressiveness became offensiveness, go-ahead-and-catch, make a good score, proselytize. There was overhanging requests in an imperative mood. Dance as a carrier of an aggressive approach, born in a wild dance performed by a frustrated and frantic woman, an instrument to recruit adherents: is that the dance of the Dancing Religion? At least it was one dance. One of them. It was also a revolutionary dance. The frantic woman acted like a man, that is why the people she met thought she was strange. DANCE AS AGGRESSIVENESS To be lost in a group and there find a deified part of the self, in the search for a better me, was to the converts an exciting and self-confidence-lifting experience that made them polish their souls on the order of Ögamisama. Inside the religious group issei could "steal back" the position they lost in Hawaiian society after the war. The rigid conformity juxtaposed with flexibility, the authority of issei over nisei, was kept when the teachings of Ögamisama were put into practice. They placed themselves between the categories of ordinary social life and the classifications on which the order in post-war Hawai'i depended was annulled inside the Dancing Religion. Perhaps, the dance can be seen as one of those symbols that "designate temporary antinomic liberation from behavioral norms and cognitive rules (Turner 1990: 273). What happened in Hawai'i after the war among those Japanese immi- grants and their children that became members of the Dancing Religion was that frustration and aggression were taken care of. Acculturation anxiety was in many cases reduced thanks to the "religious family", the sect. Different processes which underlie social conformity, like identifica- tion, compliance, and internalization, should in this case not be seen as different but overlapping. The Japanese in Hawai'i, as a group, changed their attitudes in order to become more like the Americans they respected and admired after the Second World War. That could be called identifica- tion with the enemy. When it comes to compliance, subjects go along with the majority but do not change their private beliefs, including traditional customs. The group as a whole identified with the Americans, the so-called haole, or white ones. Inside the group, the issei were directly related to the fact that the majority had power on its side, because of the war situation, which it could use through sanctions. Compliance is when a majority influ- ences a minority. But in the case of nisei it was more a matter of internali- sation. The children of the immigrants came to agree that the majority view was the more valid one. They both identified with the Americans and internalised the norms given to them at school. Even though many of them 185 Conclusion After a fire in July 1942 a spirit entered the body of a farmer's wife in the Yamaguchi prefecture in Japan. The woman, Kitamura Sayo, did not lose consciousness of her own identity but had a conversation with the deity inside, who communicated with her and ordered her around. If she did not obey, she became ill; therefore she took cold baths of penance six times a day with prayer. The only way to unite with God was through purifying one's mind and polishing one's soul. This was done by means of a magic formula na-myör-hå-ren-ge-kyö and an "ego-less" dance called muga-no-mai. After Kitamura Sayo began to receive inspiration from a deity, "the day came when Her life as the wife of Seinoshin Kitamur ended, and Her life as a Saviour gradually began. At the same time She began to forget about Her only son Yoshito, who was at the front during World War II". In the same text about "Ogamisama: The Saviour" we can read: "Lieutenant- General Fuji, with whom Ogamisama was acquainted, once said `If She had been born a male, She would now be a man of the highest rank, a premier or a general'" (Ogamisama 1968: 27-28). We have here a woman filled with aggressiveness depending on different types of aggressive motivation: inter-male (not unusual in females), sex- related, maternal, danger-induced, and irritable. Possessed, she dressed like a man and started acting like one. She became a threat to men in po- sitions of leadership, as a charismatic leader and "revealer" of so called truths. She turned dominance hierarchies upside down and she interfered in territorial fights. The family farm was successful and if the phenomenon of territoriality is a way to limit and structure inter-male aggression, she was involved in defending the space against intruders in a way that looked like warfare. Among animals inter-male aggression is sex-related in the sense that the establishment of high rank leads to opportunities to mate. In Kitamura Sayo's case it meant the opposite. As an usurper, she used her powerful position inside the family to refuse having sex. So her sex-related aggression is probably more a response to frustration. When it comes to maternal aggression, a mother will attack anything that threatens her child, and that is what Japan did. TINA I-IAMRIN TINA I-IAMRIN DANCE AS AGGRESSIVENESS Kitamura Sayo dressed in men's clothes, rode a bicycle (no women in Japan did that in the 1940s), spoke like a man, and acted aggressively — something that was only allowed for men. In other words, she overstepped the limits of what was permissible, and she madly enjoyed being angry. When she died in 1967, it was because her high blood pressure became too high. 186 Conclusion The nation, in Kitamura Sayo's eyes: the emperor, took her son away (during the war she did not know if he was dead or alive). It seems like the dancing goddess felt attacked by the barn fire, the pos- sessive element in her body, her neighbours and surroundings, as well as politicians and the nation as a whole. 187 DANCE AS AGGRESSIVENESS A move on the part of someone who is being attacked might well be to of- fer an appeasement gesture. Sometimes the dance of Kitamura Sayo is aggressive and militaristic, and sometimes it is done in a pleasing way, even though she is aroused and upset. Self-defence aggression involves sympathetic nervous system arousal, as a response to an attack, and so does irritable aggression. "Perhaps being in an aversive state gives rise to ideas associated with violence through a process of association" (Sabini 1992: 496), because after her dances Kitamura Sayo screamed like a ma- niac and scolded everyone. "God uses me as His Mouthpiece to reprimand people severely and directly... I went to Hawai'i 14 years ago to teach the people. My sermons are the same today, but they sound differently to the people there who have gradually become true human beings by practicing my teaching in their daily lives. When I visited Hawai'i last year, I did not scold the people as severely as I did during my first visit" (Ogamisama's Sermon 1966: 9). Did the coffee farmers in Hawai'i accept being yelled at by God's messen- ger? Yes. They were losers, and Kitamura Sayo told them how bad they were. "There is enough evidence that the foundress successfully manipu- lated her follower's guilt and shame as if she were aware of the dialectic interdependence between self-blame and faith in salvation. She inflated and deflated the follower's feelings of shame and guilt in such a way that drastic relief from self-blame could be attained" (Lebra 1970: 52). Even if frustration per se does not neccessarily instigate aggression, the degree to which one is frustrated can affect the degree to which one be- comes angry. The workers were frustrated and angry. They had been transgressed against and felt illigitimately frustrated. The bombing of Pearl Harbor involved on their behalf unjustified frustration. Perhaps an- ger is turned on when someone perceives frustration as unjustified. 2 In Japan, the urabon festival has been held annually since 657. It is usually held from the 13th to the 15th of the 7th month and it is a service for the repose of the dead. Popularly the festival is called bon or o-bon and it includes dancing. According to the legend, Buddha's disciple Maudgalyåyana saved his mother from suffering in the realm of hungry spirits by making offerings in accordance with some advice from Buddha. Conclusion When Kitamura Sayo scolded them and told them how worthless they were, they agreed with her and converted to her teaching. Through a ritualized marching-around-dance the male converts could canalize the emotions and "act out", regardless of whether that kind of aggressivness would remove the frustration or not. The women frequently used old bon-dance steps during muga-no-mai.2 Among the women the dance was relaxed and not military like. 188 TINA HAMRIN Since there was a general tendency among the Japanese to somatize ill- ness, most of the people that joined the Dancing Religion had complaints that were physical. Stereotypically seen, being Japanese, when you are annoyed you do not talk about it. "It is often more important to communi- cate through attitude, action, and feeling than through words... This kind of behavior in its extreme form may indeed occur in a Japanese as a kind of `acting out' behavior which is radically different from violent, explosive, or disruptive behavior observed in a Western patient", according to Hawai'ian medical journals (Rogers and Izutsu 1980: 97). Perhaps it is necessary here to make a distinction between aggressive re- actions and aggressive tendencies. Through controlling the released ten- dency with a ritualized and somewhat institutionalized "dancing- behaviour" the members are not acting aggressively. They let Kitamura Sayo arouse them with her insults and aggressiveness, but when they dance they do not think about themselves. The dancers try to be "ego-less", to reach an altered state of consciousness through repeating na-my o-ho- ren-ge-kyö in a monotone. There are both emotional and instrumental com- ponents involved. As a group "patternized" in the same Procrustean bed, I think we can leave individual factors aside. Inside this specific group they interpret the ritual situation with Kitamura Sayo (before alive, now on tape) in very much the same way, and they act in a similar way in the same situation. Therefore this is a form of aggressiveness that is sup- pressed and turned into repentance. It can be traced to the outer conditions in the specific situation that triggered off aggressive tendencies, but it was forbidden feelings. The converts should ask their divine leader to be for- given, since aggressiveness could produce evil spirits. The situation during a dancing ritual was always ambivalent. In public places it was different. When the members of the Dancing Religion were dancing with Kitamura Sayo, it was a matter of situational factors. When she was saved, she danced out of happiness, and so did Maudgalyåyana with the spirit of his mother. Bon dance is when the living are supposed to dance in the middle of the summer together with the spirits of the dead. Besides the popular legend about the Avalambana sitra and its contents, there are serious ethymological research on the bon concept made by Stephen F. Teiser in Teiser 1988 DANCE AS AGGRESSIVENESS in the world through an aggressive "ego-less" dance, as a means to prose- lytize ignorant people and make them realize that only through making a Japanese religion a world religion can there be peace on earth. It was ag- gressiveness as a drama set up by the dancing goddess, an act called proselytism. A functionalistic explanation of the dance among Japanese plantation workers in Hawai'i who were members of the Dancing Religion is that ag- gressiveness in this case had a function. And this function explains the aggressiveness. Through aggressive cues the members could increase the tendency to proselytize aggressively because of their associations with ag- gressive behaviour. Making the dancing ritual as good as possible was catching as many people as possible of those that become curious about the dance. Of course, there is no purpose or cause flitting along that can ex- plain the aggressivity inside different members. If I could explain it by telling you that there is a consciously created instituition, made especially for aggressiveness, then it world not be a functionalistic explanation, but a teleological. "Our understanding of aggression, as well as our experience of it, is literally polytextual. Altough by our social conventions about expla- nation we tend to opt for singular stories at a given moment, we are never- theless at some level aware that we use more than one story... All facts about aggression, all research findings, are inevitably contingent and lo- cated" (Rogers et al. 1995: 171). In a historical framework the dance is aggressive, the real dance acts out frustration. The ideal dance is harmony itself, always there, neither becom- ing, nor disappearing. In post-war Hawai'i, in an "ego-less" state, the danc- ers danced the ideal dance, the dance of angels, in between the marches. The dance that was observed by anthropologists in parks was real and ag- gressive. The natural world, the world in the ordinary sense of the word, was constantly there for them, as long as they looked in its direction. At the natural standpoint some researchers had another way of stating the same thing as I do, in my ideal worlds-about-me. The natural world is, of course, present to me, and I am at the natural standpoint as well as they are. But I interpret it as a background for my consciousness, as act. Conclusion The instrumental aggression that was shown when they danced in public could have been a product of their knowledge about the consequences of aggressive behaviour. On the pattern of Kitamura Sayo, taking her as a model, they knew that it is pos- sible to make friends by being aggressive, and it is possible to make peace 189 DANCE AS AGGRESSIVENESS TINA HAMRIN TINA HAMRIN DANCE AS AGGRESSIVENESS The dance I describe to you is a dance experienced and visualized with a little help from my reference frame. It is both real and ideal, depending on how I look at it. An aggressive dance for harmony. A war-coloured peace-dance. "The two worlds are present together but disconnected, apart, that is, from their relation to the Ego, in virtue of which I can freely direct my glance or my acts to the one or to the other" (Husserl 1962: 94). 190 Brinson, Peter 1988 Epilogue: Anthropology and the Study of Dance. In: Paul Spencer (ed.), Society and the Dance. The Social Anthropology of Process and Performance; pp. 206-214. Cambridge: Cambridge University Press. [1985] Buss, A. R. 1966 Instrumentality of Aggression, Feedback and Frustration as Determi- nants of Physical Aggression. Journal of Personality and Social Psy- chology 3: 153-162. gy Edwards, John 1989 Language, Society and Identity. Oxford: Basil Blackwell. [1985] Hayes, Nicky y , y 1993 Principles of Social Psychology. Hove: Lawrence Erlbaum. Hodgens, Pauline 1988 Interpreting the Dance. In: Janet Adshead (ed.), Dance Analysis. The- ory and Practice; pp. 60-89. London: Dance Books. y Husserl, Edmund 1962 Ideas: General Introduction to Pure Phenomenology. London: Mac- millan. , 1979 Building God's Kingdom: Society and Order in a Japanese Utopian Community. New York. [unpubl. thesis, Columbia University] Kimura, Yukiko 1992 Issei. Japanese Immigrants in Hawaii. Honolulu: University of Hawaii Press. [1988] Lebra, Takie Sugiyama 1967 An Interpretation of Religious Conversion: A Millennial Movement among Japanese-Americans in Hawaii. [unpubl. thesis, University of Pittsburg] 1970 Logic of Salvation: The Case of a Japanese Sect in Hawaii. The Inter- national Journal of Social Psychiatry 16/1: 45-53. 1972 Acculturation Dilemma: The Function of Japanese Moral Values for Americanization. Council on Anthropology and Education Newsletter 3/1: 6-13. Lewis, I. M. 1989 Religion in Context. Cults and Charisma. Cambridge: Cambridge Uni- versity Press. [1986] 1967 Acquisition of a Hostile Attitude and its Relationship to Aggressive Behaviour. Journal of Personality and Social Psychology 5: 335-341. Matsuno, Jonko 1972 Other Religious Organizations,1 Japanese Religion: A Survey, by the Agency for Cultural Affairs; pp. 225-232. Tokyo: Kodansha Interna- tional. 191 DANCE AS AGGRESSIVENESS Moloney, James Clark Moloney, James Clark Moloney, James Clark 1954 Understanding the Japanese Mind. New York: Philosophical Library. Ogamisama g 1968 Ogamisama: The Saviour. Voice from Heaven 5/26: 24-28. Ogamisama's Sermon g 1966 Ogamisama's Sermon. Voice from Heaven 3/16. g 1966 Ogamisama's Sermon. Voice from Heaven 3/16. 966 Oga sa a s Se o . Voice from Heaven 3/16. 1986 Ogamisama's Sermon. Voice from Heaven 133. Rogers, Rex Stanton et al. 1995 Social Psychology. A Critcal Agenda. Cambridge: Polity Press. Rogers, Terence A., and Saturu Izutsu 1980 The Japanese. In: John F. McDermott, Wen-Shing Tseng, and Thomas W. Maretzki (eds.), People and Cultures of Hawaii. A Psychocultural Profile; pp.73-99. Honolulu: University Press of Hawaii. Sabini, John 1992 Social Psychology. New York: Norton. 1992 Social Psychology. New York: Norton. 1992 Social Psychology. New York: Norton. Teiser, Stephen F. Teiser, Stephen F. 1988 The Ghost Festival in Medieval China. Princeton: Princeton Univer- sity Press. 1988 The Ghost Festival in Medieval China. Princeton: Princeton Univer- sity Press. Tensho kotai jingu kyo Tensho-kotai-jingu-kyo Tensho-kotai-jingu-kyo 1954 The Prophet of Tabuse. Tabuse: Tensho-kotai-jingu-kyo. 1960 Key to Heaven. A Concise Explanation of God's Teaching. Tabuse: Tensho-kotai-jingu-kyo. Turner, Victor Turner, Victor 1990 Dramas, Fields, and Methaphors. Symbolic Action in Human Society. Ithaca: Cornell University Press. [19741 14 14
https://openalex.org/W1986207394	https://bmcmicrobiol.biomedcentral.com/counter/pdf/10.1186/1471-2180-10-137	English	null	Vibrio chromosomes share common history	BMC Microbiology	2,010	cc-by	8,674	BioMed Central © 2010 Kirkup et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE Open Access Abstract Background: While most gamma proteobacteria have a single circular chromosome, Vibrionales have two circular chromosomes. Horizontal gene transfer is common among Vibrios, and in light of this genetic mobility, it is an open question to what extent the two chromosomes themselves share a common history since their formation. Results: Single copy genes from each chromosome (142 genes from chromosome I and 42 genes from chromosome II) were identified from 19 sequenced Vibrionales genomes and their phylogenetic comparison suggests consistent phylogenies for each chromosome. Additionally, study of the gene organization and phylogeny of the respective origins of replication confirmed the shared history. Conclusions: Thus, while elements within the chromosomes may have experienced significant genetic mobility, the backbones share a common history. This allows conclusions based on multilocus sequence analysis (MLSA) for one chromosome to be applied equally to both chromosomes. * Correspondence: bckirkup@post.harvard.edu 1 Dept. of Civil and Environmental Engineering, 15 Vassar Street, Cambridge MA 02139, USA Full list of author information is available at the end of the article Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Kirkup et al. BMC Microbiology 2010, 10:137 p gy http://www.biomedcentral.com/1471-2180/10/137 Research article Vibrio chromosomes share common history Benjamin C Kirkup Jr1, LeeAnn Chang1, Sarah Chang2, Dirk Gevers3 and Martin F Polz1 Origin of Replication Organization The second method of analysis, studying the gene organi- zation at the origins of replication (Ori), supported the finding that the two chromosomes share a single phylog- eny at the species level. This method of analysis was more advantageously applied to chromosome II than chromo- some I: Gene order in the region immediately surround- ing the chromosome I origin appears too highly conserved between species to provide robust data on its phylogeny (Figure 3; expanded in Additional files 3 and 4). However, gene content is informative in that region suggesting that the species largely conform to the expected clustering even though the tree is not well sup- ported (Figure 3). The difficulties are caused by a paucity of organizational changes that differentiate species at OriI - such as the inversion of three genes that sets apart the V. fisheri. Frequently, a change is unique to a sequenced strain and not shared by other members of its species. This can be extraordinarily disruptive of a dis- tance estimate if the number of unique differences is large. In particular, at least three obvious saltations in the gene content introduce spikes of noise. In V. cholerae B33, an apparently mobile genetic region has imposed itself very close to the origin of replication. These 18 genes, almost as large as the region to be compared, interrupt an otherwise absolutely conserved region shared by the other Vibrio cholerae. A 9 gene region in Photobacterium sp. SKA34 contains several transposon and transposase Background Page 2 of 13 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 metabolism, information processing, flagellar structure and cytoskeletal components; as such, they represent sampling points from various pathways and genomic sec- tions from around the entire genome. The concatenation of these well conserved genes provides a shared signal for the chromosomes as a whole, despite only composing a small fraction of the entire genome. The genes included in the analysis are listed under Additional files 1 and 2. The chromosome I tree is easily rooted by the various other genomes included in the analysis. All of these other clades fell together along accepted taxonomic lines. The most closely related strains in the tree are the V. cholerae strains; that clade is effectively unresolved because the internal distances are too short. The chromosome II tree cannot be rooted in the same manner as chromosome I because there is no obviously available outgroup: the chromosome II of P. atlantica is not homologous to the chromosome II of the Vibrionaceae being analyzed. How- ever, rooting it identically by using the information from the chromosome I tree preserves the branching order of each tree. Thus, the 'mean field' approximation for the phylogeny of the two chromosomes is congruent at the species level. There is insufficient resolution among V. cholerae strains and too few members of other species to make inferences at a finer phylogenetic scale. The function of a second chromosome, and of multi- chromosomality in general, has been the subject of spec- ulation [2,14,23]. That many of the genes on the Vibrio Chromosome II have specific environmental functions has been noted, and the role of the second chromosome in habitat adaptation has been tested experimentally [23]. Xu et al demonstrated that when V. cholera was grown in an animal host (rabbit ileal loop) a general shift in gene expression favored up-regulation of genes on the second chromosome relative to the gene expression profiles in exponential growth in vitro. This experimental data paired with the gross similarities among the chromosome I from all sequenced Vibrio and the great diversity of chromosome II, suggests that the second chromosome represents a collection of accessory elements and might be mobilized wholesale leading to a complete shift in hab- itat or niche [2,14]. Background 'Vibrio phylogenies' that are built using MLSA or sin- gle-copy conserved genes typically use genes located on chromosome I [15,24-34] with the exception of intra-spe- cific typing schemes for pathogens [17,22]. This is a side- effect of choosing stable, conserved, essential, single copy genes. However, it provides little assurance of represent- ing the history of the entire genome given that Chromo- some II is excluded from the analyses. Given the high degree of mobility Vibrio genetic ele- ments are presumed to have, it is possible that the two chromosomes have distinct and conflicting histories. There are currently 9 completely assembled Vibrio genomes available in the public databases and over 30 partially sequenced genomes. We explored these genomes to construct phylogenies for each of the two chromosomes using three approaches. First, single copy genes from each chromosome were assembled en suite and a phylogeny for each chromosome was inferred from these concatenated sequences. Second, the organization and gene content at the origins of replication of each chromosome (OriI and OriII for chromosomes I and II, respectively) were studied. Third, the genes from near the two chromosomal origins of replication were studied and their phylogenies estimated individually. Background Genome analysis of the Vibrios rapidly uncovers varia- tion even among closely related strains. Not only do the genome sizes differ widely [15], but even among con- served genes, there is incongruity among the inferred phylogenies. This is the well-accepted signature of hori- zontal gene transfer and homologous recombination. Gene organization also differs among sequenced strains, indicating large-scale genetic mobility. Individual genes and entire operons may be mobile among Vibrio [16-20]. In particular, Chromosome II varies widely in size and organization [14,21]. Further, many Vibrio carry (and pre- sumably exchange) plasmids. Though it may seem unusual to expect as large a quantity of DNA to be trans- ferred as an entire chromosome, there is evidence that Vibrio have experienced a transfer on that magnitude even recently: The putative V. vulnificus hybridization leading to biotype 3 involves very large quantities of DNA being transferred among V. vulnificus strains to create a hybrid strain almost evenly split in contributions from biotypes 1 and 2 [22]. However, the hybridization event involves loci from both chromosomes being transferred and appears to have preserved their associations with those chromosomes. As such, it does not appear to have been an exchange of chromosomal partners, but it raises the possibility that chromosomal exchange may have been an evolutionary mechanism within the Vibrion- aceae. g In traditional dogma, bacteria have one chromosome and a number of smaller DNA entities, like plasmids, which are propagated across generations unlinked to the chro- mosome. However, when bacteria have two chromo- somes, are they permanently paired or do these physical entities recombine frequently relative to genes on these chromosomes? Since 1998, it has been known that some gamma proteobacteria have two chromosomes [1-3]. This followed discoveries that various other proteobacte- ria, namely alpha proteobacteria [4,5] and beta pro- teobacteria [6], could have multiple chromosomes as well. An initial debate occurred over whether the second Vibrio chromosome was really a 'chromosome' or whether it was merely a 'megaplasmid' [3,7]. The argu- ments for considering the second replicon a chromosome centered on its considerable size, essential gene content [8] and consistent stoichiometry. We can now add to that a unique replication machinery [9,10] that operates inde- pendently but in a coordinated fashion [11] with synchro- nous termination and thus consistent stoichiometry [12,13]. It is now accepted that most, perhaps all, Vibrion- aceae (including the genera Vibrio and Photobacteria) have two chromosomes [14]. Results and Discussion Chromosome Phylogenies The inferred phylogenies for the two chromosomes are congruent (Figures 1 and 2) and contain the expected major features, such as Photobacterium being basal to the Vibrionaceae and V. fisheri forming the next most basal clade. There are no unexpected sister taxa. The results of this analysis are compatible with published multi-locus analyses. However, instead of using 6 or 8 genes com- monly used in MLSA, this analysis included 142 genes from chromosome I and 42 from chromosome II. These single copy genes include a range of functions including Kirkup et al. BMC Microbiology 2010, 10:137 Page 3 of 13 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 p gy http://www.biomedcentral.com/1471-2180/10/137 Figure 1 Tree (Chromosome I). Inferred mean-field phylogeny of Chromosome I derived from a sampled concatenated gene sequence of single- copy orthologs distributed around the entire Chromosome I. The species tree is fully resolved and has 100% bootstrap support on all nodes outside of V. cholerae (1000 replicates). The list of genes and included locus tags is found in Additional file 1, supplementary materials. Figure 1 Tree (Chromosome I). Inferred mean-field phylogeny of Chromosome I derived from a sampled concatenated gene sequence of single- copy orthologs distributed around the entire Chromosome I. The species tree is fully resolved and has 100% bootstrap support on all nodes outside of V. cholerae (1000 replicates). The list of genes and included locus tags is found in Additional file 1, supplementary materials. Kirkup et al. BMC Microbiology 2010, 10:137 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Page 4 of 13 p gy http://www.biomedcentral.com/1471-2180/10/137 Figure 2 Tree (Chromosome II). Inferred mean-field phylogeny of Chromosome II derived from a sampled concatenated gene sequence of single-copy orthologs distributed around the entire Chromosome II. The species tree is fully resolved and has 100% bootstrap support on all nodes (10000 replicates). The list of genes and included locus tags is found in Additional file 2, supplementary materials. Only closed ge- nomes were included in this analysis. !"#$ ! %&' (&&# !"#$ ! %&' (&&# two chromosomes overall. This third method of analysis led thus to the same conclusion as the other two. Results and Discussion Chromosome Phylogenies Table 1 lists the genes studied at each origin, focusing on their gene phylogeny, while Table 2 specifies the longer anno- tation names for the genes used in Table 1 and the type of data (DNA or AA) used to create the trees. The genes within the Ori regions are naturally subject to horizontal gene transfer and mutational noise, like all other genes. Two of them are too conserved or too noisy to present a clear phylogenetic signal over the Vibrionales. In these cases, ALrT (approximate likelihood ratio test) and boot- strap support are lacking across the entire tree (2/28 genes on chromosome I, 0 on chromosome II). Many other trees have limited support for individual clades. Clades with less than 0.05 ALrT [35] support or less than 70% bootstrap support were reduced to polytomies. In addition, the long branch of V. cholerae sometimes dis- torts other elements in the tree. In 8/28 trees from chro- mosome I and 2/12 trees derived from chromosome II, removing the cholera clade from the tree also restored a topology consistent with the mean-field tree in the other portions of the tree where previously it had been incon- sistent with the hypothesis (labeled B in the first column of the table). Finally, one clade (V. parahaemolyticus, V. alginolyticus, V. campbellii, V. harveyi) was reliably monophyletic but presented numerous permutations in its internal structure. At OriI 9/28 genes presented diverse variants in this clade; at OriII, 3/12 genes pre- sented variability within this clade. Ignoring this varia- tion, 16/28 genes from chromosome I and 10/12 genes from chromosome II confirm the chromosomal phyloge- nies inferred by the above methods (labeled A). Finally, the remaining two genes on chromosome I lead to infer- ences that conflict with the others by placing V. splendi- dus in the V. fischeri clade (basal to its expected position, see Figure 4). Genes in OriI show more variation in phy- logeny but fewer genes are available for study in OriII. Figure 2 Tree (Chromosome II). Inferred mean-field phylogeny of Chromosome II derived from a sampled concatenated gene sequence of single-copy orthologs distributed around the entire Chromosome II. The species tree is fully resolved and has 100% bootstrap support on all nodes (10000 replicates). The list of genes and included locus tags is found in Additional file 2, supplementary materials. Only closed ge- nomes were included in this analysis. genes. Conclusions Rampant horizontal gene transfer and plasmid exchange might create doubt as to the fidelity of paired chromo- somes to one another. Further, this genetic mobility can create serious difficulties for anyone reconstructing a phylogeny for something as large as a chromosome, just as they do for someone inferring organismal and species phylogenies. Here, these difficulties have been overcome by using a range of methods that operate at different tem- poral and genetic scales. At the smallest scale, a number of individual gene phylogenies were reconstructed. At an intermediate scale, the gene content of a conserved region was used to infer a phylogeny. At the largest scale, concatenation of predominantly chromosome specific genes (though they may, in other genomes, be transferred among the chromosomes) provided an estimate of the Results and Discussion Chromosome Phylogenies Similarly, 16 gene region in Vibrio splendidus MED222 interrupts an otherwise conserved region with a number of secretory system genes; it lacks apparent mobility elements which would explain its origin. Among the photobacteria, the flanking regions sometimes differ dramatically, as well, which disturbs the phylogeny with a very long branch, and the Vibrio cholerae appear to have inverted the entire region - but this would not impact a gene content analysis. Gene presence/absence data is more informative in the regions surrounding the origin of chromosome II, even though across the breadth of the Vibrionaceae, the areas adjacent to the origin of replication have been relocated to such an extent that it is not practical to reconstruct their movement from as few genomes as are currently available. This is not unexpected, given how thoroughly shuffled chromosome II is relative to chromosome I [21]; see also Additional file 5 to explore the global rearrange- ment of chromosome II. Within a relatively short dis- tance of the origin, however, genes can be reliably identified as orthologous and used in a presence/absence analysis. The origin was extended in each direction by 10 kb. As described in the methods, a gene presence/ absence tree was constructed and this led to a distance tree entirely consistent with the mean-field approxima- tion across Chromosomes I and II (i.e. Figures 1 and 2). Origin of Replication Genes ) + ! , , ) ) ) ) " " " " " -## $ .!/ $ .!0 /' &, !' & $$1' & $$ & $..2 & $..1# 3 OriI and OriII synteny figures. The two origin regions of (A) Chromosome I and (B) Chromosome II. Open reading frames called in the an- genomes are polygons pointing in the direction of their orientation. Colors label the open reading frames analyzed individually in estimating logeny of the origin. The expanded figures with all labels are found in Additional files 3 and 4, supplementary materials. Origin of Replication Genes ) * + ! , , ) ) ) ) " " " " " -## $$% & ' & ( ' & ! "$%#' &$$)' & $$! ) +# &, ) - & $$$ $ . Origin of Replication Genes The phylogenies estimated for each of the gene families near the origin support the estimations derived from the Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Kirkup et al. BMC Microbiology 2010, 10:137 Page 5 of 13 gy http://www.biomedcentral.com/1471-2180/10/137 d OriII synteny figures. The two origin regions of (A) Ch s are polygons pointing in the direction of their orientatio f the origin. The expanded figures with all labels are foun ) + ! , , ) ) ) ) " " " " " -## $$% & ' & ( ' & ! "$%#' &$$)' & $$! ) +# &, ) - & $$$ $ . Origin of Replication Genes ) + ! , , ) ) ) ) " " " " " -## $ .!/ $ .!0 /' &, !' & $$1' & $$ & $..2 & $..1# mosome I and (B) Chromosome II. Open reading frames called in the an- Colors label the open reading frames analyzed individually in estimating n Additional files 3 and 4, supplementary materials. Figure 3 OriI and OriII synteny figures. The two origin regions of (A) Chromosome I and (B) Chromosome II. Open reading frames called in the an- notated genomes are polygons pointing in the direction of their orientation. Colors label the open reading frames analyzed individually in estimating the phylogeny of the origin. The expanded figures with all labels are found in Additional files 3 and 4, supplementary materials. Kirkup et al. BMC Microbiology 2010, 10:137 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Page 6 of 13 Page 6 of 13 http://www.biomedcentral.com/1471-2180/10/137 Table 1: Gene phylogenies for OriI and OriII. Relation to hypothesized chromosome phylogeny Sequence set Resolution V. alginolyticus/V. Origin of Replication Genes parahaemolyticus clade Consistent (A) Conditionally consistent (B) Uninformative (U) Inconsistent (I) Complete(C) Incomplete (P) Full (F) Partial (N) Unresolved (U) Consistent with consensus(-) Scrambled (X) Other issues (see text) (O) Tree uninformative (n/a) C1 LysR A P N - MDR A P F - UDP I C N - Epsilon B C N O Beta B C N O Gamma B P N - Alpha A C N - Delta A C N X Bsub A C N - Csub U C U n/a Asub I C N - Isub A C N X ParB A C N - ParA A C N - GidB A C N - GidA B C N - MioC B C F X ThdF B C N X Table 1: Gene phylogenies for OriI and OriII. Table 1: Gene phylogenies for OriI and OriII. Kirkup et al. BMC Microbiology 2010, 10:137 Page 7 of 13 Page 7 of 13 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 p gy http://www.biomedcentral.com/1471-2180/10/137 YidC A C N X RnpA A C N - RpmH U P U n/a ABC1 A P N X ABC2 A P N - ABC3 A P N - DnaA A C N - DnaN A C N - RecF B C F X GyrB B C F - C2 MetC A P F X GluP B P N X PyrD A P N - GTP A P F - Hyp A P N - TraR A P N - RctB A C N - ParA2 A C N - ParB2 A C N - ChrR A C N X Poly B P F - Chlor A P F - All the genes analyzed are listed. The first column represents whether the estimated phylogeny was consistent with the hypothesized chromosome phylogeny (A and B, if removing cholera results in a consistent tree), inconsistent with the hypothesized phylogeny, or simply uninformative. The second column indicates whether all strains were represented for this locus and the third whether there were any clades outside the cholera clade at which the tree was a polytomy because of an uninformative or unsupported node. The fourth column includes i hi h V l did i t f l i th t (O) h th V h l ti /V l i l ti l d i t th i th Table 1: Gene phylogenies for OriI and OriII. Origin of Replication Genes (Continued) Table 1: Gene phylogenies for OriI and OriII. (Continued) All the genes analyzed are listed. The first column represents whether the estimated phylogeny was consistent with the hypothesized chromosome phylogeny (A and B, if removing cholera results in a consistent tree), inconsistent with the hypothesized phylogeny, or simply uninformative. The second column indicates whether all strains were represented for this locus and the third whether there were any clades outside the cholera clade at which the tree was a polytomy because of an uninformative or unsupported node. The fourth column includes cases in which V. splendidus is out of place in the tree (O) or where the V. parahaemolyticus/V. alginolyticus clade is not the same as in the consensus tree (X). Table 2: Gene names. Table 2: Gene names. Long name Short name Long name Tree type C1 LysR Transcriptional Regulator LysR AA Mdr Multidrug Resistance Protein AA UPD UDP-N-acetylglucosamine pyrophosphorylase AA Epsilon ATP synthase F1 epsilon subunit DNA Beta ATP synthase F1 Beta subunit AA Gamma ATP synthase F1 Gamma subunit AA Alpha ATP synthase F1 Alpha subunit DNA Delta ATP synthase F1 Delta subunit DNA Bsub ATP synthase F0 B subunit DNA Csub ATP synthase F0 C subunit n/a Asub ATP synthase F0 A subunit DNA Isub ATP synthase F0 I subunit DNA ParB Chromosome Partitioning Protein ParB AA ParA Chromosome Partitioning Protein ParA AA GidB Glucose inhibited division protein B AA GidA Glucose inhibited division protein A AA MioC Flavodoxin AA ThdF Thiophene and furan oxidation protein DNA YidC 60 kDa inner membrane insertion protein AA RnpA Ribonuclease P AA RpmH Ribosomal protein L34 n/a Kirkup et al. Origin of Replication Genes In each case, the observed patterns were consistent - though, while many individual genes do not present a conflicting individual history, they may not support the hypothesis for lack of signal. were entered into a MySQL database with their DNA and protein sequences as well as other identifying data. The entire suite of protein sequences were BLASTed against each other and the resulting hits were processed with orthoMCL v 1.4 to identify protein families [36]. A signif- icant parameter used in orthoMCL was an inflation value of 1.5. Genes representing single copy gene families on the different chromosomes were aligned [37], stripped of their gaps, concatenated, and 100 kb, chosen as individual random sites, was chosen as the input for PhyML [38]. This congruence between the whole of the chromo- some and the origin of replication suggests that the region around the origin of replication is either too large to relocate or is difficult to transfer because of its specific function. Individual genes in this region may experience horizontal gene transfer - witness the inclusion of a mobile genetic region in V. cholerae B33. Individual genes also appear amenable to transfer, deletion and insertion. Phylogenies for Vibrio and Photobacterium chromo- some I and II were based on the complete and incomplete published genomes with P. atlantica and Shewanella sp. ANA3 serving as the outgroup. Initially, Pseudoalteromo- nas haloplanktis was proposed as an outgroup for the chromosome II phylogeny. P. haloplanktis, unlike other sequenced pseudoalteromonads, has a second chromo- some. However, that chromosome appears to have a dis- tinct, plasmid-like origin of replication and a GC-skew that indicates unidirectional replication [39]. It shares several genes with the Vibrio chromosome II, but it is unclear how, if at all, it might share a substantive phylog- eny with the Vibrio chromosome II. It contains presum- ably essential housekeeping genes, despite its otherwise plasmid-like features and likely represents a second ori- gin of multi-chromosomality within the gamma pro- teobacteria. As a result, though genes from P. haloplanktis chromosome I were used as an outgroup to Vibrionaceae chromosome I, genes from P. haloplanktis chromosome II were not included in any analysis of Vibrionaceae chromosome II. More than being able to create a relative history for each chromosome, it appears that since the origin of the two chromosomes in the ancestral Vibrio, they have con- tinued as a pair. Origin of Replication Genes BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Page 9 of 13 ABC1 Amino acid ABC transporter, ATP-binding protein AA ABC2 Amino acid ABC transporter, permease protein AA ABC3 Amino acid ABC transporter, periplasmic amino acid-binding portion AA DnaA Chromosomal DNA replication initiator AA DnaN DNA Polymerase III, beta chain AA RecF Recombination Protein F DNA GyrB DNA Gyrase DNA C2 MetC methyl-accepting chemotaxis protein AA GluP glucose-1-phosphate adenylyl transferase AA PyrD pyridoxamine 5'-phosphate oxidase DNA GTP GTP cyclohydrolase II DNA Hyp Hypothetical Protein AA TraR transcriptional repressor DNA RctB Putative Translation Elongation Factor AA ParA2 ATPases involved in Chromosome Partitioning AA ParB2 Predicted Transcriptional Regulator ParB AA ChrR Transcriptional Activator ChrR AA Poly Polymerase, hypothetical cytosolic protein AA Chlor Chloramphenicol Acetyltransferase AA The genes surrounding the origins of replication otherwise identified by short names are described by their longer annotation names. In addition, the data (DNA or AA) used to create the trees is listed. This relates to the degree of conservation in the data; more conserved sequences require DNA trees to provide signal, less conserved sequences require AA trees to avoid excessive noise. Table 2: Gene names. (Continued) ABC1 Amino acid ABC transporter, ATP-binding protein AA ABC2 Amino acid ABC transporter, permease protein AA ABC3 Amino acid ABC transporter, periplasmic amino acid-binding portion AA DnaA Chromosomal DNA replication initiator AA DnaN DNA Polymerase III, beta chain AA RecF Recombination Protein F DNA GyrB DNA Gyrase DNA C2 MetC methyl-accepting chemotaxis protein AA GluP glucose-1-phosphate adenylyl transferase AA PyrD pyridoxamine 5'-phosphate oxidase DNA GTP GTP cyclohydrolase II DNA Hyp Hypothetical Protein AA TraR transcriptional repressor DNA RctB Putative Translation Elongation Factor AA ParA2 ATPases involved in Chromosome Partitioning AA ParB2 Predicted Transcriptional Regulator ParB AA ChrR Transcriptional Activator ChrR AA Poly Polymerase, hypothetical cytosolic protein AA Chlor Chloramphenicol Acetyltransferase AA The genes surrounding the origins of replication otherwise identified by short names are described by their longer annotation names. In addition, the data (DNA or AA) used to create the trees is listed. This relates to the degree of conservation in the data; more conserved sequences require DNA trees to provide signal, less conserved sequences require AA trees to avoid excessive noise. Table 2: Gene names. (Continued) Table 2: Gene names. (Continued) Page 10 of 13 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 history of the whole chromosome. Chromosome Phylogenies Mean field approximation refers to the generalized phy- logeny of the entire chromosome, regardless of differing histories. This was accomplished conceptually by means of concatenated gene trees for single copy homologous genes whose relatives are most easily determined and whose chromosomal affiliation is most certain. The restriction that the genes had to be single copy is meant to limit the analysis to orthologs while excluding paral- ogs. To select the genes for this analysis, a database of genomes was created. All the available Vibrionaceae (Vibrio and Photobacterium) genomes as well as an assortment of other gamma proteobacterial genomes (Additional file 6) were selected for analysis. All 62 genomes were broken down into lists of ORFs, which Origin of Replication Genes This suggests that they have also followed the cell itself; that we have a consistent phylogeny for the Vibrio species themselves - at least, the ones included in this analysis. Further genome sequencing would allow a similar analysis to provide the 'definitive' phylogeny of the Vibrio, but at much greater effort per strain than for MLSA [33]. MLSA schemes currently devised provide a mean field estimate of the phylogeny of Chromosome I; thus, as they are expanded to include increasing numbers of genes, those phylogenies are expected to agree with the phylogenies derived from studying the origins of replica- tion. This suggests several genes that might be used in an MLSA of the Vibrionaceae, including Alpha, DnaN, and YidC from Chromosome 1 and ParA2 and GluP from Chromosome 2. These genes have potential primer sequences that are hypothetically capable of creating phy- logenetic trees with the highest resolution and consistent signal so that they are comparable to the trees found in this study. It is a pleasing conclusion that separate MLSA schemes will not have to be executed for each chromo- some independently. Initially, only completed Vibrionaceae genomes were analyzed for phylogeny of chromosome II. The incom- plete genomes were then added to the analysis; genes rep- resented multiple times in these genomes were excluded from the analysis. Incomplete genomes of Vibrio cholerae B33, Vibrio harveyi HY01, Vibrio cholera MZO-2, and Vibrio angustum S14 were excluded from this tree because they appeared to be missing members of gene families shared by the other genomes, even quite closely related conspecific strains. Finally, all the selected genes were processed as above, under the assumption that in the incompletely sequenced strains, genes particular to chromosome II in the complete genomes remained on chromosome II. With significantly fewer taxa in chromo- some II than chromosome I, comparison for phylogenetic congruence involved eliminating a given taxa from the comparison if it was missing from one of the trees, and only using taxa present in both trees. Origin of Replication Organization For unfinished genomes, the origin could not be used in this analysis if it was broken up over several small contigs, but when the entire region was readily assembled in an unmistakable fashion, those contigs were included in the analysis. In the incomplete genomes, orthologous regions were identified by both gene content and skew. When the expected gene families and gene order coincided with appropriate shifts in skew, the origin was identified. For unfinished genomes, the origin could not be used in this analysis if it was broken up over several small contigs, but when the entire region was readily assembled in an unmistakable fashion, those contigs were included in the analysis. Dist A B A = − ( ) / Where: A = the smaller number of labeled genes in either of the two regions (i.e. in genome 1 or 2) The gene families derived from the above database were used to identify orthologs. Four core genes present in virtually all the genomes immediately at the origin were identified and used to anchor the analysis. From their furthest start and stop codons, regions 10 kb (OriII) and 20 kb (OriI) stretching outward were defined. These distances were chosen to balance issues of signal and noise. Particularly for OriI, a shorter region was uninfor- mative because there were too few differences in gene content. For both of the chromosomes, as the regions grew larger, genome rearrangements were encountered that would wash out any signal from similarities in gene content at the origins themselves. B = the number of families shared by the two regions (i.e. in the 10 or 20 kb regions on both genomes) These pairwise distances were used to construct a square matrix; neighbor.exe from PHYLIP [40] was used to construct a neighbor-joining tree (settings; 10000 jum- bles, root, otherwise default). Origin of Replication Organization The origins of replication were studied first in the com- plete genomes, where they are identifiable by GC skew, annotation, and common gene content and organization. Page 11 of 13 Kirkup et al. BMC Microbiology 2010, 10:137 http://www.biomedcentral.com/1471-2180/10/137 Figure 4 Aberrant tree. Tree inferred from the gene Asub on Chromosome I that is inconsistent with the trees inferred by other methods as de- scribed in this paper, including the trees for the individual gene phylogenies at other nearby genes. In this tree, the V. splendidus clade is found next to the V. fisheri clade, making it basal to its expected position. This tree is also referred to as "I" in Table 1, column 1. As shown, the tree is not fully resolved and branches with low support have been collapsed. % % % % !"# $ % & $'() $'*+" , , --3"((( --(. 34# %,/ % 45 /# &'4# -44% -/65 #,% %%, 7'6 ( 4 8 ! / 001 001 , , Figure 4 Aberrant tree. Tree inferred from the gene Asub on Chromosome I that is inconsistent with the trees inferred by other methods as de- scribed in this paper, including the trees for the individual gene phylogenies at other nearby genes. In this tree, the V. splendidus clade is found next to the V. fisheri clade, making it basal to its expected position. This tree is also referred to as "I" in Table 1, column 1. As shown, the tree is not fully resolved and branches with low support have been collapsed. The genes within the selected regions were labeled by family and this data was used to produce a list of genes present in each region. This data was used to produce a pairwise distance estimate. In the incomplete genomes, orthologous regions were identified by both gene content and skew. When the expected gene families and gene order coincided with appropriate shifts in skew, the origin was identified. Additional material Additional file 1 Chromosome I core table. A key for the core genes on Chromosome I and their related locus tags from GenBank. Additional file 2 Chromosome II core table. A key for the core genes on Chromosome II and their related locus tags from GenBank. Additional file 3 OriI synteny figure. An expanded figure for OriI. Additional file 4 OriII synteny figure. An expanded figure for OriII Additional file 5 Colinearity of Chromosome II. The regions of homol- ogy among strains on chromosome II are not generally conserved in order or direction. Additional file 1 Chromosome I core table. A key for the core genes on Chromosome I and their related locus tags from GenBank. Additional file 1 Chromosome I core table. A key for the core genes on Chromosome I and their related locus tags from GenBank. 10. Fogel MA, Waldor MK: A dynamic, mitotic-like mechanism for bacterial chromosome segregation. Genes & Development 2006, 20:3269-3282. 10. Fogel MA, Waldor MK: A dynamic, mitotic-like mechanism for bacterial chromosome segregation. Genes & Development 2006, 20:3269-3282. Additional file 2 Chromosome II core table. A key for the core genes on Chromosome II and their related locus tags from GenBank. Additional file 2 Chromosome II core table. A key for the core genes on Chromosome II and their related locus tags from GenBank. 11. Rasmussen T, Jensen RB, Skovgaard O: The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle. The EMBO Journal 2007, 26:3124-3131. 11. Rasmussen T, Jensen RB, Skovgaard O: The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle. The EMBO Journal 2007, 26:3124-3131. Additional file 3 OriI synteny figure. An expanded figure for OriI. Additional file 4 OriII synteny figure. An expanded figure for OriII Additional file 3 OriI synteny figure. An expanded figure for OriI. Additional file 4 OriII synteny figure. An expanded figure for OriII 12. Egan ES, Løbner-Olesen A, Waldor MK: Synchronous replication initiation of the two Vibrio cholerae chromosomes. Current Biology 2004, 14:R501-R502. Additional file 5 Colinearity of Chromosome II. The regions of homol- ogy among strains on chromosome II are not generally conserved in order or direction. Additional file 5 Colinearity of Chromosome II. The regions of homol- ogy among strains on chromosome II are not generally conserved in order or direction. 13. Origin of Replication Genes Yamaichi Y, Fogel MA, Waldor MK: par genes and the pathology of chromosome loss in Vibrio cholerae. Proceedings of the National Academy of Sciences USA 2007, 104:630-635. 9. Duigou S, Knudsen KG, Skovgaard O, Egan ES, Løbner-Olesen A, Waldor MK: Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB. Journal of Bacteriology 2006, 188:6419-6424. Acknowledgements Funding was provided by The Woods Hole Center for Oceans and Human Health (NSF&NIEHS), the Moore Foundation and DOE-Genomes to Life; com- putational support was provided by the Darwin Cluster at MIT. Health (NSF&NIEHS), the Moore Foundation and DOE-Genomes to Life; com- putational support was provided by the Darwin Cluster at MIT. 18. Kasai S, Okada K, Hoshino A, Iida T, Honda T: Lateral transfer of the lux gene cluster. Journal of Biochemistry 2007, 141:231-237. 19. Urbanczyk H, Ast JC, Kaeding AJ, Oliver JD, Dunlap PV: Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae. Journal of Bacteriology 2008, 190:3494-3504. Author Details 1Dept. of Civil and Environmental Engineering, 15 Vassar Street, Cambridge MA 02139, USA, 2Infectious Diseases, Childrens' Hospital Boston, 200 Longwood Ave., Boston MA 02115, USA and 3Microbial Sequencing Center, The Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge MA 02142, USA 20. Hunt DE, David LA, Gevers D, Preheim SP, Alm EJ, Polz MF: Resource Partitioning and Sympatric Differentiation Among Closely Related Bacterioplankton. Science 2008, 320:1081-1085. 21. Reen F, Almagro-Moreno S, Ussery D, Boyd E: The genomic code: inferring Vibrionaceae niche specialization. Nature Reviews: Microbiology 2006, 4:697-704. Received: 27 August 2009 Accepted: 10 May 2010 Published: 10 May 2010 This article is available from: http://www biomedcentral com/1471 2180/10/137 © 2010 Kirkup 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 distributio BMC Microbiology 2010 10:137 Received: 27 August 2009 Accepted: 10 May 2010 Published: 10 May 2010 This article is available from: http://www biomedcentral com/1471-2180/10/137 © 2010 Kirkup 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 BMC Microbiology 2010 10:137 22. Bisharat N, Cohen DI, Harding RM, Falush D, Crook DW, Peto T, Maiden MC: Hybrid Vibrio vulnificus. Emerging Infectious Diseases 2005, 11:30-35. Additional material Srivastava P, Fekete RA, Chattoraj DK: Segregation of the replication terminus of the two Vibrio cholerae chromosomes. Journal of Bacteriology 2006, 188:1060-1070. Additional file 6 Strains included table. All the genomes included in the manuscript are listed with their genome sizes. gy 14. Okada K, Iida T, Kita-Tsukamoto K, Honda T: Vibrios commonly possess two chromosomes. Journal of Bacteriology 2005, 187:752-757. Authors' contributions BCK conceived of the project, generated the methods and drafted the manu- script. LC performed the final version of the analysis for each section and par- ticipated in writing the manuscript. SC performed an initial version of the first two analyses. DG developed the database for the research and reviewed drafts of the manuscript. MFP contributed ongoing critical review of the research aims and methods, extensively reviewed and edited the manuscript. All authors have read and approved the final manuscript. 15. 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https://openalex.org/W4300537354	https://zenodo.org/records/49136/files/neutro-chapter-IGI-Global-Multi-Attribute-Decision-Making-Trapezoid-Linguistic-Aggregation.pdf	Latin	null	Multi-Attribute Decision Making Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators	Zenodo (CERN European Organization for Nuclear Research)	2,016	cc-by	19,200	Handbook of Research on Generalized and Hybrid Set Structures and Applications for Soft Computing Sunil Jacob John National Institute of Technology Calicut, India A volume in the Advances in Computational Intelligence and Robotics (ACIR) Book Series Published in the United States of America by Information Science Reference (an imprint of IGI Global) 701 E. Chocolate Avenue Hershey PA, USA 17033 Tel: 717-533-8845 Fax: 717-533-8661 E-mail: cust@igi-global.com Web site: http://www.igi-global.com Published in the United States of America by Information Science Reference (an imprint of IGI Global) 701 E. Chocolate Avenue Hershey PA, USA 17033 Tel: 717-533-8845 Fax: 717-533-8661 E-mail: cust@igi-global.com Web site: http://www.igi-global.com Published in the United States of America by Information Science Reference (an imprint of IGI Global) 701 E. Chocolate Avenue Hershey PA, USA 17033 Tel: 717-533-8845 Fax: 717-533-8661 E-mail: cust@igi-global.com Web site: http://www.igi-global.com Copyright © 2016 by IGI Global. All rights reserved. No part of this publication may be reproduced, stored or distributed in any form or by any means, electronic or mechanical, including photocopying, without written permission from the publisher. Product or company names used in this set are for identification purposes only. Inclusion of the names of the products or companies does not indicate a claim of ownership by IGI Global of the trademark or registered trademark. Library of Congress Cataloging-in-Publication Data Library of Congress Cataloging-in-Publication Data Names: John, Sunil Jacob, 1973- editor. Names: John, Sunil Jacob, 1973- editor. Title: Handbook of research on generalized and hybrid set structures and Title: Handbook of research on generalized and hybrid set structures and applications for soft computing / Sunil Jacob John editor Title: Handbook of research on generalized and hybrid set structures and applications for soft computing / Sunil Jacob John, editor. g y applications for soft computing / Sunil Jacob John, editor. Description: Hershey, PA : Information Science Reference, [2016] \| Includes bibli hi l f d i d Description: Hershey, PA : Information Science Reference, [2016] \| Includes bibliographical references and index. g p Identifiers: LCCN 2015043853\| ISBN 9781466697980 (hardcover) \| ISBN 9781466697997 (ebook) 9781466697997 (ebook) Subjects: LCSH: Soft computing--Mathematics--Handbooks, manuals, etc. \| Set theory--Handbooks, manuals, etc. Classification: LCC QA76 9 S63 H348 2016 \| DDC 006 3--dc23 LC record available at http://lccn loc gov/2015043853 Subjects: LCSH: Soft computing--Mathematics--Handbooks, manuals, etc. \| Set y , , Classification: LCC QA76.9.S63 .H348 2016 \| DDC 006.3--dc23 LC record available at http://lccn.loc.gov/2015043853 Classification: LCC QA76.9.S63 .H348 2016 \| DDC 006.3--dc23 LC record available at http://lccn.loc.gov/20 This book is published in the IGI Global book series Advances in Computational Intelligence and Robotics (ACIR) (ISSN: 2327-0411; eISSN: 2327-042X) This book is published in the IGI Global book series Advances in Computational Intelligence and Robotics (ACIR) (ISSN: 2327-0411; eISSN: 2327-042X) British Cataloguing in Publication Data A Cataloguing in Publication record for this book is available from the British Library. Published in the United States of America by Information Science Reference (an imprint of IGI Global) 701 E. Chocolate Avenue Hershey PA, USA 17033 Tel: 717-533-8845 Fax: 717-533-8661 E-mail: cust@igi-global.com Web site: http://www.igi-global.com British Cataloguing in Publication Data A Cataloguing in Publication record for this book is available from the British Library. All work contributed to this book is new, previously-unpublished material. The views expressed in this book are those of the authors, but not necessarily of the publisher. For electronic access to this publication, please contact: eresources@igi-global.com. All work contributed to this book is new, previously-unpublished material. The views expressed in this book are those of the authors, but not necessarily of the publisher. All work contributed to this book is new, previously-unpublished material. The views expressed in this book are those of the authors, but not necessarily of the publisher. For electronic access to this publication, please contact: eresources@igi-global.com. 344 ABSTRACT Multi-attribute decision making (MADM) play an important role in many applications, due to the ef- ficiency to handle indeterminate and inconsistent information, interval neutrosophic sets is widely used to model indeterminate information. In this paper, a new MADM method based on interval neutrosophic trapezoid linguistic weighted arithmetic averaging aggregation (INTrLWAA) operator and interval neutrosophic trapezoid linguistic weighted geometric aggregation (INTrLWGA) operatoris presented. A numerical example is presented to demonstrate the application and efficiency of the proposed method. Chapter 17 Multi-Attribute Decision Making Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Broumi Said University of Hassan II, Morocco Florentin Smarandache University of New Mexico, USA Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited. 1. INTRODUCTION Smarandache (1998) proposed the neutrosophic set (NS) by adding an independent indeterminacy- membership function. The concept of neutrosophic set is a generalization of classic set, fuzzy set (Zadeh, 1956), intuitionistic fuzzy set (Atanassov, 1989), interval intuitionistic fuzzy set(Atanassov et al., 1989; Atanassov, 1994) and so on. In NS, the indeterminacy is quantified explicitly and truth-membership, inde- terminacy membership, and false-membership are completely independent. From scientific or engineering point of view, the neutrosophic set and set- theoretic view, operators need to be specified. Otherwise, it will be difficult to apply in the real applications. Therefore, Wang, et al.(2010) defined a single valued neutrosophic set (SVNS) and then provided the set theoretic operations and various properties of single valued neutrosophic sets. Furthermore, Wang, et al.(2005) proposed the set theoretic operations on an DOI: 10.4018/978-1-4666-9798-0.ch017 Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators instance of neutrosophic set called interval valued neutrosophic set (IVNS) which is more flexible and practical than NS. The works on neutrosophic set (NS) and interval valued neutrosophic set (IVNS), in theories and application have been progressing rapidly (e.g, Kharal, 2013; Ansaria et al., 2013; Saha et al, 2013; Rabounski et al, 2005 ; Lupiáñez, 2008 ; Wang et al, 2010; Deli et al, 2014 ; Deli et al, 2014a, Ye, 2014 ; Ye, 2014a ; Ye, 2014b ; Ye, 2014c ; Ye, 2014d ; Ye, 2014e ; Ye, 2014f ; Ye, 2014g ; Zeng, 2006; Peide et al. 2014 ; Arora et al, 2011; Arora et al, 2010 ; Chi et al. 2013 ; Liu et al. 2014 ;Biswas et al. 2014 ; Şahin et al . 2014 ; Aggarwal et al, 2010 ; Broumi et al, 2013; Broumi et al, 2013a; Broumi et al, 2013b; Broumi et al, 2013c; Broumi et al, 2014d; Broumi et al, 2013e; Broumi et al, 2014f; Broumi et al, 2014g ; Broumi et al, 2014h, Broumi et al, 2015i ; Broumi et al, 2014j ; Broumi et al, 2015k ; Broumi et al, 2015l ; Broumi et al, 2015m) Multiple attribute decision making (MADM) problem are of importance in most kind of fields such as engineering, economics, and management. In many situations decision makers have incomplete, inde- terminate and inconsistent information about alternatives with respect to attributes. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators In order to process incomplete, indeterminate and inconsistent information more efficiency and pre- cisely, it is necessary to make a further study on the extended form of the interval neutrosophic uncertain linguistic variables by combining trapezoid fuzzy linguistic variables and interval neutrosophic set. For example, we can evaluate the investment alternatives problem by the linguistic set: S= {s0 (extremely low); s1 (very low); s2 (low); s3 (medium); s4 (high); s5 (very high); s6 (extermley high). Perhaps, we can use the trapezoid fuzzy linguistic [sθ, sρ, sμ, sv], (0 ≤ θ ≤ ρ ≤ μ ≤ v ≤ l – 1) to describe the evaluation result, but this is not accurate, because it merely provides a linguistic range. In this paper, we can use interval neutrosophic trapezoid linguistic (INTrL), <[sθ(x), sρ(x), sμ(x), sv(x)], (TA(x), IA(x), FA(x))>to describe the investment problem giving the membership degree, indeterminacy degree, and non-membership degree interval to [sθ, sρ, sμ, sv]. This is the motivation of our study. As a fact, INTrL avoids the informa- tion distortions and losing in decision making process, and overcomes the shortcomings of the interval neutrosophic linguistic variables by Ye (2014) and interval neutrosophic uncertain linguistic variables by Ye (2015). To achieve the above purposes, The remainder of this paper is organized as follows: some basic definitions of trapezoid linguistic term set, neutrosophic set, and interval neutrosophic set are briefly reviewed in section 2. In section3, the concept, operational laws, score function, accuracy function and certainty function of including interval neutrosophic trapezoid linguistic elements are defined. In section 4, some interval neutrosophic trapezoid linguistic aggregation operators are proposed, such as interval neutrosophic trapezoid linguistic weighted average (INTrLWAA) operator, interval neutrosophic trapezoid linguistic weighted average (INTrLWGA) operators, then some desirable properties of the proposed operators are investigated. In section 5, we develop an approach for multiple attribute decision making problems with interval neutrosophic trapezoid linguistic information based on the proposed operators. In section 6, a numerical example is given to illustrate the application of the proposed method. The paper is concluded in section 7. 2. PRELIMINARIES In the following section, we shall introduce some basic concepts related to trapezoidal fuzzy linguistic variables, neutrosophic set, single valued neutrosophic set, interval neutrosophic sets, Interval neutro- sophic linguistic sets and interval neutrosophic uncertain linguistic sets. 1. INTRODUCTION It is well known that the conventional and fuzzy or intuitionistic fuzzy decision making analysis (Zeng, 2006; Broumi et al., 2015; Broumi et al., 2014) using different techniques tools have been found to be inadequate to handle indeterminate an inconsistent data. So, Recently, neutrosophic multicriteria decision making problems have been proposed to deal with such situation. In addition, because the aggregation operators are the important tools to process the neutrosophic decision making problems. Lately, Research on aggregation methods and multiple attribute decision making theories under neutrosophic environment is very active and lot of results have been obtained from neutrosophic information. Based on the aggregation operators, Ye (2013) developed some new weighted arithmetic averaging and weighted geometric averaging operators for simplified neutrosophic sets. Peide et al.(2014) present the generalized neutrosophic Hamacher aggregation operators such as Generalized neutrosophic number Hamacher weighted averaging (GNNHWA) operator, Generalized neutrosophic number Hamacher ordered weighted averaging (GNNHOWA) operator, and Generalized neutrosophic number Hamacher hybrid averaging (GNNHA) operator and studied some properties of these operators and analyzed some special cases and gave a decision-making method based on these operators for multiple attribute group decision making with neutrosophic numbers. Based on the idea of Bonferroni mean, Peide et al. (2014) proposed some Bonferroni mean operators such as the single- valued neutrosophic normalized weighted Bonferroni mean. Based on the linguistic variable and the concept of interval neutrosophic sets, Ye (2014) defined interval neutrosophic linguistic variable, as well as its operation principles, and developed some new aggregation operators for the interval neutrosophic linguistic information, including interval neutrosophic linguistic arithmetic weighted average(INLAWA) operator, linguistic geometric weighted average(INLGWA) operator and discuss some properties. Furthermore, he proposed thedecision making method for mul- tiple attribute decision making (MAGDM) problems with an illustrated example to show the process of decision making and the effectiveness of the proposed method. In order to deal with the more complex neutrosophic information. Ye (2013), further proposed the interval neutrosophic uncertain linguistic variables by extending uncertain linguistic variables with an interval neutrosophic set, and proposed the operational rules, score function, accuracy function and certainty function of interval neutrosophic uncertain variables. Then, the interval neutrosophic uncer- tain weighted arithmetic averaging operator and interval neutrosophic neutrosophic uncertain linguistic weighted geometric averaging operator are developed, and a multiple attribute decision making method with interval neutrosophic linguistic information is proposed. 345 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators In particular, if any two of sθ, sρ, sμ, sv are equal, then ˆs is reduced to triangular fuzzy linguistic vari- able; if any three of sθ, sρ, sμ, sv are equal, then ˆs is reduced to uncertain linguistic variable 2.3 Neutrosophic Sets Definition 2.2 (Smarandache, 1998): Let U be a universe of discourse then the neutrosophic set A is an object having the form A = {< x: TA(x), IA(x),FA(x) >, x ∈X }, where the functions T, I, F: U→]-0,1+[define respectively the degree of membership, the degree of indeterminacy, and the degree of non-membership of the element x ∈ X to the set A with the condition. −0 ≤s up TA(x) +sup IA(x) +sup FA(x) ≤ 3+. where the functions T, I, F: U→]-0,1+[define respectively the degree of membership, the degree of indeterminacy, and the degree of non-membership of the element x ∈ X to the set A with the condition. −0 ≤s up TA(x) +sup IA(x) +sup FA(x) ≤ 3+. −0 ≤s up TA(x) +sup IA(x) +sup FA(x) ≤ 3+. From philosophical point of view, the neutrosophic set takes the value from real standard or non- standard subsets of ]−0,1+[. So instead of ]−0,1+[ we need to take the interval [0,1] for technical ap- plications, because ]−0,1+[ will be difficult to apply in the real applications such as in scientific and engineering problems. 2.2 The Expected Value of Trapezo Fuzzy Linguistic Variable Let ˆs = ([sθ, sρ, sμ, sv]) be a trapezoid fuzzy linguistic variable, then the expected value E( ˆs ) of ˆs is defined as: E s (ˆ) = + + + θ ρ µ ν 4 2.1 Trapezoid Fuzzy Linguistic Variables A linguistic set is defined as a finite and completely ordered discreet term set, S = (s0, s1, …, sl–1), where l is the odd value. For example, when l =7, the linguistic term set S can be defined as follows: S={s0 (extremely low); s1 (very low); s2 (low); s3 (medium); s4 (high); s5 (very high); s6 (extremely high)} Definition 2.1 (Chen et al.,(2011): Let S = {sθ \| s0 ≤ sθ ≤ sl–1 θ∈ [0, l-1]}, which is the continuous form of linguistic set S. sθ, sρ, sμ, sv are four linguistic terms in, and s0 ≤ sθ≤ sρ≤ sμ≤ sv ≤ sl–1 if 0 ≤ θ ≤ ρ ≤ μ ≤ v ≤ l–1, then the trapezoid fuzzy linguistic variable is defined as ˆs = [sθ, sρ, sμ, sv], and ˆS denotes a set of the trapezoid fuzzy linguistic variables. 346 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators For two IVNS, A x T x T x I x I x F x F x A L A U A L A U A L A U IVNS = ( ) ( )   ( ) ( )   ( ) , , , , , , ( )   ∈ { } \| x X and B x T x T x I x I x F x F x B L B U B L B U B L B U IVNS = ( ) ( )   ( ) ( )   ( ) , , , , , , ( )   ∈ { } \| x X the two relations are defined as follows: 1. AIVNS⊆BIVNS if and only if T x T x A L B L ( ) ≤ ( ), T x T x A U B U ( ) ≤ ( ),I x I x A L B L ( ) ≥ ( ), I x I x A U B U ( ) ≥ ( ), F x F x A L B L ( ) ≥ ( ), F x F x A U B U ( ) ≥ ( ). 2. AIVNS = BIVNS if and only if, TA(x) = TB(x), IA(x) = IB(x), FA(x) = FB(x) for any x∈X. For two IVNS, The complement of AIVNS is denoted by AIVNS o and is defined by A x IVNS o L = ( ) ( )   − ( ) − ( )   ( ) , , , , , , F x F x I x I x T x A L A U A U A A L 1 1 T x A U ( )   ∈ { } \| x X A B T x T x T x T x I x A L B L A U B U A L ∩ = ( ) ( ) ( ) ( ) ( ) ( )   { ( ) min , ,min , , max , I x I x I x F B L A U B U A L ( ) ( )  ( ) ( ) ( ) , max , , max x F x F x F x x X B L B U ( ) ( ) ( ) ( ) ( ) ( )   ∈ } , ,max , : A B T x T x T x T x I x A L B L A U B U A L ∪ = ( ) ( ) ( ) ( ) ( ) ( )   { ( ) max , ,max , , min , I x I x I x F B L A U B U A L ( ) ( )  ( ) ( ) ( ) , min , , min x F x F x F x x X B L A U B U ( ) ( ) ( ) ( ) ( ) ( )   ∈ } , ,min , : 2.4 Single Valued Neutrosophic Sets Definition 2.3 (Wang, et al., 2010): Let X be an universe of discourse then the neutrosophic set A is an object having the form A = {< x: TA(x), IA(x),FA(x) >, x ∈X }, where the functions μ, v, ω: U→[0,1]define respectively the degree of membership, the degree of in- determinacy, and the degree of non-membership of the element x ∈ X to the set A with the condition. where the functions μ, v, ω: U→[0,1]define respectively the degree of membership, the degree of in- determinacy, and the degree of non-membership of the element x ∈ X to the set A with the condition. 0 ≤s up TA(x) +sup IA(x) +sup FA(x) ≤ 3 347 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Definition 2.4 (Wang, et al., 2010): A single valued neutrosophic set A is contained in another single valued neutrosophic set B i.e. A ⊆ B if ∀x ∈ U, TA(x) ≤ TB(x), IA(x) ≥ IB(x), FA(x) ≥ FB(x). Definition 2.4 (Wang, et al., 2010): A single valued neutrosophic set A is contained in another single valued neutrosophic set B i.e. A ⊆ B if ∀x ∈ U, TA(x) ≤ TB(x), IA(x) ≥ IB(x), FA(x) ≥ FB(x). 2.5 Interval Valued Neutrosophic Sets Definition 2.5 (Wang, et al., 2005): Let X be a space of points (objects) with generic elements in X denoted by x. An interval valued neutrosophic set (for short IVNS) A in X is characterized by truth-membership function TA(x), indeteminacy-membership function IA(x) and falsity-membership function FA(x). For each point x in X, we have that TA(x), IA(x), FA(x)∈ [0, 1]. 2.6 Interval Neutrosophic Linguistic Set Based on interval neutrosophic set and linguistic variables, (Ye, 2014) presented the extension form of the linguistic set, i.e, interval neutrosophic linguistic set, which is shown as follows: 348 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Definition2.6 (Ye, 2014): An interval neutrosophic linguistic set A in X can be defined as Definition2.6 (Ye, 2014): An interval neutrosophic linguistic set A in X can be defined as A ={<x, sθ(x), (TA (x), IA (x), FA (x))>\| x ∈ X} where s s x θ( ) ∈ˆ, TA (x) = T T A L A U ( ) ( ) , x x   ⊆ [0.1], IA (x) = I I A L A U ( ) ( ) , x x   ⊆ [0.1], and FA (x) = F F A L A U ( ) ( ) , x x   ⊆ [0.1] with the condition 0≤ T I F x x x A U A U A U ( ) ( ) ( ) + + ≤3 for any x ∈ X. The func- tion TA (x), IA (x) and FA (x) express, respectively, the truth-membership degree, the indeterminacy – membership degree, and the falsity-membership degree with interval values of the element x in X to the linguistic variable sθ(x). 2.7 Single Valued Neutrosophic Trapezoid Linguistic Sets Definition2.7 (Broumi, et al., 2014). A single valued neutrosophic trapezoid linguistic set A in X can be defined as A ={<x, [sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA(x), FA (x))>: x ∈ X} A ={<x, [sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA(x), FA (x))>: x ∈ X} where sθ(x), sρ(x), sμ(x), sν(x)∈ IA ˆs , TA (x) ∈ [0.1], (x) ∈ [0.1], and FA (x) ∈ [0.1] with the condition 0 ≤TA (x)+ IA (x)+ FA (x) ≤3 for any x ∈ X. [sθ(x), TA sρ(x), sμ(x), sν(x)] is a trapezoid fuzzy linguistic term, The function (x), IA (x) and FA (x) express, respectively, the truth-membership degree, the indeterminacy – membership degree, and the falsity-membership degree of the element x in X belonging to the linguis- tic term [sθ(x), sρ(x), sμ(x), sν(x)]. Definition2.8: Let A ={<x, [sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA (x), FA (x))>: x ∈ X} be an SVNTrLN. Then the eight tuple <[ sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA (x), FA (x)) > is called a SVNTrLV and A can be viewed as a collection of SVNTrLVs. Thus, the SVNTrLVS can also be expressed as Definition2.8: Let A ={<x, [sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA (x), FA (x))>: x ∈ X} be an SVNTrLN. Then the eight tuple <[ sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA (x), FA (x)) > is called a SVNTrLV and A can be viewed as a collection of SVNTrLVs. Thus, the SVNTrLVS can also be expressed as A ={<x, [sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA (x), FA (x)) >: x ∈ X} For any two SVNTrLNs If E a E a i j ( ) ( ) > , then a a i j > ; 2. If E a E a i j ( ) ( ) = , then If H a H a i j ( ) ( ) > , then a a i j > , If H a H a i j ( ) ( ) = , then a a i j = , If H a H a i j ( ) ( ) < , then a a i j < . 1. If E a E a i j ( ) ( ) > , then a a i j > ; 2. If E a E a i j ( ) ( ) = , then If H a H a i j ( ) ( ) > , then a a i j > , If H a H a i j ( ) ( ) = , then a a i j = , If H a H a i j ( ) ( ) < , then a a i j < . 1. If E a E a i j ( ) ( ) > , then a a i j > ; 2. If E a E a i j ( ) ( ) = , then If H a H a i j ( ) ( ) > , then a a i j > , If H a H a i j ( ) ( ) = , then a a i j = , If H a H a i j ( ) ( ) < , then a a i j < . For any two SVNTrLNs a T a I a s s s s a a a a 1 1 1 1 1 1 1 =   ( ) ( ) ( ) ( ) θ ρ µ ν , , , ( ), ( ) , , ( ) F a1 ( ) and and a T a I a s s s s a a a a 2 2 2 2 2 2 2 =   ( ) ( ) ( ) ( ) θ ρ µ ν , , , ( ), ( ) , , ( ) F a2 ( ) and λ≥ 0, (Broumi et al. For any two SVNTrLNs 2014) defined the following operational rules: 349 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊕ = ( )+ ( ) ( )+ ( ) ( )+ ( ) θ θ ρ ρ µ µ ν , , , ( )+ ( )   + − ( ) ν a T a T a T a T a I a I a 2 1 2 1 2 1 2 ( ) ( ) ( ), ( ) , ( ), ( ) , ( ), ( ) F a F a 1 2 ( ) a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊗ = ( )× ( ) ( )× ( ) ( )× ( ) θ θ ρ ρ µ µ ν , , , ( )× ( )   + − ( ) ν a T a T a I a I a I a I a 2 1 2 1 2 1 2 ( ) ( ), ( ) ( ) ( ) ( ) , F a F a F a F a ( ) ( ) ( ) ( ) 1 2 1 2 + − ( ) ( ) λ λθ λρ λµ λν λ a a s s s s T a a a a 1 1 1 1 1 1 1 =   − ( ) ( ) ( ) ( ) ( ) ( ) , , , , , I F a a 1 1 ( ) ( ) ( ) ( ) ( ) λ λ , a a s s s s T a a a a 1 1 1 1 1 1 1 λ θ ρ µ ν λ λ λ λ λ =   ( ) ( ) ( ) ( ) ( ) ( ) , , , , , −( ) ( ) − ( ) ( ) ( ) I F a a 1 1 1 λ λ , a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊕ = ( )+ ( ) ( )+ ( ) ( )+ ( ) θ θ ρ ρ µ µ ν , , , ( )+ ( )   + − ( ) ν a T a T a T a T a I a I a 2 1 2 1 2 1 2 ( ) ( ) ( ), ( ) , ( ), ( ) , ( ), ( ) F a F a 1 2 ( ) a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊗ = ( )× ( ) ( )× ( ) ( )× ( ) θ θ ρ ρ µ µ ν , , , ( )× ( )   + − ( ) ν a T a T a I a I a I a I a 2 1 2 1 2 1 2 ( ) ( ), ( ) ( ) ( ) ( ) , F a F a F a F a ( ) ( ) ( ) ( ) 1 2 1 2 + − ( ) ( ) λ λθ λρ λµ λν λ a a s s s s T a a a a 1 1 1 1 1 1 1 =   − ( ) ( ) ( ) ( ) ( ) ( ) , , , , , I F a a 1 1 ( ) ( ) ( ) ( ) ( ) λ λ , a a s s s s T a a a a 1 1 1 1 1 1 1 λ θ ρ µ ν λ λ λ λ λ =   ( ) ( ) ( ) ( ) ( ) ( ) , , , , , −( ) ( ) − ( ) ( ) ( ) I F a a 1 1 1 λ λ , a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊗ = ( )× ( ) ( )× ( ) ( )× ( ) θ θ ρ ρ µ µ ν , , , ( )× ( )   ν a2 a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊗ = ( )× ( ) ( )× ( ) ( )× ( ) θ θ ρ ρ µ µ ν , , , ( )× ( )   ν a2 + − ( ) T a T a I a I a I a I a 1 2 1 2 1 2 ( ) ( ), ( ) ( ) ( ) ( ) , F a F a F a F a ( ) ( ) ( ) ( ) 1 2 1 2 + − ( ) ( ) λ λθ λρ λµ λν λ a a s s s s T a a a a 1 1 1 1 1 1 1 =   − ( ) ( ) ( ) ( ) ( ) ( ) , , , , , I F a a 1 1 ( ) ( ) ( ) ( ) ( ) λ λ , a a s s s s T a a a a 1 1 1 1 1 1 1 λ θ ρ µ ν λ λ λ λ λ =   ( ) ( ) ( ) ( ) ( ) ( ) , , , , , −( ) ( ) − ( ) ( ) ( ) I F a a 1 1 1 λ λ , Definition 2.9: Let a T a I a F s s s s i i i a a a a i i i i =   ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( ), ( ) ai ( ) be a SVNTrFLN, the expected function E( ai ) and the accuracy H( ai ) of are define as follows: Definition 2.9: Let a T a I a F s s s s i i i a a a a i i i i =   ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( ), ( ) ai ( ) be a SVNTrFLN, the expected function E( ai ) and the accuracy H( ai ) of are define as follows: E a T a I a F a S a a a a ( ) ( ) ( ) ( ) = − − × + ( )+ ( )+ ( )+ ( )      1 3 2 4 θ ρ µ ν  + ( )−( )−( ) ( )× ( )+ ( )+ ( )+   = S T I F a a a a a a 1 12 2 θ ρ µ ν a H a a F a ( ) ( ) = − ( )) ( ) T( ) + ( )−( )−( ) ( )× ( )+ ( )+ ( )+ = S T I F a a a a a a 1 12 2 θ ρ µ ν a H a a F a ( ) ( ) = − ( )) ( ) T( ) θ ρ µ ν a a a a S ( )+ ( )+ ( )+ ( )       × 4 = ( )−( )× ( )+ ( )+ ( )+ ( ) ( ) S T F a a a a a a 1 4( θ ρ µ ν Assume that ai and a j are two SVNTrLNs, they can be compared by the following rules: Assume that ai and a j are two SVNTrLNs, they can be compared by the following rules: 1. be an INTrL. Then the eight tuple s s s s T T I I x x x x x x x A L A U A L A U θ ρ µ ν ( ) ( ) ( ) ( )     , , , , ( ), ( ) , ( ), ( ) , ( ), ( ) x x x F F A L A U     ( ) is called an INTrLV and A can be viewed as a collection of INTrLvs. Thus, the INTrLS can also be expressed as is called an INTrLV and A can be viewed as a collection of INTrLvs. Thus, the INTrLS can also be expressed as A x x x x s s s s T T I x x x x A L A U A L =     ( ) ( ) ( ) ( ) , , , , , ( ), ( ) , ( θ ρ µ ν ), ( ) , ( ), ( ) \| I F F x x x x X A U A L A U     ∈ { } ( ) A x x x x s s s s T T I x x x x A L A U A L =     ( ) ( ) ( ) ( ) , , , , , ( ), ( ) , ( θ ρ µ ν ), ( ) , ( ), ( ) \| I F F x x x x X A U A L A U     ∈ { } ( ) Definition 3.2: Let A = x s s s s T T I x x x x x x x A L A U A L , , , , , ( ), ( ) , ( ), θ ρ µ ν ( ) ( ) ( ) ( )     I F F x x x x X A U A L A U ( ) , ( ), ( ) \|     ∈ { } ( ) 3 INTERVAL NEUTROSOPHIC TRAPEZOID LINGUISTIC SETS Based on the concept of INS and trapezoid fuzzy linguistic variable, we extend the NTrLS to define the INTrLS and INTrLNs. The operations and ranking method of INTrLN s are also given in this section Definition3.1: Let X be a finite universal set and [sθ(x), sρ(x), sμ(x), sν(x)] ∈ˆs be trapezoid fuzzy linguistic variable. An INTrLS in X is defined as Definition3.1: Let X be a finite universal set and [sθ(x), sρ(x), sμ(x), sν(x)] ∈ˆs be trapezoid fuzzy linguistic variable. An INTrLS in X is defined as 350 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators A ={〈x, [sθ(x), sρ(x), sμ(x), sν(x)], (TA (x), IA (x), FA (x))〉\| x ∈ X} where [sθ(x), sρ(x), sμ(x), sν(x)], ∈ˆs , TA (x) = T T x x A L A U ( ), ( )   ⊆ [0.1], IA (x) = I I x x A L A U ( ), ( )   ⊆ [0.1], and FA (x) = F F x x A L A U ( ), ( )   ⊆ [0.1] with the condition 0 ≤sup TA (x)+sup IA (x)+sup FA (x) ≤3 for any x ∈ X. The function TA (x), IA (x) and FA (x) express, respectively, the truth-membership degree interval, the indeterminacy –membership degree interval, and the falsity-membership degree interval of the element x in X belonging to the trapezoid linguistic variable [sθ(x), sρ(x), sμ(x), sν(x)] ∈ˆs . where [sθ(x), sρ(x), sμ(x), sν(x)], ∈ˆs , TA (x) = T T x x A L A U ( ), ( )   ⊆ [0.1], IA (x) = I I x x A L A U ( ), ( )   ⊆ [0.1], and FA (x) = F F x x A L A U ( ), ( )   ⊆ [0.1] with the condition 0 ≤sup TA (x)+sup IA (x)+sup FA (x) ≤3 for any x ∈ X. The function TA (x), IA (x) and FA (x) express, respectively, the truth-membership degree interval, the indeterminacy –membership degree interval, and the falsity-membership degree interval of the element x in X belonging to the trapezoid linguistic variable [sθ(x), sρ(x), sμ(x), sν(x)] ∈ˆs . Theorem 3.4: Let a T a T a s s s s a a a a L U 1 1 1 1 1 1 1 =    ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( )      ( ) , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 1 1 1 1 and and a T a T a s s s s a a a a L U 2 2 2 2 2 2 2 =    ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( )      ( ) , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 2 2 2 2 be any two interval neutrosophic trapezoid linguistic variables, and λ, λ1, λ2≥0, then the characterstics of interval neutrosophic trapezoid linguistic variables are shown as follows: be any two interval neutrosophic trapezoid linguistic variables, and λ, λ1, λ2≥0, then the characterstics of interval neutrosophic trapezoid linguistic variables are shown as follows: 1. a a a a 1 2 2 1 ⊕ ⊕ = ; 2. a a a a 1 2 2 1 ⊗ ⊗ = ; 3. λ λ λ a a a a 1 2 1 2 ⊕ ⊕ ( ) = ; 4. λ λ λ λ a a a 1 2 1 2 1 ⊕ + = ( ) ; 5. a a a 1 1 1 1 2 1 2 λ λ λ λ ⊗ = + ; 6. a a a a 1 2 1 2 1 1 1 λ λ λ ⊗ = ⊗ ( ) . 1. a a a a 1 2 2 1 ⊕ ⊕ = ; 2. a a a a 1 2 2 1 ⊗ ⊗ = ; 3. λ λ λ a a a a 1 2 1 2 ⊕ ⊕ ( ) = ; 4. λ λ λ λ a a a 1 2 1 2 1 ⊕ + = ( ) ; 5. a a a 1 1 1 1 2 1 2 λ λ λ λ ⊗ = + ; 6. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators a a s s s s a a a a a a 1 2 1 2 1 2 1 2 ⊗ = ( )× ( ) ( )× ( ) ( )× ( ) θ θ ρ ρ µ µ ν , , , a a L L U U L T a T a T a T a I 1 2 1 2 1 2 ( )× ( )     ν , ( ) ( ), ( ) ( ) , ( a I a I a I a I a I a I a I L L L U U U 1 2 1 2 1 2 1 ) ( ) ( ) ( ), ( ) ( ) ( )  ( + − + − + U L L L L U U a F a F a F a F a F a F a ( ) , ( ) ( ) ( ) ( ), ( ) ( 2 1 2 1 2 1 2  + −  + ) ( ) ( ) − ) F a F a U U 1 2 λ λθ λρ λµ λν λ a a s s s s T a a a a L 1 1 1 1 1 1 1 =   − ( ) ( ) ( ) ( ) ( ) ( ) , , , , , 1 1 1 1 1 − ( ) ( ) ( ) ( ) ( ) ( )   ( )  ( ) T I I F F a a a a U L U L U λ λ λ λ , , , , a1 ( ) ( )   ( ) λ a a s s s s T T a a a a L U 1 1 1 1 1 1 λ θ ρ µ ν λ λ λ λ λ =   ( ) ( ) ( ) ( ) ( ) ( ) , , , , , a a a a I I F L U L 1 1 1 1 1 1 1 ( ) ( ) − ( ) ( ) − ( ) ( ) − ( ) ( )     λ λ λ λ , , , , 1 1 − ( ) ( )   ( ) F a U λ Obviously, the above operational results are still INTrLvs. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Obviously, the above operational results are still INTrLvs. Definition 3.3: Let a T a T a s s s s a a a a L U 1 1 1 1 1 1 1 =    ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( )      ( ) , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 1 1 1 1 and a x T a T a s s s s a a a a L U 2 2 2 2 2 2 2 =   ( ) ( ) ( ) ( ) , , , , , ( ), ( ) θ ρ µ ν       ( ) { , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 2 2 2 2 a T a T a s s s s a a a a L U 1 1 1 1 1 1 1 =    ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( )      ( ) , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 1 1 1 1 and a x T a T a s s s s a a a a L U 2 2 2 2 2 2 2 =   ( ) ( ) ( ) ( ) , , , , , ( ), ( ) θ ρ µ ν       ( ) { , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 2 2 2 2 a x T a T a s s s s a a a a L U 2 2 2 2 2 2 2 =   ( ) ( ) ( ) ( ) , , , , , ( ), ( ) θ ρ µ ν       ( ) { , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 2 2 2 2 a x T a T a s s s s a a a a L U 2 2 2 2 2 2 2 =   ( ) ( ) ( ) ( ) , , , , , ( ), ( ) θ ρ µ ν       ( ) { , ( ), ( ) , ( ), ( ) I a I a F a F a L U L U 2 2 2 2 be two INTrLvs and λ≥ 0, then the operational laws of INTrLvs are defined as follows: a a s s s s a a a a a a a 1 2 1 2 1 2 1 2 1 ⊕ = ( )+ ( ) ( )+ ( ) ( )+ ( ) θ θ ρ ρ µ µ ν , , , ( )+ ( )   + − ( ν a L L L L U T a T a T a T a T a 2 1 2 1 2 1 , ( ) ( ) ( ) ( ), ( ) + −  T a T a T a I a I a I a I a U U U L L U U ( ) ( ) ( ) , ( ) ( ), ( ) ( ) 2 1 2 1 2 1 2   ) , ( ) ( ), ( ) ( ) F a F a F a F a L L U U 1 2 1 2 351 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Definition 3.5: Let Based on definition 3.5, a ranking method between INTrLvs can be given as follows. Based on definition 3.5, a ranking method between INTrLvs can be given as follows Based on definition 3.5, a ranking method between INTrLvs can be given as follows. Definition 3.6 Let a1 and a2 be two INTrLNs. Then, the ranking method can be defined as follows: Definition 3.6 Let a1 and a2 be two INTrLNs. Then, the ranking method can be defined as follows: If then E a E a a a ( ) ( ) 1 2 1 2 > > , If and then E a E a H a H a a a ( ) ( ) ( ) ( ) 1 2 1 2 1 2 = > > , If and and then E a E a H a H a C a C a ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 2 1 2 = = > a a 1 2 > , If and and then E a E a H a H a C a C a ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 2 1 2 = = = a a 1 2 = . Definition 3.5: Let a T a T a I s s s s a a a a L U L =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , ( ), ( ) , ( a I a F a F a U L U ), ( ) , ( ), ( )     ( ) be an INTrFLV. Then, the score function, accuracy function and certainty function of an INTrFLN a are defined, respectively, as follows: be an INTrFLV. Theorem 3.4: Let a a a a 1 2 1 2 1 1 1 λ λ λ ⊗ = ⊗ ( ) . Theorem 3.4 can be easily proven according to definition 3.3 (omitted). To rank INTrLNs, we define the score function, accuracy function and certainty function of an IN- TrFLN based on (Ye, 2013 g), which are important indexes for ranking alternatives in decision making problems. 352 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Definition 3.5: Let Definition 3.5: Let Then, the score function, accuracy function and certainty function of an INTrFLN a are defined, respectively, as follows: E a T I F T I F a a a a a a L L L U U U ( ) ( ) ( ) ( ) ( ) ( ) ( ) = + − − + − − ( ) 1 6 4 × = ( )+ ( )+ ( )+ ( )       + ( )− S S a a a a a T I L L θ ρ µ ν 4 1 24 4 a a a a a a a a a F T I F L U U U ( )− ( )+ ( )− ( )− ( ) ( )× ( )+ ( )+ ( )+ ( ) θ ρ µ ν ( ) (1) H a T F F T S a a a a L U L U a ( ) ( ) ( ) ( ) ( ) = − + − ( ) × ( )+ 1 2 θ ρ a a a a a a a T F T F S L L U U ( )+ ( )+ ( )       ( )− ( )+ ( )− ( ) ( )× = µ ν θ 4 1 8 a a a a ( )+ ( )+ ( )+ ( ) ( ) ρ µ ν (2) C a T T S S a a L U a a a a ( ) ( ) ( ) = + ( ) × = ( )+ ( )+ ( )+ ( )       1 2 4 θ ρ µ ν 1 8 T T a a a a a a L U ( )+ ( ) ( )× ( )+ ( )+ ( )+ ( ) ( ) θ ρ µ ν (3) (1) (2) S a × ( )+ θ ρ a a a a a a a T F T F S L L U U ( )+ ( )+ ( )       ( )− ( )+ ( )− ( ) ( )× = µ ν θ 4 1 8 a a a a ( )+ ( )+ ( )+ ( ) ( ) ρ µ ν (2) C a T T S S a a L U a a a a ( ) ( ) ( ) = + ( ) × = ( )+ ( )+ ( )+ ( )       1 2 4 θ ρ µ ν 1 8 T T a a a a a a L U ( )+ ( ) ( )× ( )+ ( )+ ( )+ ( ) ( ) θ ρ µ ν (3) (3) Based on definition 3.5, a ranking method between INTrLvs can be given as follows. INTrLWAA INTrLWAA a a a s s s n a a j n j j j n j j j n j 1 2 1 1 1 , ,..., , , ( ) = ∑ ∑ ∑ = = = ( ) ( ) ω θ ω ρ ω µ a a j n L j j n j j n j j j s T a ( ) ( ) = = =∑ − − ( ) ( ) −       ∏ , , , 1 1 1 1 1 1 ω ν ω ∏ ∏ ∏ − ( ) ( ) ( ) ( ) ( ) ( )       = = 1 1 1 T I I a a a U j j n L j j n U j j j j ω ω ω , ,      ( ) ( ) ( ) ( ) = = ∏ ∏ , , j n L j j n U j F F a a j j 1 1 ω ω (5) j j n = −    1 1∏ ∏ ∏ − ( ) ( ) ( ) ( ) ( ) ( )       = = 1 1 1 T I I a a a U j j n L j j n U j j j j ω ω ω , ,      ( ) ( ) ( ) ( ) = = ∏ ∏ , , j n L j j n U j F F a a j j 1 1 ω ω (5) (5) where, ωj= (ω1, ω2, …, ωn)T is the weight vector of a j (j= 1,2,…,n), ωj∈ [0,1]and j n j =∑ 1 ω =1. where, ωj= (ω1, ω2, …, ωn)T is the weight vector of a j (j= 1,2,…,n), ωj∈ [0,1]and j n j =∑ 1 ω =1. 4. INTERVAL NEUTROSOPHIC TRAPEZOID LINGUISTIC AGGREGATION OPERATORS Based on the operational laws in definition 3.3, we can propose the following weighted arithmetic ag- gregation operator and weighted geometric aggregation operator for INTrLNs, which are usually utilized in decision making. 353 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 4.1 Interval Neutrosophic Trapezoid Linguistic Weighted Arithmetic Averaging Operator Definition 4.1: Let a s s s s T T I I j a L a U a L a a a a j j j =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , , , , a U a L a U j j j F F     ( ) , , (j=1,2,…,n) Definition 4.1: Let a s s s s T T I I j a L a U a L a a a a j j j =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , , , , a U a L a U j j j F F     ( ) , , (j=1,2,…,n) be a collection of INTrLNs. The interval neutrosophic trapezoid linguistic weighted arithmetic averaging average (INTrLWAA) operator can be defined as follows and INTrLWAA: Ωn→Ω INTrLWAA a a a a n j n j j 1 2 1 , ,..., ( ) = =∑ω INTrLWAA a a a a n j n j j 1 2 1 , ,..., ( ) = =∑ω (4) a a n j n j j 1 ., ) = =∑ω (4) (4) where, ωj= (ω1, ω2, …, ωn)T is the weight vector of a j (j= 1, 2,…, n), ωj∈ [0,1] and j n j =∑ 1 ω =1. Theorem 4.2: Let a s s s s T T j L U a a a a a a j j j j j j =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , , , I I F F a a a a L U L U j j j j , , ,     ( ) (j=1, 2,…,n) be a collection of INTrLNs, Then by Equation (4) and the operational l 2,…,n) be a collection of INTrLNs, Then by Equation (4) and the operational laws in Definition 3.3, we have the following result , , ) , y q ( ) p 3.3, we have the following result 3.3, we have the following result INTrLWAA Proof: The proof of Equation (5) can be done by means of mathematical induction When n=2, then When n=2, then When n=2, then ω ω θ ω ρ µ ν 1 1 1 1 1 1 1 1 1 1 1 a a s s s s T a a a a L =   − − ( ) ( ) ( ) ( ) ( ) , , , , ( ) − ( ) ( )   ( ) ( ) − ( ) ω ω ω 1 1 1 1 1 1 1 1 , , , T I I a a a U L U ( ) ( ) ( ) ( )   ( )   ω ω ω 1 1 1 1 1 , , F F a a L U ω ω θ ω ρ µ ν 2 2 2 2 2 2 2 2 2 1 1 a a s s s s T a a a a L =   − − ( ) ( ) ( ) ( ) ( ) , , , , ( ) − ( ) ( )   ( ) ( ) − ( ) ω ω ω 2 2 2 1 1 2 2 2 , , , T I I a a a U L U ( )   ( )   ( ) ( ) ( ) ω ω ω 2 2 2 2 2 , , F F a a L U 354 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Thus, INTrLWAA INTrLWAA a a a a S S S j j j j j j j a a 1 2 1 1 2 2 1 2 1 2 1 2 , , , ( ) = = ⊕ ∑ ∑ ∑ = = = ( ) ( ) ω ω ω θ ω ρ ω µ ω ν ω j j j j j a a L S T a ( ) ( ) =∑ − ( ) ( )      , , 1 2 1 1 1 + −( ) − ( ) ( ) − ( ) ( ) − ( ) ( ) − ( ) ( ) 1 1 1 1 2 1 2 1 2 1 2 T T T T a a a a L L L U ω ω ω ω , 1 2 1 2 1 1 1 2 1 2 + −( ) − ( ) ( ) − ( ) ( ) − ( ) ( ) T T T a a a U u U ω ω ω ,  ( ) ( ) ( ) ( )   ( ) ( ) ( ) ( ) , , , I I I I a a a a L L U U 1 2 1 2 1 2 1 2 ω ω ω ω F F F F a a a a L L U U 1 2 1 2 1 2 1 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ω ω ω ω ,   = ∑ ∑ ∑ = = = ( ) ( ) S S S j j j j j j j j j a a a 1 2 1 2 1 2 ω θ ω ρ ω µ , , ( ) ( ) =∑ − ( ) ( ) − ( ) ( )    ( )   , , , S T T a a j j j a L L 1 2 1 2 1 1 1 2 ω ν ω ω 1 1 1 2 1 2 1 2 − ( ) ( ) − ( ) ( ) ( )  =∏ T T I a a a u U j L ω ω , j j U j j L j j j I F a a ( ) ( ) ( ) ( )     = = ∏ ∏ ω ω , , 1 2 1 2 ( ) ( ) ( ) ( )     =∏ ω ω j j j U j F a , 1 2 (6 INTrLWAA a a a a S S S j j j j j j j a a 1 2 1 1 2 2 1 2 1 2 1 2 , , , ( ) = = ⊕ ∑ ∑ ∑ = = = ( ) ( ) ω ω ω θ ω ρ ω µ ω ν ω j j j j j a a L S T a ( ) ( ) =∑ − ( ) ( )      , , 1 2 1 1 1 + −( ) − ( ) ( ) − ( ) ( ) − ( ) ( ) − ( ) ( ) 1 1 1 1 2 1 2 1 2 1 2 T T T T a a a a L L L U ω ω ω ω , 1 2 1 2 1 1 1 2 1 2 + −( ) − ( ) ( ) − ( ) ( ) − ( ) ( ) T T T a a a U u U ω ω ω ,  ( ) ( ) ( ) ( )   ( ) ( ) ( ) ( ) , , , I I I I a a a a L L U U 1 2 1 2 1 2 1 2 ω ω ω ω F F F F a a a a L L U U 1 2 1 2 1 2 1 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ω ω ω ω ,   = ∑ ∑ ∑ = = = ( ) ( ) S S S j j j j j j j j j a a a 1 2 1 2 1 2 ω θ ω ρ ω µ , , ( ) ( ) =∑ − ( ) ( ) − ( ) ( )    ( )   , , , S T T a a j j j a L L 1 2 1 2 1 1 1 2 ω ν ω ω 1 1 1 2 1 2 1 2 − ( ) ( ) − ( ) ( ) ( )  =∏ T T I a a a u U j L ω ω , j j U j j L j j j I F a a ( ) ( ) ( ) ( )     = = ∏ ∏ ω ω , , 1 2 1 2 ( ) ( ) ( ) ( )     =∏ ω ω j j j U j F a , 1 2 (6) 2. 3. When n=k+1, by applying Equation (6) and Equation (7), we can get INTrLWAA When n=k, by applying Equation (5), we get 2. When n=k, by applying Equation (5), we get 2. When n=k, by applying Equation (5), we get 2. When n=k, by applying Equation (5), we get INTrLWAA a a a S S S k a a j k j j j k j j j k j 1 2 1 1 1 , ,..., , , ( ) = ∑ ∑ ∑ = = = ( ) ( ) ω θ ω ρ ω µ a a j k L j j j k j j j S T a ( ) ( ) = =∑ − − ( ) ( )      ∏ , , , 1 1 1 1 ω ν ω 1 1 1 1 − − ( ) ( )   = = ∏ ∏ j k U j j k L j T I a a j ω , ( ) ( ) ( ) ( )     =∏ ω ω j j j k U j I a , , 1 j k L j j k U j F F a a j j = = ∏ ∏ ( ) ( ) ( ) ( )     1 1 ω ω , (7) (7) 3. INTrLWAA When n=k+1, by applying Equation (6) and Equation (7), we can get 355 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators INTrLWAA a a a a S S k k a a j k j j k k j k 1 2 1 1 1 1 1 , ,..., , , + ( )+ ( ) ( ) = ∑ ∑ = + + = ω θ ω θ ω ρ ω ρ ω µ ω µ ω j j k k j k j j k k j k a a a a S S ( )+ ( ) ( )+ ( ) + + = + + = ∑ ∑ 1 1 1 1 1 1 , , j j k k j a a j k L j L k T T a a ν ω ν ω ( )+ ( ) = + +     + − − − ( ) ( ) − ∏ 1 1 1 1 1 1 1 , + = + ( ) ( ) − − ( ) ( ) − ( ) ( )       + + ∏ 1 1 1 1 1 1 1 1 ω ω ω k j k j k L j L k T T a a ,    + −     − − ( ) ( ) − ( ) ( ) − = + = ∏ + , , 1 1 1 1 1 1 1 1 j k U j U k j T T a a j k ω ω 1 1 1 1 1 1 1 k U j U k j k L j T T I a a a j k ∏ ∏ − ( ) ( ) − ( ) ( ) ( ) ( )     + = + + ω ω ω , j j j j k U j j k L j j k U I F F a a a , , , = + = + = + ∏ ∏ ∏ ( ) ( ) ( ) ( )     1 1 1 1 1 1 ω ω j a a j j k j j j k j j j k S S S ( ) ( ) ∑ ∑     = = + = + = + ( ) ( ) ω ω θ ω ρ 1 1 1 1 1 1 , , ∑ ∑ − − ( ) ( )     ( ) ( ) = + = + ∏ ω µ ω ν ω j j j k j j j a a j k L j S T a , , 1 1 1 1 1 1 , , , 1 1 1 1 1 1 1 1 − − ( ) ( )     ( ) ( ) = + = + = + ∏ ∏ ∏ j k U j j k L j j k T I I a a j j ω ω U j j k L j j k U j a a a F F j j j ( ) ( ) ( ) ( ) ( ) ( )       = + = + ∏ ∏ ω ω ω , , 1 1 1 1    INTrLWAA INTrLWAA a a a a S S k k a a j k j j k k j k 1 2 1 1 1 1 1 , ,..., , , + ( )+ ( ) ( ) = ∑ ∑ = + + = ω θ ω θ ω ρ ω ρ ω µ ω µ ω j j k k j k j j k k j k a a a a S S ( )+ ( ) ( )+ ( ) + + = + + = ∑ ∑ 1 1 1 1 1 1 , , j j k k j a a j k L j L k T T a a ν ω ν ω ( )+ ( ) = + +     + − − − ( ) ( ) − ∏ 1 1 1 1 1 1 1 , + = + ( ) ( ) − − ( ) ( ) − ( ) ( )       + + ∏ 1 1 1 1 1 1 1 1 ω ω ω k j k j k L j L k T T a a ,    + −     − − ( ) ( ) − ( ) ( ) − = + = ∏ + , , 1 1 1 1 1 1 1 1 j k U j U k j T T a a j k ω ω 1 1 1 1 1 1 1 k U j U k j k L j T T I a a a j k ∏ ∏ − ( ) ( ) − ( ) ( ) ( ) ( )     + = + + ω ω ω , j j j j k U j j k L j j k U I F F a a a , , , = + = + = + ∏ ∏ ∏ ( ) ( ) ( ) ( )     1 1 1 1 1 1 ω ω j a a j j k j j j k j j j k S S S ( ) ( ) ∑ ∑     = = + = + = + ( ) ( ) ω ω θ ω ρ 1 1 1 1 1 1 , , ∑ ∑ − − ( ) ( )     ( ) ( ) = + = + ∏ ω µ ω ν ω j j j k j j j a a j k L j S T a , , 1 1 1 1 1 1 , , , 1 1 1 1 1 1 1 1 − − ( ) ( )     ( ) ( ) = + = + = + ∏ ∏ ∏ j k U j j k L j j k T I I a a j j ω ω U j j k L j j k U j a a a F F j j j ( ) ( ) ( ) ( ) ( ) ( )       = + = + ∏ ∏ ω ω ω , , 1 1 1 1    1. Since a a j = for j=1, 2,…,n. we have INTrLWAA Since a a a a min n = ( ) min , ,..., 1 2 and a a a a max n = ( ) max , ,..., 1 2 for j=1,2,…,n, there is a a a min j max ≤ ≤ . Thus, there exist is j n j min j n j j j n j max a a a = = = ∑ ∑ ∑ ≤ ≤ 1 1 1 ω ω ω . This is a a a min j n j j max ≤ ≤ =∑ 1 ω . i.e., amin ≤INTrLWAA a a a a n max 1 2 , ,..., ( ) ≤ . 2. Since a a a a min n = ( ) min , ,..., 1 2 and a a a a max n = ( ) max , ,..., 1 2 for j=1,2,…,n, there is a a a min j max ≤ ≤ . Thus, there exist is j n j min j n j j j n j max a a a = = = ∑ ∑ ∑ ≤ ≤ 1 1 1 ω ω ω . This is a a a min j n j j max ≤ ≤ =∑ 1 ω . i.e., amin ≤INTrLWAA a a a a n max 1 2 , ,..., ( ) ≤ . 3. Since a a j j ≤ * for j= 1, 2,…, n. There is j n j j j n j j a a = = ∑ ∑ ≤ 1 1 ω ω * Then INTRLWAA a a an 1 2 , ,..., ( ) ≤INTrLWAA a a an 1 2 * * * , ,..., ( ). 3. Since a a j j ≤ * for j= 1, 2,…, n. There is j n j j j n j j a a = = ∑ ∑ ≤ 1 1 ω ω * Then INTRLWAA a a an 1 2 , ,..., ( ) ≤INTrLWAA a a an 1 2 * * * , ,..., ( ). Thus, we complete the proofs of these properties Thus, we complete the proofs of these properties Thus, we complete the proofs of these properties INTrLWAA Therefore, considering the above results, we have Equation (5) for any. This completes the proof. T Especially when ω= 1 1 1 n n n T , , , …     , then INTrLWAA operator reduces to an interval neutro- Especially when ω= n n n , , , …     , then INTrLWAA operator reduces to an interval neutro- sophic trapezoid linguistic arithmetic averaging operator for INTrLvs. n n n   sophic trapezoid linguistic arithmetic averaging operator for INTrLvs. sophic trapezoid linguistic arithmetic averaging operator for INTrLvs. zoid linguistic arithmetic averaging operator for INTrLvs. It is obvious that the INTrLWAA operator satisfies the following properties bvious that the INTrLWAA operator satisfies the following properties: It is obvious that the INTrLWAA operator satisfies the following properties: 1. Idempotency: Let a j (j=1, 2,…, n) be a collection of INTrLvs. If a j (j=1, 2,…,n) is equal, i.e a a j = for j=1,2,…,n, then INTrLWAA a a a a n 1 2 , ,..., . ( ) = 2. Boundedness: Let a j (j=1, 2,…, n) be a collection of INTrLvs and a a a a min n = ( ) min , ,..., 1 2 and a a a a max n = ( ) max , ,..., 1 2 for j=1,2,…,n, amin ≤INTrLWAA a a a a n max 1 2 , ,..., ( ) ≤ then be a collection of INTrLvs. 3. Monotonity: Let a j (j=1, 2,…, n) be a collection of INTrLvs. If a a j j ≤ * for j= 1,2,…,n. Then INTrLWAA a a an 1 2 , ,..., ( )≤INTrLWAA a a an 1 2 * * * , ,..., ( ). Proof: 1. Since a a j = for j=1, 2,…,n. we have 1. Since a a j = for j=1, 2,…,n. INTrLWAA we have 356 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators INTrLWAA a a a s s s n a a j n j j j n j j j n j 1 2 1 1 1 , ,..., , , ( ) = ∑ ∑ ∑ = = = ( ) ( ) ω θ ω ρ ω µ a a j n L j j j n j j j s T a ( ) ( ) = =∑ − − ( ) ( )       ∏ , , , 1 1 1 1 ω ν ω 1 1 1 1 −   − ( ) ( ) = = ∏ ∏ j n U j j n L T I a a j ω , j j n U j j j I a ( ) ( ) ( ) ( )     =∏ ω ω , , 1 j n L j j n U j a F F s s a a j j j n j = = ( ) ∏ ∏ ( ) ( ) ( ) ( ) = ∑     = 1 1 1 ω ω θ ω ρ , , a a a L j n j j n j j n j s s T a ( ) ( ) ( ) = = = ∑ ∑ ∑ − − ( )     1 1 1 1 1 ω µ ω ν ω , , , ( )∑ − ( ) ( )∑     ( ) ( )∑ − = = = j n j j n j j n T I a a u L 1 1 1 1 1 ω ω ω , , j j n j j n j j n j I F F a a a U L U , , , ( ) ( )∑ ( ) ( )∑ ( ) ( )∑      = = = 1 1 1 ω ω ω    =     ( ) ( ) ( ) ( ) s s s s T T I a a a a a a L U θ ρ µ ν , , , , , , a a a a L U L U I F F a , , ,     ( ) = MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 2. Definition 4.3: Let Definition 4.3: Let a s s s s T T I j a L a U a a a a a j j j j j j =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , , , j j j j L a U a L a U I F F , , ,     ( ) (j=1,2,…,n) a s s s s T T I j a L a U a a a a a j j j j j j =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , , , j j j j L a U a L a U I F F , , ,     ( ) (j=1,2,…,n) be a collection of INTrLNs. The interval neutrosophic trapezoid linguistic weighted geometric averaging (INTrLWGA) operator can be defined as follows and INTrLWGA: Ωn→Ω be a collection of INTrLNs. The interval neutrosophic trapezoid linguistic weighted geometric averaging (INTrLWGA) operator can be defined as follows and INTrLWGA: Ωn→Ω be a collection of INTrLNs. The interval neutrosophic trapezoid linguistic weighted geom be a collection of INTrLNs. The interval neutrosophic trapezoid linguistic w (INTrLWGA) operator can be defined as follows and INTrLWGA: Ωn→Ω (INTrLWGA) operator can be defined as follows and INTrLWGA: Ωn→Ω INTrLWGA a a a a n j n j j 1 2 1 , ,... ( ) = =∏ ω (8) 357 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators where, ωj= (ω1,ω2,…,ωn)T is the weight vector of a j (j= 1,2,…,n), ωj∈ [0,1]and j n j =∑ 1 ω =1. Theorem 4.4: Let a s s s s T T j L U a a a a a a j j j j j j =     ( ) ( ) ( ) ( ) θ ρ µ ν , , , , , , I I F F a a a a L U L U j j j j , , ,     ( ) (j=1,2,…,n) be a collection of INTrLs, Then by Equation (8) and the operational laws in Definition 3.3, we have the following result be a collection of INTrLs, Then by Equation (8) and the operational laws in Definition 3.3, we have the following result INTrLWGA G a a a s s s n a a j n j j j n j j j n j 1 2 1 1 1 , ,..., , , ( ) = ∏ ∏ ∏ = = = ( ) ( ) θ ρ µ ω ω ω a a j n L j j n U j j j n j j j s T T a a ( ) ( ) = = =∏ ( ) ( )     ∏ ∏ , , , 1 1 1 ν ω ω ω j j j n L j I a     − − − ( ) ( ) =∏ , , 1 1 1 1 ω j n U j j n I a j = = ∏ ∏ − ( ) ( )     − 1 1 1 1 ω , 1 1 1 1 − ( ) ( ) − ( ) ( )     − =∏ F F a a L j j n U j j j ω ω , (9) n (9) where, ω= (ω1,ω2,…,ωn)T is the weight vector of a j (j= 1, 2,…,n), ωj∈ [0,1]and j n j =∑ 1 ω =1. By a similar proof manner of theorem 4 2 we can also give the proof of theorem 4 4 (omitte where, ω= (ω1,ω2,…,ωn)T is the weight vector of a j (j= 1, 2,…,n), ωj∈ [0,1]and j n j =∑ 1 ω =1. j=1 By a similar proof manner of theorem 4.2, we can also give the proof of theorem 4.4 (omitted) T j By a similar proof manner of theorem 4.2, we can also give the proof of theorem 4.4 (om Especially when ω= 1 1 1 n n n T , , , …    , then INTrLWGA operator reduces to an interval neutrosoph- i t id li i ti t i i t f INT L ic trapezoid linguistic geometric averaging operator for INTrLvs. c trapezoid linguistic geometric averaging operator for INTrLvs. It is obvious that the INTrLWGA operator satisfies the following properties: It is obvious that the INTrLWGA operator satisfies the following properties: 1. Idempotency: Let a j (j=1, 2,…,n) be a collection of INTrLvs. INTrLWGA If a j (j=1, 2,…, n) is equal, i.e a a j = for j=1, 2,…,n, then INTrLWGA a a a a n 1 2 , ,..., ( ) = . 1. Idempotency: Let a j (j=1, 2,…,n) be a collection of INTrLvs. If a j (j=1, 2,…, n) is equal, i.e a a j = for j=1, 2,…,n, then INTrLWGA a a a a n 1 2 , ,..., ( ) = . 2. Boundedness: Let a j (j=1, 2,…,n) be a collection of INTrLvs and a a a a min n = ( ) min , ,..., 1 2 and a a a a max n = ( ) max , ,..., 1 2 for j=1, 2,…,n, amin ≤INTrLWGA a a a a n max 1 2 , ,..., ( ) ≤ then be a collection of INTrLvs. 3. Monotonity: Let a j (j=1, 2,…,n) be a collection of INTrLvs. If a a j j ≤ * for j= 1,2,…,n. Then INTrLWGA a a an 1 2 , ,..., ( )≤INTrLWGA a a an 1 2 * * * , ,..., ( ). Since the proof process of these properties is similar to the above proofs, we do not repeat it here. Since the proof process of these properties is similar to the above proofs, we do not repeat it here. 358 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 5. DECISION MAKING METHOD BY INTRLWAA AND INTRLWGA OPERATORS. This section presents a method for multi attribute decision making problems based on the INTrLWAA and INTrLWGA operators ant the score, accuracy, and certainty functions of INTrLvs under interval neutrosophic trapezoid linguistic variable environment. In a multiple attribute decision-making problem, assume that A={A1, A2, A3,…,Am} is a setoff alter- natives and C ={C1, C2,…,Cn} is a set of attributes. The weight vector of the attributes Cj (j=1, 2,…,n), entered by the decision maker, is ω= (ω1,ω2,…,ωn)T where ωj∈ [0,1]and j n j É =∑ 1 =1. In the decision process, the evaluation information of the alternatives Ai (i=1, 2,…,m) with respect to the attribute Cj (j=1, 2,…,n) is represented by the form of an INTrLS: A s s s s T C I C F i C C C C A j A j A i j i j i j i j i i =   ( ) ( ) ( ) ( ) θ ρ µ ν , , , ( ), ( ), , i C C C j j ( ) \| ( ) ∈ where where s s s s s i j i j i j i j C C C C θ ρ µ ν ( ) ( ) ( ) ( )    ∈ , , , ˆ , T T T C x C A A L A U j j i i i ( ) ( ), ( ) [ . ] =  ⊆0 1 , I C I C I C A j A L j A U j i i i ( ) ( ), ( ) [ . ] =  ⊆0 1 , and F C F C F C A j A L j A U j i i i ( ) ( ), ( ) [ . ] =  ⊆0 1 with the condition with the condition with the condition with the condition ≤ T C I C F C A U j A U j A U j i i i ( ) ( ) ( ) + + ≤3 0≤ T C I C F C A U j A U j A U j i i i ( ) ( ) ( ) + + ≤3 for j=1,2,..,n and i=1,2,…,m. For convenience, an INTrLv is an INTrLS is denoted by for j=1,2,..,n and i=1,2,…,m. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators Step2: Calculate the score function E( di ) (i=1, 2,…, m) (accuracy function H( di ) and certainty func- tion C( di ) by applying Equation (1) (Equations (2) and (3)). Step 3: Rank the alternatives according to the values of E( di ) (H( di ) and C( di )) ((i=1,2,…,m) by the ranking method in Defintion 3.3, and then select the best one(s). St 4 E d Step 3: Rank the alternatives according to the values of E( di ) (H( di ) and C( di )) ((i=1,2,…,m) by the ranking method in Defintion 3.3, and then select the best one(s). Step4: End. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators d S S S S T T I I F F i i L i U i L i U i L i U i i i i =       θ ρ µ ν , , , , , , , , ,   ( ) ( ) = ∑ = =INTrLWAA d d d s s i i in j n j ij 1 2 1 , ,..., , ω θ j n j ij j n j ij j n j ij s s T j n ij L = = = ∑ ∑ ∑ − −     =∏ 1 1 1 1 1 1 ω ρ ω µ ω ν , , , ( ) − ( )     ( ) − = = = ∏ ∏ ∏ ω ω ω j j j j n ij U j n ij L j n T I I , , 1 1 1 1 1 ij U j n ij L j n ij U j j j F F ( ) ( ) ( )         = = ∏ ∏ ω ω ω , , 1 1 (10) d S S S S T T I I F F i i L i U i L i U i L i U i i i i =       θ ρ µ ν , , , , , , , , ,   ( ) ( ) =INTrLWAA d d d i i in 1 2 , ,..., = ∑ = s s j n j ij 1 , ω θ j n j ij j n j ij j n j ij s s T j n ij L = = = ∑ ∑ ∑ − −     =∏ 1 1 1 1 1 1 ω ρ ω µ ω ν , , , ( ) − ( )     ( ) − = = = ∏ ∏ ∏ ω ω ω j j j j n ij U j n ij L j n T I I , , 1 1 1 1 1 ij U j n ij L j n ij U j j j F F ( ) ( ) ( )         = = ∏ ∏ ω ω ω , , 1 1 (10) or or or d S S S S T T I I F F i i L i U i L i U i L i U i i i i =       θ ρ µ ν , , , , , , , , ,   ( ) = ( ) = ∏ = = INTrLWGA d d d s s i i in j n ij j j 1 2 1 , ,..., , θ ω 1 1 1 1 1 n ij j j n ij j j n ij j j s s T j n ij L j ∏ ∏ ∏     = = = = ∏ ρ µ ν ω ω ω ω , , , , ( ) n ij U j n ij L j T I j j ∏ ∏          − − − ( ) = = ( ) , , ω ω 1 1 1 1 1 1 1 1 1 1 1 1 n ij U j n ij L j n ij U I F F j j j ∏ ∏ ∏ − ( )   − − ( ) − ( )    − = = ω ω ω , ,     (11) d S S S S T T I I F F i i L i U i L i U i L i U i i i i =       θ ρ µ ν , , , , , , , , ,   ( ) = ( ) INTrLWGA d d d i i in 1 2 , ,..., = ∏ = = s s j n ij j j 1 , θ ω 1 1 1 1 1 n ij j j n ij j j n ij j j s s T j n ij L j ∏ ∏ ∏     = = = = ∏ ρ µ ν ω ω ω ω , , , , ( ) n ij U j n ij L j T I j j ∏ ∏          − − − ( ) = = ( ) , , ω ω 1 1 1 1 1 1 1 1 1 1 1 1 n ij U j n ij L j n ij U I F F j j j ∏ ∏ ∏ − ( )   − − ( ) − ( )    − = = ω ω ω , ,     (11) (11) Step2: Calculate the score function E( di ) (i=1, 2,…, m) (accuracy function H( di ) and certainty func- tion C( di ) by applying Equation (1) (Equations (2) and (3)). 5. DECISION MAKING METHOD BY INTRLWAA AND INTRLWGA OPERATORS. For convenience, an INTrLv is an INTrLS is denoted by d S S S S T T I I ij ij L ij U ij L ij U ij ij ij ij =       θ ρ µ ν , , , , , , , , , F F ij L ij U   ( ) (i=1=1,2,..m) j=1,2,…,n) thus, one can establish an interval neutrosophic trapezoid linguistic decision matrix D= dij ( ) × m n . The decision steps are described as follows thus, one can establish an interval neutrosophic trapezoid linguistic decision matrix D= dij ( ) × m n . The decision steps are described as follows thus, one can establish an interval neutrosophic trapezoid linguistic decision matrix D= dij ( ) × m n . ( The decision steps are described as follows Step1: Calculate the individual overall value of the INTrLv d j for Ai (i=1,2,…,m) by the following aggregation formula: Step1: Calculate the individual overall value of the INTrLv d j for Ai (i=1,2,…,m) by the following aggregation formula: 359 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 35 375,0.5275 , 0.1000, 0.2603 , 0.1933, 0.3565 , , , , [0.5216, 0.6565 , 0.000, 0.1569 , 0.1189, 0.2213 s s s d s = [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] 1.275 2.645 4.195 5.315 1.305 2.445 3.015 5.320 1.745 2.900 3.875 5.4 0 3 9 1 2 , , , , [0.3268, 0.4590 , 0.1275, 0.2305 , 0.3325, 0.4704 , , , , [0.5271, 0.7000 , 0.1320, 0.2759 , 0.1516, 0.3519 , , , , [0.4 s s s s s s d d s s s s d s s = = = ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) 1.430 2.530 4.3 5 5 4 6 5. 35 375,0.5275 , 0.1000, 0.2603 , 0.1933, 0.3565 , , , , [0.5216, 0.6565 , 0.000, 0.1569 , 0.1189, 0.2213 s s s d s = [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] 1.275 2.645 4.195 5.315 1.305 2.445 3.015 5.320 1.745 2.900 3.875 5.4 0 3 9 1 2 , , , , [0.3268, 0.4590 , 0.1275, 0.2305 , 0.3325, 0.4704 , , , , [0.5271, 0.7000 , 0.1320, 0.2759 , 0.1516, 0.3519 , , , , [0.4 s s s s s s d d s s s s d s s = = = ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) 1.430 2.530 4.3 5 5 4 6 5. 35 375,0.5275 , 0.1000, 0.2603 , 0.1933, 0.3565 , , , , [0.5216, 0.6565 , 0.000, 0.1569 , 0.1189, 0.2213 s s s d s = Step2: By applying Equation (1), we can obtain the score value of E( di ) (i=1,2,3,4) Step2: By applying Equation (1), we can obtain the score value of E( di ) (i=1,2,3,4) E d s E d s E d s E d s ( ) , ( ) , ( ) , ( ) . . . . MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators The evaluation information of an alternative Ai (i=1,2,3,4) with respect to an attribute Cj (j=1,2,3) can be given by the expert. For example, the INTrL value of an alternative A1 with respect to an attribute C1 is given as <[s1.4, s2.7, s3, s5.3], ([0.4, 0.5], [0.2, 0.3], [0.3, 0.4])> by the expert, which indicates that the mark of the alternative A1 with respect to the attribute C1 is about the trapezoid fuzzy linguistic value [s1.4, s2.7, s3, s5.3] with the satisfaction degree interval [0.4, 0.5], indeterminacy degree interval [0.2, 0.3], and dissatisfaction degree interval [0.3, 0.4]. similarly, the four possible alternatives with respect to the three attributes can be evaluated by the expert, thus we can obtain the following interval neutrosophic trapezoid linguistic decision matrix: ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] m n 1.8 3.4 4.5 5.5 1.3 2.3 4.4 5.4 0.8 2.2 3.8 5.1 1.4 2.8 3.8 5.1 , , , , [0.4,0.5 , 0.2,0.3 , 0.3, 0.4 , , , , [0.4, 0.6 , 0.1, 0.2 , 0.2, 0.4 , , , , [0.2, 0.3 , 0.1, 0.2 , 0.5, 0.6 , , , , [0.5, 0.7 , 0.1, 0.2 , 0 ij d D s s s s s s s s s s s s s s s s × = [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] 1.5 2.5 4.5 5.5 1.1 2.1 1.4 5.4 2.1 3.2 4.5 5.7 1.8 2.8 4.4 5.5 .2, 0.3 , , , , [0.6, 0.7 , 0.1, 0.2 , 0.2, 0.3 , , , , [0.5, 0.7 , 0.2, 0.2 , 0.1, 0.2 , , , , [0.3, 0.5 , 0.1, 0.2 , 0.3, 0.4 , , , , [0.5, 0.6 , 0.1, 0. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators s s s s s s s s s s s s s s s s [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] ( ] 1.4 2.7 3 5.3 1.5 3.1 4.7 5.9 1.7 2.9 4 5.4 1.2 1.8 4.3 5.3 3 , 0.3, 0.4 , , , , [0.5, 0.6 , 0.1, 0.3 , 0.1, 0.3 , , , , [0.7, 0.8 , 0.0, 0.1 , 0.1, 0.2 , , , , [0.5, 0.7 , 0.1, 0.2 , 0.2, 0.3 , , , , [0.3, 0.4 , 0.1, 0. s s s s s s s s s s s s s s s s [ ] [ ] ( ) 2 , 0.1, 0.2                           The proposed decision making method can handle this decision making problem according to the following calculation steps: Step1: By applying Equation (10), we can obtain the individual overall value of the INTrLV di for Ai (i=1,2,.3,4) Step1: By applying Equation (10), we can obtain the individual overall value of the INTrLV di for Ai (i=1,2,.3,4) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] 1.275 2.645 4.195 5.315 1.305 2.445 3.015 5.320 1.745 2.900 3.875 5.4 0 3 9 1 2 , , , , [0.3268, 0.4590 , 0.1275, 0.2305 , 0.3325, 0.4704 , , , , [0.5271, 0.7000 , 0.1320, 0.2759 , 0.1516, 0.3519 , , , , [0.4 s s s s s s d d s s s s d s s = = = ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) 1.430 2.530 4.3 5 5 4 6 5. 6. ILLUSTRATIVE EXAMPLE An illustrative example about investment alternatives problem adapted from (Ye, 2015) is used to dem- onstrate the applications of the proposed decision making method under interval neutrosophic trapezoid linguistic environment. There is an investment company, which wants to invest a sum of money in the best option. To invest the money, there is a panel with four possible alternatives: (1) A1 is car company; (2) A2 is food company; (3) A3 is a computer company; (4) A4 is an arms company. The investement company must take a decision according to the three attributes: (1) C1 is the risk; (2) C2 is the growth; (3) C3 is a the environmental impact. The weight vector of the attributes is ω= (0.35, 0.25, 0.4)T.The expert evaluates the four possible alternatives of Ai (i=1, 2, 3, 4) with respect to the three attributes of Cj (i=1,2,3), where the evaluation information is expressed by the form of INTrLV values under the linguistic term set S={s0=extremely poor, s1=very poor, s2= poor, s3= medium, s4= good, s5= very good, s6= extremely good}. 360 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 1 1 937 2 2 207 3 2 332 4 2 556 = = = = Step 3: Since E d E d E d E d ( ) ( ) ( ) ( ) 4 3 2 1 > > > , the ranking order of four alternatives. Therefore, we can see that the alternative A4 is the best choice among all the alternative. Step 3: Since E d E d E d E d ( ) ( ) ( ) ( ) 4 3 2 1 > > > , the ranking order of four alternatives. Therefore, we can see that the alternative A4 is the best choice among all the alternative. Obviously, we can see that the above two kinds of ranking orders of the alternatives are the same and the most desirable choice is the alternative A3. The INTrLS is a further generalization of interval neutrosophic linguistic set and interval neutrosophic uncertain linguistic set proposed by (Ye, 2015; Ye, 2013 g). So the decision –making method proposed in this paper is more typical in applications. Furthermore, the decision making approach proposed in this paper can be used to solve not only the interval neutrosophic linguistic information and interval neutro- sophic uncertain linguistic information but also decision making problems with interval neutrosophic triangular and trapezoidal linguistic information. Therefore, the decision making method proposed in the paper is a generalization of existing decision method with interval neutrosophic linguistic information and interval neutrosophic uncertain linguistic information. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 1 2 028 2 2 173 3 2 390 4 2 703 = = = = Step 3: Since E d E d E d E d ( ) ( ) ( ) ( ) 4 3 2 1 > > > , the ranking order of four alternatives. Therefore, we can see that the alternative A4 is the best choice among all the alternative. Step 3: Since E d E d E d E d ( ) ( ) ( ) ( ) 4 3 2 1 > > > , the ranking order of four alternatives. Therefore, we can see that the alternative A4 is the best choice among all the alternative. 361 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators On the other hand, we can also utilize the INTrLWGA operator as the following computational steps: Step1: By applying Equation (11), we can obtain the individual overall value of the INTrLV di for Ai (i=1,2,.3,4) On the other hand, we can also utilize the INTrLWGA operator as the following computational steps: Step1: By applying Equation (11), we can obtain the individual overall value of the INTrLV di for Ai (i=1,2,.3,4) [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] 1.200 2.591 4.182 5.312 1.293 2.426 2.659 5.317 1.718 2.892 3.805 5.4 7 3 8 1 2 , , , , [0.3031, 0.4266 , 0.1363, 0.2365 , 0.3674, 0.4898 , , , , [0.5233, 0.7000 , 0.1414, 0.1635 , 0.1614, 0.2279 , , , , [0.4 s s s s s s d d s s s s d s s = = = ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) 1.416 2.453 4.3 6 5 4 5 5. MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators 28 181,0.5629 , 0.1000, 0.2665 , 0.2260, 0.3618 , , , , [0.4585, 0.5864 , 0.0662, 0.1663 , 0.1261, 0.2263 s s s d s = [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] 1.200 2.591 4.182 5.312 1.293 2.426 2.659 5.317 1.718 2.892 3.805 5.4 7 3 8 1 2 , , , , [0.3031, 0.4266 , 0.1363, 0.2365 , 0.3674, 0.4898 , , , , [0.5233, 0.7000 , 0.1414, 0.1635 , 0.1614, 0.2279 , , , , [0.4 s s s s s s d d s s s s d s s = = = ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) 1.416 2.453 4.3 6 5 4 5 5. 28 181,0.5629 , 0.1000, 0.2665 , 0.2260, 0.3618 , , , , [0.4585, 0.5864 , 0.0662, 0.1663 , 0.1261, 0.2263 s s s d s = [ ] ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) [ ] 1.200 2.591 4.182 5.312 1.293 2.426 2.659 5.317 1.718 2.892 3.805 5.4 7 3 8 1 2 , , , , [0.3031, 0.4266 , 0.1363, 0.2365 , 0.3674, 0.4898 , , , , [0.5233, 0.7000 , 0.1414, 0.1635 , 0.1614, 0.2279 , , , , [0.4 s s s s s s d d s s s s d s s = = = ( ] [ ] [ ] ( ) [ ] ( ] [ ] [ ] ( ) 1.416 2.453 4.3 6 5 4 5 5. 28 181,0.5629 , 0.1000, 0.2665 , 0.2260, 0.3618 , , , , [0.4585, 0.5864 , 0.0662, 0.1663 , 0.1261, 0.2263 s s s d s = Step2: By applying Equation (1), we can obtain the score value of E( di ) (i=1,2,3,4) Step2: By applying Equation (1), we can obtain the score value of E( di ) (i=1,2,3,4) E d s E d s E d s E d s ( ) , ( ) , ( ) , ( ) . . . . 7. CONCLUSION In this paper, we have proposed some interval neutrosophic trapezoidal linguistic operators such as interval neutrosophic trapezoid linguistic weighted arithmetic averaging INTrLWAA and interval neutrosophic trapezoid fuzzy linguistic weighted geometric averaging INTrLWGA. We have studied some desirable properties of the proposed operators, such as commutativity, idempotency and monotonicity, and applied the INTrLWAA and INTrLWGA operator to decision making with interval neutrosophic trapezoidal linguistic information. Finally, an illustrative example has been given to show the developed operators. 362 MADM Based on Interval Neutrosophic Trapezoid Linguistic Aggregation Operators REFERENCES Aggarwal, S., Biswas, R. & Ansari, A. Q. (2010). Neutrosophic Modeling and Control. Computer and Communication Technology, 718-723. Ansaria, A. Q., Biswas, R., & Aggarwal, S. (2013). Neutrosophic classifier: An extension of fuzzy clas- sifer. 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https://openalex.org/W2597804221	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0175166&type=printable	English	null	High prevalence of curable sexually transmitted infections among pregnant women in a rural county hospital in Kilifi, Kenya	PloS one	2,017	cc-by	8,476	RESEARCH ARTICLE Abstract Citation: Masha SC, Wahome E, Vaneechoutte M, Cools P, Crucitti T, Sanders EJ (2017) High prevalence of curable sexually transmitted infections among pregnant women in a rural county hospital in Kilifi, Kenya. PLoS ONE 12(3): e0175166. https://doi.org/10.1371/journal. pone.0175166 Background Women attending antenatal care (ANC) in resource-limited countries are frequently screened for syphilis and HIV, but rarely for other sexually transmitted infections (STIs). We assessed the prevalence of curable STIs, defined as infection with either Chlamydia tracho- matis or Neisseria gonorrhoeae or Trichomonas vaginalis, from July to September 2015. Editor: David N. Fredricks, Fred Hutchinson Cancer Research Center, UNITED STATES Received: December 16, 2016 Accepted: March 21, 2017 Published: March 31, 2017 Copyright: © 2017 Masha 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. Methods In a cross-sectional study, women attending ANC at the Kilifi County Hospital, Kenya, had a urine sample tested for C. trachomatis/N. gonorrhoeae by GeneXpert® and a vaginal swab for T. vaginalis by culture. Bacterial vaginosis (BV) was defined as a Nugent score of 7–10 of the Gram stain of a vaginal smear in combination with self-reported vaginal discharge. Genital ulcers were observed during collection of vaginal swabs. All women responded to questions on socio-demographics and sexual health and clinical symptoms of STIs. Predic- tors for curable STIs were assessed in multivariable logistic regression. Copyright: © 2017 Masha 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: All relevant data are within the paper and its Supporting Information files. * schengo@kemri-wellcome.org Simon Chengo Masha1,2,3, Elizabeth Wahome1, Mario Vaneechoutte2, Piet Cools2, Tania Crucitti4, Eduard J. Sanders1,5 Simon Chengo Masha1,2,3, Elizabeth Wahome1, Mario Vaneechoutte2, Piet Cools2, Tania Crucitti4, Eduard J. Sanders1,5 1 Centre for Geographic Medicine Research–Coast, Kenya Medical Research Institute (KEMRI), Kilifi, Kenya, 2 Laboratory Bacteriology Research, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium, 3 Pwani University, Faculty of Pure and Applied Sciences, Department of Biological Sciences, Kilifi, Kenya, 4 HIV/STI Reference Laboratory, Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium, 5 Nuffield Department of Medicine, University of Oxford, Headington, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 High prevalence of curable sexually transmitted infections among pregnant women in a rural county hospital in Kilifi, Kenya Simon Chengo Masha1,2,3, Elizabeth Wahome1, Mario Vaneechoutte2, Piet Cools2, Tania Crucitti4, Eduard J. Sanders1,5 Conclusion Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. One in five women attending ANC had a curable STI. These infections were associated with genital ulcers, hygiene practices, early sexual debut and bacterial vaginosis. Curable sexually transmitted infections among pregnant women in Kilifi, Kenya 4.1, 95% CI:1.5–11.3), women who reported having sexual debut 17 years compared to women having sexual debut 18 years (AOR = 2.7, 95% CI:1.1–6.6), and BV-positive women (AOR = 2.7, 95% CI:1.1–6.6) compared to BV-negative women. 4.1, 95% CI:1.5–11.3), women who reported having sexual debut 17 years compared to women having sexual debut 18 years (AOR = 2.7, 95% CI:1.1–6.6), and BV-positive women (AOR = 2.7, 95% CI:1.1–6.6) compared to BV-negative women. Research-Kilifi is supported by core funding from the Wellcome Trust (#077092). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Sexually transmitted infections (STIs) are a major public health concern globally and especially among women in resource-limited countries [1]. Common curable STIs among women in sub-Saharan Africa (sSA) include Chlamydia trachomatis, Neisseria gonorrhoeae and Tricho- monas vaginalis [2]. Additionally, bacterial vaginosis (BV), which has been associated with an increased risk of incident infection with C. trachomatis and N. gonorrhoeae [3, 4], is highly prevalent [5]. These curable STIs and BV have been associated with a number of adverse preg- nancy outcomes such as spontaneous abortion, ectopic pregnancy, preterm delivery, low birth weight, stillbirth, postpartum sepsis, and congenital infection [6]. Infants born of mothers with genital C. trachomatis are at an increased risk of suffering from neonatal pneumonia and oph- thalmia neonatorum [7]. At present the efficacy of neonatal ocular prophylaxis with erythro- mycin for prevention of C. trachomatis ophthalmia is not clear [8]. These associated sequelae of STIs hence necessitate prevention and management of curable STIs and BV, especially dur- ing pregnancy. Pregnant women attending antenatal care (ANC) in resource-limited countries are fre- quently screened for syphilis [9] and HIV [10], but rarely for other curable STIs or BV. More- over, curable STIs and BV are associated with increased acquisition and transmission of HIV [11–13]. Among pregnant HIV positive women C. trachomatis is associated with increased risk of HIV mother-to-child transmission [14]. Many resource-limited countries have adopted the World Health Organization’s syndromic approach for the management of curable STI [15]. This syndromic approach is deficient in both sensitivity and specificity because STIs may be asymptomatic or have non-specific clinical signs and symptoms [16, 17]. There is a paucity of epidemiological and risk factor data on curable STIs among women residing in rural areas, including among pregnant women in coastal Kenya. Most of the recent information about the prevalence of curable STIs among women in Kenya has been from stud- ies done among high-risk individuals [18], women residing in cities [19, 20] or from regions with high incidence of HIV [13]. Consequently, we assessed the prevalence and predictors of curable STIs in pregnant women attending ANC at a rural county hospital in Coastal Kenya. Results A total of 42/202 (20.8%, 95% confidence interval (CI):15.4–27.0) women had a curable STI. The prevalence was 14.9% for C. trachomatis (95% CI:10.2–20.5), 1.0% for N. gonor- rhoeae (95% CI: 0.1–3.5), 7.4% for T. vaginalis (95% CI:4.2–12.0), 19.3% for BV (95% CI: 14.1–25.4) and 2.5% for genital ulcers (95% CI: 0.8–5.7). Predictors for infection with cur- able STIs included women with a genital ulcer (adjusted odds ratio (AOR) = 35.0, 95% CI: 2.7–461.6) compared to women without a genital ulcer, women who used water for cleaning after visiting the toilet compared to those who used toilet paper or other solid means (AOR = Funding: SCM is supported for his PhD by the Belgian Development Cooperation. SCM received Investigator initiated research funds from International AIDS Vaccine Initiative, who are funded by USAID. The Kenya Medical Research Institute-Wellcome Trust Research Programme (KWTRP) at the Centre for Geographical Medicine 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Methods This was a cross-sectional study conducted from July to September 2015, at the ANC clinic of the Kilifi County Hospital (KCH), Kenya, a clinic attended to by approximately 4,000 women annually [21]. We aimed to enroll a convenience sample of 350 consecutive pregnant women to identify 20–25 women with T. vaginalis for detailed molecular studies of T. vaginalis and assessment of the vaginal microbiome. Here, we report on a sub-study of 202 women who were tested for curable STIs, i.e. C. trachomatis, N. gonorrhoeae, T. vaginalis, and for BV. PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 2 / 16 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Procedures Recruitment: A nurse at the ANC clinic provided general information about the study to women who had completed their ANC visit. Women interested to know more about the study were invited to participate and up to nine consecutive women were enrolled per day. Inclusion criteria: Women were eligible if they met the following criteria: age 18–45 years, gestation 14 weeks, resident of the Kilifi Health and Demographic Surveillance area [22] and willing to undergo free STI and BV screening procedures. Study procedures: Consenting women underwent a short private interview with the nurse at the ANC clinic using a structured questionnaire capturing symptoms of vaginal infections. This was followed by a collection of vaginal swabs by the ANC nurse. During the collection of vaginal swabs, the nurse observed for abnormal vaginal discharge and genital ulcers. Three vaginal swabs were collected, by inserting one swab at a time approximately 2 inches into the vaginal opening and gently turning around twice ensuring rubbing the swab against the vagi- nal wall. The first swab was used for BV diagnosis and vaginal smears were prepared by rolling the swab onto a microscope glass slide. Slides were air-dried at the clinic before being transported to the laboratory for heat fixation followed by Gram staining and microscopy using the scoring system described by Nugent [23]. Women who self-reported discharge and had a Nugent score of 7–10 were considered BV-positive, and were treated as recommended [8]. Women with a Nugent score of 7–10 but without discharge were considered BV-negative. The second swab was used to diagnose T. vaginalis using a commercially available T. vagi- nalis InPouchTM system (BioMed Diagnostics, White City, Oregon, USA). The vaginal swab was inoculated in the upper-chamber of the InPouch at the clinic. The inoculated InPouch was transferred to the laboratory for direct microscopy of the upper chamber, after which the upper chamber was merged with the lower chamber and incubated at 37˚C. Daily microscopy was performed and samples with motile trichomonads within 5 days of culture were consid- ered positive for T. vaginalis. The third swab was placed in a sterile labeled 2 ml Eppendorf tube, the swab shaft was broken by bending the shaft against the neck of the Eppendorf tube. The bottom portion of the swab with the specimen was transported to the laboratory where it was immediately stored at -80˚C for molecular studies of the vaginal microbiome. PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Ethical considerations The study was approved by the KEMRI Scientific and Ethics Review Unit (#3022). All partici- pants provided written informed consent for study participation. Permission to use hospital records on syphilis and HIV status was obtained from Kilifi County Health department. Results From June to September 2015, a total of 373 women were invited to participate in the study. Twenty-three (6.2%) women declined to participate citing no interest in research, lacking time or having no permission from spouse/partner as the main reasons. Of the 350 women enrolled, 148 (42.3%) women did not have C. trachomatis and N. gonorrhoeae tests performed due the unavail- ability of the tests at the beginning of the study. Therefore, a total of 202 women were tested for C. trachomatis, N. gonorrhoeae, T. vaginalis and BV and were included in this sub-analysis (Fig 1). Characteristics of the study population The median age and gestation age including the interquartile range (IQR) for the participants was 26 (22–31) years and 24 (20–28) weeks respectively. The majority of the participants were married (93.6%) and Christian (72.3%). A quarter of the participants had secondary school education and above. The median age and IQR of sexual debut was 18 (16–20) years, although 20.8% of the participants were not sure or did not respond to this question. Forty-two percent of the participants had given birth before and the number of children ranged from 1–7. Sixty- two percent of the participants had previously experienced signs/symptoms associated with reproductive tract infection and one third had ever received syndromic treatment for genital signs or symptoms of infection. The socio-demographic characteristics of the participants are summarized in Table 1. Curable sexually transmitted infections among pregnant women in Kilifi, Kenya After clinical procedures were completed, all study participants were interviewed. The interviews were conducted in Kiswahili by a trained female fieldworker. The questionnaire covered sociodemographic, hygienic and sexual behavior questions. These included age, edu- cation level, marital status, number of children, occupation, consumption of alcohol, con- sumption of tobacco and other substances, age at first sex, number of sexual partners, history of STIs, and hygiene and sanitary practices. Data analysis Data were entered in the REDCapTM electronic data capture tool, version 6.5.0 (Vanderbilt University, Nashville, Tennessee). Prevalence of STIs was determined as the number of partici- pants with either C. trachomatis, N. gonorrhoeae and/or T. vaginalis out of 202 women that could be tested, expressed as a percentage. Exact 95% binomial confidence intervals (CIs) were calculated for prevalence estimates. Bivariable logistic regression was used to determine associ- ations of sociodemographic, hygienic, behavioral and clinical characteristics with curable STIs. Variables significant at p 0.1 in bivariable analysis were entered into a multivariable model to identify independent associations. Crude odds ratios (COR) and adjusted odds ratios (AOR) were calculated. These statistical analyses were done using STATA version 13.1 (Stata- Corp, College Station, Texas). Procedures No transport or freezing medium was added. The third swab was placed in a sterile labeled 2 ml Eppendorf tube, the swab shaft was broken by bending the shaft against the neck of the Eppendorf tube. The bottom portion of the swab with the specimen was transported to the laboratory where it was immediately stored at -80˚C for molecular studies of the vaginal microbiome. No transport or freezing medium was added. Women provided fresh first catch urine for C. trachomatis and N. gonorrhoeae testing, that was performed by the GeneXpert1 CT/NG Assay (Cepheid, Sunnyvale, California), according to the manufacturer’s instructions. the specimen was transported to the laboratory where it was immediately stored at -80˚C for molecular studies of the vaginal microbiome. No transport or freezing medium was added. Women provided fresh first catch urine for C. trachomatis and N. gonorrhoeae testing, that was performed by the GeneXpert1 CT/NG Assay (Cepheid, Sunnyvale, California), according to the manufacturer’s instructions. Women provided fresh first catch urine for C. trachomatis and N. gonorrhoeae testing, that was performed by the GeneXpert1 CT/NG Assay (Cepheid, Sunnyvale, California), according to the manufacturer’s instructions. Results for C. trachomatis, N. gonorrhoeae, for T. vaginalis direct microscopy and for BV were reported on the same day of the visit, and STI and BV treatment was offered according to Kenyan national guidelines [24], except for treatment of N. gonorrhoeae where ceftriaxone was used in place of quinolones [25]. Women who were negative on T. vaginalis direct microscopy but positive on culture were contacted to return to the ANC clinic for treatment immediately after the culture turned positive. Treatment was also offered for the partners of the women with curable STIs. Syphilis and HIV test results were obtained from each participant’s ANC records. Syphilis results were based on testing for treponemal antibodies only, done at the hospital laboratory using a commercial test kit Advanced Quality One Step Anti-TP (InTec products INC, Xia- men, China). Rapid HIV testing was done by the Alere Determine HIV-1/2 kit (Alere Medical Co Ltd, Chiba, Japan), the First response HIV 1-2-0 kit (Premier Medical Corporation Lim- ited, Daman, India) was used to confirm positive results, and the Uni-Gold kit (Trinity Bio- tech, Wicklow, Ireland) was used to resolve any discordant test results. 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Prevalence of curable STIs, BV, HIV and syphilis A total of 20.8% (42/202) (95% confidence interval (CI): 15.4–27.0) women had a curable STI and 19.4% (39/202) (95% CI: 14.2–25.6) women had BV i.e. Nugent score of 7–10 and reported PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 4 / 16 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Fig 1. Flow chart of women attending antenatal care clinic at Kilifi County Hospital invited to participate in the study, June- September 2015 https://doi.org/10.1371/journal.pone.0175166.g001 Fig 1. Flow chart of women attending antenatal care clinic at Kilifi County Hospital invited to participate in the study, June- September 2015 Fig 1. Flow chart of women attending antenatal care clinic at Kilifi County Hospital invited to participate in the study, June- September 2015 https://doi.org/10.1371/journal.pone.0175166.g001 https://doi.org/10.1371/journal.pone.0175166.g001 vaginal discharge. The prevalence was 14.9% for C. trachomatis (95% CI: 10.2–20.5), 1.0% for N. gonorrhoeae (95% CI: 0.1–3.5) and 7.4% for T. vaginalis (95% CI: 4.2–12.0) (Fig 1). Five (11.9%) women had more than one STI, while 13 (31%) out of the 42 women with a curable STI also had BV. Only two women (1.0%) had asymptomatic BV and none of them had a cur- able STI (data not shown). vaginal discharge. The prevalence was 14.9% for C. trachomatis (95% CI: 10.2–20.5), 1.0% for N. gonorrhoeae (95% CI: 0.1–3.5) and 7.4% for T. vaginalis (95% CI: 4.2–12.0) (Fig 1). Five (11.9%) women had more than one STI, while 13 (31%) out of the 42 women with a curable STI also had BV. Only two women (1.0%) had asymptomatic BV and none of them had a cur- able STI (data not shown). Thirteen women were HIV-positive, a prevalence of 6.4% (95% CI: 3.4–10.8), including four (2.0%; 95% CI: 0.5–5.0) women who were newly diagnosed. Of note, HIV prevalence in 82 women aged 18–24 vs. 120 women aged 25 years was 0% vs. 10.8% (p = 0.002). All nine HIV positive women who knew their status were on anti-retroviral therapy. Among the 13 women with HIV, three women had curable STIs, i.e. a prevalence of 23.1% (95% CI: 5.0– 53.8). All women with HIV and a curable STI also had BV. The prevalence of curable STIs, or BV in the 13 women with HIV was not statistically significantly different from that of the 189 women without HIV (data not shown). Two women had treponemal antibodies, a prevalence of 1.0% (95% CI: 0.1–3.6). Prevalence of curable STIs, BV, HIV and syphilis Prevalence of HIV, treponemal antibodies and T. vaginalis for the 148 women who were not tested for C. trachomatis and N. gonorrhoeae was not statistically significantly different than the prevalence of HIV, syphilis, and T. vaginalis in the 202 tested for C. trachomatis and N. gonorrhoeae in the sub-study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Clinical signs and symptoms The most commonly self-reported symptoms amongst all participants were vaginal discharge (73.8%) and lower abdominal pain (46.5%). However, during collection of specimen by the study nurse, only 17.3% of the participants had an abnormal discharge (defined as excess 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Table 1. Socio-demographic, hygienic, and behavioral characteristics of 202 women attending ante- natal care and tested for sexually transmitted infections and bacterial vaginosis in Kilifi County Hos- pital, Kenya, July-September 2015. natal care and tested for sexually transmitted infections and bacterial vaginosis in Kilifi County Hos- pital, Kenya, July-September 2015. Total n = 202 Positive for curable STIs n = 42 (%) Demographic characteristics Age group (Years) 18–24 82 24 (29.3) 25 120 18 (15.0) Religion Christian 146 28(19.2) Muslim 28 7 (25.0) Other/None 28 7 (25.0) Education None 35 7 (20.0) Primary 118 26 (22.0) Secondary/Tertiary 49 9 (18.4) Marital status Single 13 5 (38.5) Married 189 37 (19.6) Residency Living with partner 138 27 (19.6) Not living with the partner 64 15 (23.4) Employment status Employed/self-employed 115 20 (17.4) Unemployed 87 22 (25.3) Parity 0 58 15 (25.9) 1–2 69 17 (24.6) 3+ 75 10 (13.3) Gestational age (weeks)* 14–25 114 24 (21.1) 26 87 18 (20.7) Hygiene characteristics Toilet type Flushing toilet 75 15 (20.0) Pit latrine 105 22 (21.0) Bush/Other 22 5 (22.7) Mode of cleaning after visiting the toilet Tissue paper/other solid materials 64 7 (10.9) Water 138 35 (25.4) Vaginal washing when bathing ‡ External 32 6 (18.8) Internal 166 35 (21.1) Behavioral characteristics Sexual debut age (years) 17 64 21 (32.8) 18 96 14 (14.6) Don’t know/no response 42 7 (16.7) Number of lifetime sex partners 1 96 16 (16.7) (Continued) 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Table 1. (Continued) Table 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Clinical signs and symptoms (Continued) Total n = 202 Positive for curable STIs n = 42 (%) 2+ 106 26 (24.5) Partner had other sexual partners in the last 6 months No 182 38 (20.9) Yes 20 4 (20.0) Alcohol consumption ever No 160 35 (21.9) Yes 42 7 (16.7) Tobacco smoking/chewing ever No 191 41 (21.5) Yes 11 1 (9.1) Drugs/substance use ever No 194 39 (20.1) Yes 8 3 (37.5) Sexually transmitted infections/ reproductive tract infection HIV Negative 189 39 (20.6) Positive 13 3 (23.1) Syphilis*** Negative 198 42 (21.2) Positive 2 0 (0.0) Bacterial vaginosis* Negative 162 29 (17.9) Positive 39 13 (33.3) Clinical signs and symptoms of STI¥ Previous history of vaginal discharge No 75 13 (17.3) Yes 127 29 (22.8) Previous syndromic treatment of genital infection No 136 28 (20.6) Yes 66 14 (21.2) Current vaginal discharge (self-reported) No 52 10 (19.2) Yes 149 32 (21.5) Abnormal vaginal discharge foul smell/color (observed) No 165 32 (19.4) Yes 36 10 (27.8) Dysuria No 159 32 (20.1) Yes 42 10 (23.8) Dyspareunia No 150 29 (19.3) Yes 51 13 (25.5) Vaginal itching No 146 31 (21.2) Yes 55 11 (20.0) (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 7 / 16 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Table 1. (Continued) Total n = 202 Positive for curable STIs n = 42 (%) Lower abdominal pain No 107 24 (22.4) Yes 94 18 (19.1) Genital warts No 196 40 (20.4) Yes 5 2(40.0) Genital ulcer (observed) No 196 38 (19.4) Yes 5 4 (80.0) Vaginitis No 195 40 (20.5) Yes 6 2 (33.3) Curable STIs = Infection with any of the following sexually transmitted infectious agents: Chlamydia trachomatis, Neisseria gonorrheae and/or Trichomonas vaginalis ‡ Four participants did not respond to this question Bacterial vaginosis result for one participant was not available * Gestational age for one participant was missing *** Syphilis hospital records for two participants were unavailable. ¥Clinical signs and symptoms of STI records for one participant were missing. https://doi.org/10.1371/journal.pone.0175166.t001 Table 1. (Continued) Total n = 202 Positive for curable STIs n = 42 (%) Lower abdominal pain No 107 24 (22.4) Yes 94 18 (19.1) Genital warts No 196 40 (20.4) Yes 5 2(40.0) Genital ulcer (observed) No 196 38 (19.4) Yes 5 4 (80.0) Vaginitis No 195 40 (20.5) Yes 6 2 (33.3) Table 1. (Continued) Table 1. (Continued) discharge/foul smelling discharge/colored discharge) upon examination. Clinical signs and symptoms Five (2.5%) women had genital ulcers upon examination, of which 4 (9.6%) of the 42 women with a curable STI vs. 1 (0.6%) of the 159 women without a curable STI (p = 0.001). None of the women with genital ulcers had HIV or treponemal antibodies. Other self-reported clinical signs or symptoms were not associated with curable STIs. Based on symptoms routinely used in syndromic management of STIs (i.e. genital ulcer, lower abdominal pain or abnormal vaginal discharge), 45.2% of the 42 women with curable STIs were asymptomatic. Risk factors/predictors In bivariable analysis, curable STIs were more common among participants who were 24 years, had less than 3 children, had a sexual debut 17 years, used water to clean themselves after visiting the toilet, had a genital ulcer or were BV positive (Table 2). In multivariable analysis, independent predictors associated with curable STIs included women with a genital ulcer (AOR = 35.0, 95% CI: 2.7–461.6) compared to women without a genital ulcer, women who used water for cleaning after visiting the toilet compared to those who used toilet paper or other solid materials (AOR = 4.1, 95% CI: 1.5–11.3), women who reported having sexual debut 17 years compared to women having sexual debut 18 years (AOR = 2.7, 95% CI: 1.1–6.6), and BV-positive women (AOR = 2.7, 95% CI: 1.1–6.6) com- pared to BV-negative women (Table 2). https://doi.org/10.1371/journal.pone.0175166.t001 Discussion We found that one in five out of 202 pregnant women attending the ANC at the Kilifi County Hospital, Kenya, during the period July to September 2015 had at least one curable STI. The 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Table 2. Bivariable and multivariable analysis of socio-demographic, hygienic, behavioral, and clinical characteristics of pregnant women with curable sexually transmitted infections attending antenatal clinic at Kilifi County Hospital, July—September 2015 (n = 202). CurableSTIs n(%) Bivariable analysis Multivariable analysis Demographic characteristics COR (95% C.I) P- value AOR (95% C.I) P-value Age group (Years) 18–24 24 (29.3) 2.3 (1.2–4.7) 0.016 1.8 (0.7–4.4) 0.226 25 18 (15.0) Ref Religion Christian 28 (19.2) Ref Muslim 7 (25.0) 1.4 (0.5–3.6) 0.483 - - Other/None 7 (25.0) 1.4 (0.5–3.6) 0.483 - - Education None 7 (20.0) Ref Primary 26 (22.0) 1.1 (0.4–2.9) 0.797 - - Secondary/Tertiary 9 (18.4) 0.9 (0.3–2.7) 0.851 - - Marital status Single 5 (38.5) Ref Married 37 (19.6) 0.4 (0.1–1.3) 0.115 - - Residency Living with partner 27 (19.6) Ref Not living with partner 15 (23.4) 1.3 (0.6–2.6) 0.529 - - Employment status Employed/self-employed 20 (17.4) Ref Unemployed 22 (25.3) 1.6 (0.8–3.2) 0.173 - - Parity 0 15 (25.9) 2.3 (0.9–5.5) 0.071 2.7 (0.8–8.9) 0.111 1–2 17 (24.6) 2.1 (0.9–5.0) 0.087 1.9 (0.7–5.2) 0.237 3+ 10 (13.3) Ref Gestational age (weeks) 14–25 23 (20.5) Ref 26 18 (20.7) 1.0 (0.5–2.0) 0.979 - - Hygiene characteristics Toilet type Flushing toilet 15 (20.0) Ref Pit latrine 22 (21.0) 1.1 (0.5–2.2) 0.876 - - Bush/Other 5 (22.7) 1.2 (0.4–3.7) 0.781 - - Mode of cleaning after visiting the toilet Tissue paper/other solid material 7 (10.9) Ref Water 35 (25.4) 2.8 (1.2–6.6) 0.022 4.1 (1.5–11.3) 0.007 Vaginal washing when bathing* External 6 (18.8) Ref Internal 35 (21.1) 1.2 (0.4–3.0) 0.766 - - Behavioral characteristics Sexual debut age (years) 17 21 (32.8) 2.9 (1.3–6.2) 0.007 2.7 (1.1–6.6) 0.026 18 14 (14.6) Ref Don’t know/no response 7 (16.7) 1.2 (0.4–3.2) 0.754 - - Number of lifetime sex partners 1 16 (16.7) Ref (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 9 / 16 one.0175166 March 31, 2017 10 / 16 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Table 2. (Continued) CurableSTIs n(%) Bivariable analysis Multivariable analysis Demographic characteristics COR (95% C.I) P- value AOR (95% C.I) P-value Genital ulcer (observed) No 38 (19.4) Ref Yes 4 (80.0) 16.6 (1.8–153.1) 0.013 35.0 (2.7–461.6) 0.031 Vaginitis No 40 (20.5) Ref Yes 2 (33.3) 1.9 (0.3–11.0) 0.454 - - Curable STI = Infection with any of the following sexually transmitted infectious agents: Chlamydia trachomatis, Neisseria gonorrheae, Trichomonas vaginalis Table 2. (Continued) CurableSTIs n(%) Bivariable analysis Multivariable analysis Demographic characteristics COR (95% C.I) P- value AOR (95% C.I) P-value Genital ulcer (observed) No 38 (19.4) Ref Yes 4 (80.0) 16.6 (1.8–153.1) 0.013 35.0 (2.7–461.6) 0.031 Vaginitis No 40 (20.5) Ref Yes 2 (33.3) 1.9 (0.3–11.0) 0.454 - - Curable STI = Infection with any of the following sexually transmitted infectious agents: Chlamydia trachomatis, Neisseria gonorrheae, Trichomonas vaginalis AOR: Adjusted Odds Ratio CI: Conﬁdence interval COR: Crude Odds Ratio *One participant did not respond to this question https://doi.org/10.1371/journal.pone.0175166.t002 Table 2. (Continued) owing sexually transmitted infectious agents: Chlamydia trachomatis, Neisseria gonorrheae, Trichomonas high burden of curable STIs was mostly due to cases of C. trachomatis (14.9% of women) and T. vaginalis (7.4% of women). We also found approximately 19.4% of the women to have BV, defined as the presence of vaginal discharge in combination with a Nugent score 7. Four out of the 5 women with genital ulcers also had a curable STI. Our study documented that having genital ulcers, cleaning with water as opposed to use of toilet paper after visiting the toilet, early sexual debut, and BV were strongly associated with the presence of curable STIs. The high prevalence (14.9%) of C. trachomatis found in this study is comparable to a preva- lence of 13% (95% CI: 8.6–16.8) among women seeking family planning services in Nairobi, Kenya [20] and 9.9% (95% CI: 7.2–13.2) among women in a multi-country (Kenya, Rwanda and South Africa) cross-sectional study [26]. Two other recent studies reported lower preva- lence rates of C. trachomatis: 5.6% (95% CI: 4.4–7.0) among 1300 HIV-seronegative ANC- attending women at two district hospitals in rural western Kenya [13], and 6.0% (95% CI: 3.6– 9.3) in another study among 300 women attending outpatient clinics in Nairobi [27]. Since the high prevalence of C. trachomatis among pregnant women may likely reflect ongoing trans- mission of C. Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Table 2. (Continued) CurableSTIs n(%) Bivariable analysis Multivariable analysis Demographic characteristics COR (95% C.I) P- value AOR (95% C.I) P-value 2 26 (24.5) 1.6 (0.8–3.3) 0.171 - - Partner has had other sexual partner(s) in the last 6 months No 38 (20.9) 1.1 (0.3–3.3) 0.927 - - Yes 4 (20.0) Ref Alcohol consumption ever No 35 (21.9) 1.4 (0.6–3.4) 0.461 - - Yes 7 (16.7) Ref Tobacco smoking/chewing ever No 41 (21.5) 2.7 (0.3–22.0) 0.344 - - Yes 1 (9.1) Ref Drugs/Substance use ever No 39 (20.1) 0.4 (0.1–1.8) 0.248 - - Yes 3 (37.5) Ref Clinical characteristics HIV status Negative 39 (20.6) Ref Positive 3 (23.1) 1.2 (0.3–4.4) 0.834 - - Bacterial vaginosis Negative 29 (17.9) Ref Positive 13 (33.3) 2.3 (1.1–5.0) 0.036 2.7 (1.1–6.6) 0.031 Clinical signs and symptoms of STI Previous history of vaginal discharge No 13 (17.3) Ref Yes 29 (22.8) 1.4(0.7–2.9) 0.353 - - Previous syndromic treatment of genital infection No 28 (20.5) Ref Yes 14 (21.2) 1.0 (0.5–2.1) 0.918 - - Current vaginal discharge (self-reported) No 10 (19.2) Ref Yes 32 (21.5) 1.1 (0.5–2.5) 0.732 - - Abnormal vaginal discharge (observed) No 32 (19.4) Ref Yes 10 (27.8) 1.6 (0.7–3.6) 0.265 - - Dysuria No 32 (20.1) Ref Yes 10 (23.8) 1.2 (0.6–2.8) 0.602 - - Dyspareunia No 29 (19.3) Ref Yes 13 (25.5) 1.4 (0.7–3.0) 0.352 - - Vaginal itching No 31 (21.2) Ref Yes 11 (20.0) 0.9 (0.4–2.0) 0.848 - - Lower abdominal pain No 24 (22.4) Ref Yes 18 (19.1) 0.8 (0.4–1.6) 0.568 - - Genital warts No 40 (20.4) Ref Yes 2 (40.0) 2.6 (0.4–16.1) 0.304 - - (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 10 / 16 Table 2. (Continued) 10 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya A relatively low prevalence of N. gonorrhoeae (1%) and treponemal antibodies (1%) were found in our study. The prevalence of treponemal antibodies was similar to the study among HIV-seronegative ANC-attending women in rural western Kenya, [13] while the N. gonor- rhoeae prevalence in the latter study was 3.0%. BV diagnosis in our study was based on an a priori self-report of vaginal discharge in combi- nation with a Nugent score of 7 (while only 2 (1%) women had asymptomatic BV). Women with BV had an almost 3-fold higher risk of infection with a curable STIs than women without BV, comparable to other studies [4, 33]. It might be advisable to better document the presence of BV. However, general screening for BV among all women attending ANC, using the Nugent score [23] or Amsel’s criteria [34], would require significant commitment of resources and staff. Using simple point of care testing, Madhivanan et al. [35] demonstrated that a vaginal pH >4.5 and a positive whiff test had a sensitivity of 83% but a specificity of only 47% for diagnosing BV. Although, not specific this approach may be adopted for BV screening in resource poor settings. Alternatively, presumptive treatment with intravaginal metronidazole (750 mg) plus micona- zole (200 mg) for five consecutive nights each month for 12 months was shown to be effective to improve vaginal health and to decrease susceptibility to bacterial STIs, in a recent trial among non-pregnant women [36], but the frequency of treatment would make it challenging to upscale this intervention. Our findings reinforce the need to integrate STI services at ANC. Screening for T. vaginalis can be achieved at a relatively affordable cost (US $ 5), making screening for T. vaginalis po- tentially feasible. Recent acquisition by the Kenyan government of over 120 GeneXpert IV machines for county and sub-county hospitals for tuberculosis testing [37], has potentially expanded the diagnostic capacity for curable STIs using the same testing platform at larger hospitals. However, the cost of the C. trachomatis and N. gonorrhoeae test kits are currently high. Without assay costs becoming considerably less expensive, it seems unlikely that ANC- attending women will be routinely tested for C. trachomatis and N. gonorrhoeae. Cost-effective studies have played an important role of informing policy on the gains of syphilis screening in pregnancy while minimizing on the expenditure [38]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 trachomatis among the general rural population in Kilifi, further studies con- firming the high prevalence and ongoing C. trachomatis transmission among this population would be helpful. Chlamydia infection has been found to be an independent predictor of HIV- 1 acquisition in a recent study from western Kenya, with also syphilis, yeast infection/colonisa- tion and BV showing strong associations with incident HIV-1 infection [13]. We documented a prevalence of T. vaginalis (7.4%) comparable to 6.0% among HIV-sero- negative ANC-attending women in rural western Kenya [13], and 7.7% among women attend- ing a child-health clinic in Mombasa, Kenya [28]. In vitro evidence suggests that T. vaginalis may alter the vaginal microbiome towards BV [29]. T. vaginalis has also been associated with increased viral shedding and increased acquisition of HIV [30, 31], potentially contributing to a significant number of additional HIV infections globally [31]. The prevalence of HIV-1 (6.4%) in this study was substantial, with all known and newly diag- nosed cases occurring amongst the 120 women 25 years. In Coastal Kenya, HIV-1 prevalence estimates in women aged 15–24 years decreased from 5.7% in 2007 to 2.0% in 2012 (p = 0.026) [32]. Our results suggest a further decline in HIV-1 prevalence, among women 24 years. PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 11 / 16 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya bacterial species and loads between the vagina and rectum of pregnant women [44]. McClelland et.al [31] showed that vaginal washing increased risk of HIV-1 acquisition among African women. Although, we did not assess whether water used by our study participants was shared, sharing of water may potentially lead to contamination of the vagina [45]. Pereira-Neves and Benchimol [46] showed in vitro that T. vaginalis remained viable and infective in swimming pool water samples for several hours. Further research is required to investigate the potential role that sanitary and hygiene practices may play in women with high prevalence of curable STIs and BV. Observed genital ulcers had the strongest association with curable STIs, and were found in 2.5% of study participants. Presumptively treating curable STIs on the basis of observed genital ulcers in pregnant women would have treated ~10% of the curable STIs in our study. Studies examining microbial aetiology of genital ulcer disease, have shown that genital ulcers can be attributed to several sexually transmitted pathogens with most cases being attributed to Herpes Simplex virus type 2 [47, 48]. Genital ulcers were also identified as the strongest predictor of acute HIV acquisition in attendees of STD clinics who initially had HIV-seronegative or dis- cordant rapid test results [49]. While in our study no women with genital ulcers had HIV, observing genital ulcers in women may help initiate risk reduction during ANC counselling and targeted acute HIV screening [50]. Our study had some limitations. First, our study only included a relatively small sample of pregnant women limiting the precision of our prevalence estimates. Second, we only included adult women and were not able to enroll pregnant women who were minors. Minors are potentially more vulnerable and may have presented with different STIs. Third, we included only women residing within the Kilifi Health and Demographic Surveillance Area [22], and therefore will have excluded women from outside the surveillance area who may have different health characteristics. Fourth, our hygiene and sanitary questions were few, and limited [51]. Finally, this was a cross-sectional study, only allowing for an assessment of associations and we did not collect data on pregnancy outcomes yg y q Finally, this was a cross-sectional study, only allowing for an assessment of associations and we did not collect data on pregnancy outcomes. Acknowledgments We would like to thank the study participants and a special thanks to all nurses at the ANC clinic of the Kilifi County Hospital and to Sarah Kioko (field worker). This manuscript was submitted for publication with permission from the Director of the Kenya Medical Research Institute (KEMRI). Despite these limitations, we showed that there is high prevalence of curable STIs among pregnant women attending an antenatal clinic at Kilifi County Hospital, Kenya. Curable STIs were associated with genital ulcers, hygienic practices, early sexual debut and BV. We recom- mend that antenatal care programs consider strengthening their diagnostic screening for cur- able STIs and BV. Such cost-effectiveness studies of ANC screening and treatment of C. trachomatis, N. gonorrhoeae, and T. vaginalis should be con- ducted in resource poor settings. Treatment of STIs in both infected persons and their partner is pivotal for STI management, ensuring reduction in cases of reinfection or non-resolving infections and prevention of adverse outcomes [8]. In our study a presumptive patient delivered partner treatment approach was adopted. Despite the fact that we did not collect information on the number of partners who were treated, this approach has been shown to be acceptable and feasible among pregnant women in Kenya [39]. Early sexual debut was strongly associated with curable STIs in our study population, simi- lar to findings in other studies [40, 41]. Kilifi county is one of the poorest counties in Kenya, characterized by low literacy levels mostly among women, young age at marriage of women, and high fertility rates [42]. Structural changes focusing on the economic empowerment of communities, including improved access to education, and legislation protecting women from early marriage will be necessary to reduce the high burden of curable STIs among pregnant women. A salient predictor of curable STIs in our study population was the use of water to clean after visiting the toilet as compared to those women who reported to use toilet paper or other solid materials. Unfortunately, our hygiene question did not distinguish urinating from defecating, or inquired whether the cleaning mode included both anus and vagina. It is possible that such cleaning transported bacteria from the perianal area to the vagina, as pathogens causing curable STIs may be found in paragenital areas [43]. 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Journal of the International AIDS Society. 2016; 19(1). PubMed Central PMCID: PMCPMC4718968. 11. Conceptualization: SCM MV EJS. Conceptualization: SCM MV EJS. Data curation: SCM EW. Formal analysis: SCM EW. Formal analysis: SCM EW. Funding acquisition: SCM MV EJS. Investigation: SCM. 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0175166 March 31, 2017 Curable sexually transmitted infections among pregnant women in Kilifi, Kenya Writing – review & editing: SCM EW MV PC TC EJS. Writing – review & editing: SCM EW MV PC TC EJS. Project administration: SCM EJS. Supervision: SCM EJS. Validation: SCM. Validation: SCM. Visualization: SCM EW. Writing – original draft: SCM. References Van Der Pol B, Kwok C, Pierre-Louis B, Rinaldi A, Salata RA, Chen PL, et al. Trichomonas vaginalis infection and human immunodeficiency virus acquisition in African women. The Journal of infectious diseases. 2008; 197(4):548–54. Epub 2008/02/16. https://doi.org/10.1086/526496 PMID: 18275275 12. 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https://openalex.org/W3094114963	https://www.frontiersin.org/articles/10.3389/fimmu.2020.585034/pdf	English	null	Identification of Immune Cell Infiltration and Immune-Related Genes in the Tumor Microenvironment of Glioblastomas	Frontiers in immunology	2,020	cc-by	8,614	ORIGINAL RESEARCH published: 20 October 2020 doi: 10.3389/fimmu.2020.585034 Identiﬁcation of Immune Cell Inﬁltration and Immune-Related Genes in the Tumor Microenvironment of Glioblastomas Sicong Huang 1†, Zijun Song 2†, Tiesong Zhang 1, Xuyan He 3, Kaiyuan Huang 1, Qihui Zhang 4,5, Jian Shen 1* and Jianwei Pan 1* 1 Department of Neurosurgery, The First Afﬁliated Hospital, Zhejiang University School of Medicine, Hangzhou, China, 2 The First Afﬁliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China, 3 The First Afﬁliated Hospital, School of Public Health, Zhejiang University School of Medicine, Hangzhou, China, 4 Department of Neurology, Dong Fang Hospital, Beijing University of Chinese Medicine, Beijing, China, 5 Department of Clinical Neurosciences, University of Calgary, Calgary, AB, Canada Keywords: glioblastoma, tumor microenvironment, immune inﬁltration, immune therapy, TCGA Edited by: Reviewed by: Rongjing Ge, Bengbu Medical College, China Xianli Lv, Tsinghua University, China Reviewed by: Rongjing Ge, Bengbu Medical College, China Xianli Lv, Tsinghua University, China *Correspondence: Jian Shen sjzju@126.com Jianwei Pan 1202053@zju.edu.cn †These authors have contributed equally to this work Specialty section: This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology Received: 19 July 2020 Accepted: 28 September 2020 Published: 20 October 2020 Specialty section: This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology Received: 19 July 2020 Accepted: 28 September 2020 Published: 20 October 2020 Edited by: Edited by: Xiaoxing Xiong, Renmin Hospital of Wuhan University, China Edited by: Xiaoxing Xiong, Renmin Hospital of Wuhan University, China Glioblastoma (GBM) is one of the most prevalent malignant brain tumors with poor prognosis. Increasing evidence has revealed that inﬁltrating immune cells and other stromal components in the tumor microenvironment (TME) are associated with prognosis of GBM. The aim of the present study was to identify immune cells and immune-related genes extracted from TME in GBM. RNA-sequencing and clinical data of GBM were downloaded from The Cancer Genome Atlas (TCGA). Four survival-related immune cells were identiﬁed via Kaplan-Meier survival analysis and immune-related differentially expressed genes (DEGs) screened. Functional enrichment and protein- protein interaction (PPI) networks for the genes were constructed. In addition, we identiﬁed 24 hub genes and the expressions of 6 of the genes were signiﬁcantly associated with prognosis of GBM. Finally, the genes were validated in single-cell sequencing studies of GBM, and the immune cells validated in an independent GBM cohort from the Chinese Glioma Genome Atlas (CGGA). Overall, 24 immune-related genes inﬁltrating the tumor microenvironment were identiﬁed in the present study, which could serve as novel biomarkers and immune therapeutic targets. Glioblastoma (GBM) is one of the most prevalent malignant brain tumors with poor prognosis. Increasing evidence has revealed that inﬁltrating immune cells and other stromal components in the tumor microenvironment (TME) are associated with prognosis of GBM. The aim of the present study was to identify immune cells and immune-related genes extracted from TME in GBM. RNA-sequencing and clinical data of GBM were downloaded from The Cancer Genome Atlas (TCGA). Four survival-related immune cells were identiﬁed via Kaplan-Meier survival analysis and immune-related differentially expressed genes (DEGs) screened. Functional enrichment and protein- protein interaction (PPI) networks for the genes were constructed. In addition, we identiﬁed 24 hub genes and the expressions of 6 of the genes were signiﬁcantly associated with prognosis of GBM. Finally, the genes were validated in single-cell sequencing studies of GBM, and the immune cells validated in an independent GBM cohort from the Chinese Glioma Genome Atlas (CGGA). Overall, 24 immune-related genes inﬁltrating the tumor microenvironment were identiﬁed in the present study, which could serve as novel biomarkers and immune therapeutic targets. Citation: Glioblastoma (GBM) is the most common primary malignant brain tumor accounting for approximately 80% of all primary malignant brain tumors, and has a dismal prognosis and poor quality of life, with a median overall survival (OS) often < 1 year. Hereditary syndromes and ionizing radiation are the most common risk factors for GBM (1). The standard care of GBM is surgical resection followed by concomitant radiation therapy and chemotherapy with temozolomide (TMZ). Although multiple treatments have improved due to the development of gene therapy, immunotherapy, vaccine therapy, and others (2), therapeutic options for managing Huang S, Song Z, Zhang T, He X, Huang K, Zhang Q, Shen J and Pan J (2020) Identiﬁcation of Immune Cell Inﬁltration and Immune-Related Genes in the Tumor Microenvironment of Glioblastomas. Front. Immunol. 11:585034. doi: 10.3389/fimmu.2020.585034 October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org The Immune Inﬁltration of Glioblastoma Huang et al. recurrence in GBM are limited. Immune checkpoint inhibitors (ICIs) such as anti-programmed cell death protein-1 (PD-1)/ programmed death ligand-1 (PD-L1) and anti-cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) have been extensively studied for both primary and recurrent glioblastomas in medical research. However, most of the clinical studies for GBM based on ICIs and trials with vaccine therapies have been unsuccessful. The cause of the failure in clinical trials of GBM via immunotherapy is attributed to several factors, including a highly immunosuppressive environment and multiple mechanisms of therapeutic resistance. GBM induces local immune dysfunction and systemic immunosuppression, which causes more complex coupling relationships between GBM and the surrounding tumor microenvironment (TME). Studying the mechanisms of GBM immunosuppression enhances our understanding on development of immunotherapy strategies (3). 22 types of inﬁltrating immune cells in the 166 patients were estimated using the method of estimating relative subsets of RNA transcripts (CIBERSORT) (6). Subsequently, four survival-related immune cells were identiﬁed from the survival analyses of 22 types of immune cells. Immune-related genes were ranked through differential gene expression analyses and 24 hub genes selected from the protein-protein interaction (PPI) network established using Cytoscape (7). Six hub genes associated with overall survival were identiﬁed. Finally, immune cells were validated in an independent GBM cohort from the Chinese Glioma Genome Atlas (CGGA), and hub genes veriﬁed in single-cell sequencing studies of GBM. All analyses were conducted using R software. Citation: The ﬁndings of the present study provide valuable information that will guide patient-speciﬁc clinical immunotherapeutic strategies, and further construction of prediction models for prognosis of GBM. Moreover, immune cells inﬁltrating in the tumor microenvironment could act as therapeutic targets for the clinical treatment of GBM. TME is one of the crucial factors of local immune dysfunction, which establishes a niche for cancer cells, multiple stromal cells (endothelial cells, immune cells, etc.) and extracellular components (extracellular matrix, cytokines, growth factors, etc.). TME plays a critical role in the establishment of speciﬁc conditions, thereby interfering with angiogenesis, cell death, oxidative stress, and immune escape (4). Increasing studies have revealed that TME is notonlypivotalintumorinitiation,progression,andmigration,but it also affects generation of therapeutic resistance and malignancy. Cellular composition of TME and accessibility of immune cells exhibit large variations among GBM subtypes and patients. Such factors contribute to immunosuppression of GBM, which in turn lead to immunotherapeutic treatment failure (5). Identiﬁcation of actively involved types of immune genes and immune cells associated with the TME facilitates elucidation of the general mechanisms of GBM immunosuppression. Raw Data Collection RNA-Seq expression proﬁles of immune cells and corresponding clinical data of 166 patients with GBM were downloaded from TCGA database. The ﬁle format of RNA-seq expression was FPKM. The expression proﬁle of each sample included age, gender, expression subclass, and MGMT promoter status. RNA-Seq expression information of immune cells from CGGA were also downloaded for the validation. Data acquisition and analyses were performed using R software (8).The entire research data analysis process is presented in Figure 1. Therefore, the present study investigated survival-related immune cells in GBM and identiﬁed hub genes associated with immunecellinﬁltration. WeacquiredRNA-sequencing (RNA-seq) expressiondataandcorrespondingclinicaldataof166patientswith GBM from The Cancer Genome Atlas (TCGA) database. A total of Identiﬁcation of Survival-Related Tumor-Inﬁltrating Immune Cells CIBERSORT is an analytical algorithm, which can characterize cell composition of complex tissues based on normalized gene Identiﬁcation of Survival-Related Tumor-Inﬁltrating Immune Cells l l l h h h CIBERSORT is an analytical algorithm, which can characterize cell composition of complex tissues based on normalized gene FIGURE 1 \| Flow chart of the whole analysis process. Frontiers in Immunology \| www.frontiersin.org October 2020 \| Volume 11 \| Article 585034 2 FIGURE 1 \| Flow chart of the whole analysis process. FIGURE 1 \| Flow chart of the whole analysis process. October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 2 The Immune Inﬁltration of Glioblastoma Huang et al. expression proﬁles (9). We used CIBERSORT to estimate the ratio of 22 inﬁltrating immune cell types based on each GBM sample. Afterward, 57 samples with P ≤0.05 were selected and correlation analyses conducted to analyze contents of the 22 immune cells (10). Survival analyses of the ﬁltered immune cells in the tumor microenvironment were performed by the Kaplan- Meier survival analysis, with a cut-off level set at the median value. The results were tested by log-rank test. All the analyses were conducted using R software. genes and 22 immune cells was determined using Person’s correlation analysis and CIBERSORT to reveal the relationship between hub genes and immune cells (17). Afterward, comprehensive correlation analysis between six selected survival-related hub genes and tumor-inﬁltrating immune cell signatures for GBM were performed using Tumor Immune Estimation Resource (TIMER 1.0, https://cistrome.shinyapps. io/timer/) (18). Clinical Data Correlated With Survival-Related Immune Cells To determine the effect of immune cells on the clinical characteristics of GBM, relevant GBM clinical data were downloaded to investigate correlation with the abundance ratios of survival-related immune cells. The clinical characteristics included age, gender, expression subclass, and MGMT promoter status. The odds ratio of monocytes and eosinophils increased in neural and proneural types and was higher in males than in females (Figure 3). Data Source and Identiﬁcation of Survival-Related Immune Cells The workﬂow of the study is presented in Figure 1. Publicly available data for the 166 cases of GBM, including RNA-Seq (FPKM and counts format) and clinical data were downloaded from TCGA database. The abundance ratios of 22 immune cells in the 57 samples are presented in the Figure 2A, and the relationship between abundance ratios of the immune cells is presented in Figure 2B. Consequently, the correlations between abundance ratios of immune cells were analyzed using Kaplan-Meier survival analysis to elucidate the potential role of the abundance ratios of immune cells in overall survival. The four immune cells that were associated with survival are presented in Figures 2C–F. The results of survival analyses indicated that there was a signiﬁcant negative correlation between M0 Macrophages, while monocytes, activated NK cells, and eosinophils predicted positive overall survival. Construction of PPI Network, Selection and Analysis of Hub Genes PPI networks of immune-related genes were predicted using the Search Tool for the Retrieval of Interacting Genes (STRING, https://string-db.org/) (14). An interaction combined score of >0.4 was considered statistically signiﬁcant. Cytoscape is an open-access software platform designed to analyze and visualize complex interaction networks (7). Molecular Complex Detection(MCODE) plugin of Cytoscape was used to cluster the networks based on topology to identify densely connected regions with MCODE score > 5, degree cut-off = 2, node score cut-off = 0.2, max depth = 100, and k-score = 2 (15). Hub genes were deﬁned based on module connectivity (16). RESULTS Immune related-genes were analyzed using survival-related cells that had been obtained previously. Data analysis was performed using the edgeR R package, and \|logFC\| ≥1.0 and P < 0.05 were set as the cut-offs to screen for immune-related genes. Subsequently, a Venn diagram was used to visualize genes displayed by the online tool (http://bioinformatics.psb.ugent. be/webtools/Venn/) (11). DAVID software (https://david. ncifcrf.gov/) was used to analyze immune-related genes in the Gene Ontology(GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (12). Results of GO analysis revealed the functions of immune-related genes in biology process, cell component, and molecular function (13). KEGG pathway analyses results revealed the role of development- related signaling pathways. Relationship Between Clinical Characteristics and Survival-Related Immune Cells Data for the single cell GBM analysis was derived from the paper “An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma”, and the Seurat R package was used to reprocess the count matrix in which the dimensional reduction plot and cell type annotation were both retrieved from published meta data (19). The distribution of expressions of the hub genes was created using the Feature Plot function. To determine the relationship between survival-related immune cells and clinical features such as age, gender, expression subclass, and MGMT promoter status, 57 samples were analyzed. An independent sample t-test was used to compare means of two groups, while one-way analysis of variance (ANOVA) test was used to compare the means of four groups. Screening of Immune-Related Genes The immune-related genes were categorized into high- and low- expression groups in GBM to identify genes that associated with the four survival-related immune cells. Unique genes expression proﬁles of the four survival-related immune cells are presented Kaplan-Meier plots were used to identify immune-related genes in relation to the overall survival of patients. These results were analyzed by long-rank test. The correlation between 24 hub October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 3 The Immune Inﬁltration of Glioblastoma Huang et al. A B D E F C GURE 2 \| The abundance ratios of 22 immune cells and overall survival analysis. (A) The abundance ratios of immune cells in the 57 samples. The speciﬁc 22 mune cells corresponded to to one sample by different colors as shown in barplot. (B) The abundance ratios matrix of 22 immune cells. The value represents the rrelation value, green represents the positive correlation while brown represents negative correlation. (C–F) Overall survival analysis of four immune cells based on aplan Meier-plotter from the comparison of groups of high (yellow line) and low (blue line) genes expression. (p<0.05). A A B C C B D D E E F E F FIGURE 2 \| The abundance ratios of 22 immune cells and overall survival analysis. (A) The abundance ratios of immune cells in the 57 samples. The speciﬁc 22 immune cells corresponded to to one sample by different colors as shown in barplot. (B) The abundance ratios matrix of 22 immune cells. The value represents the correlation value, green represents the positive correlation while brown represents negative correlation. (C–F) Overall survival analysis of four immune cells based on Kaplan Meier-plotter from the comparison of groups of high (yellow line) and low (blue line) genes expression. (p<0.05). by volcano plots in Figure 4. A total of 1,107 genes were identiﬁed in monocytes, 1,137 genes in macrophages M0, 1,742 genes in activated NK cells, and 1,336 genes in eosinophils (Figures 4A–D). In addition, 38 identical genes expressed in inﬁltration of the four immune cells are presented by Venn diagrams in Figure 4E. of immune-related genes (Figure 5). GO term analysis revealed that immune-related genes were signiﬁcantly enriched in the biological processes (BP) of nervous system development, cell adhesion, extracellular matrix organization, and chemical synaptic transmission (Figure 5A). Screening of Immune-Related Genes Genes in the cellular components (CC) groups (Figure 5B) were primarily enriched in the plasma membrane, extracellular exosome, extracellular space, and extracellular region; the molecular functions (MF) were enriched in protein binding, calcium binding, structural constituent of cytoskeleton, and microtubule binding (Figure 5C). Moreover, the KEGG analysis revealed that immune-related Modular Analysis Based on PPI Network Modular Analysis Based on PPI Network Considering the limitation of the PPI networks regarding the number of genes, we screened all the differentially expressed genes but selected the genes only co-expressed in at least two immune cells. Overall, we identiﬁed 920 genes from 4,122 genes. These genes were imported into the online STRING tool to elucidate the interaction of immune-related genes. Finally, we got the PPI network with 357 genes which the combined-score was set to ≥0.4 (Figure 6A). We selected the most signiﬁcant module for further functional enrichment analysis (Figure 6B). Validation of the Correlation Between Immune Cell Inﬁltration and Survival- Related Hub Genes The correlation between survival-related hub genes and immune cell inﬁltration in GBM was analyzed after determining the prognostic value of hub genes (Figure 9). Functional Enrichment Analysis of Immune-Related Genes Functional enrichment analysis of immune-related genes was performed via DAVID website to reveal the potential functions October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org The Immune Inﬁltration of Glioblastoma Huang et al. A B D C FIGURE 3 \| Relationship between four survival-related immune cells and clinical features. (A–D) The relationship between four survival-related immune cells and age, gender, expression subclass, and MGMT status. A B C A B C C D D FIGURE 3 \| Relationship between four survival-related immune cells and clinical features. (A–D) The relationship between four survival-related immune cells and age, gender, expression subclass, and MGMT status. FIGURE 3 \| Relationship between four survival-related immune cells and clinical features. (A–D) The relationship between four su gender, expression subclass, and MGMT status. FIGURE 3 \| Relationship between four survival-related immune cells and clinical features. (A–D) The relationship between four survival-related immune cells and age, gender expression subclass and MGMT status onship between four survival-related immune cells and clinical features. (A–D) The relationship between four survival-related immune subclass, and MGMT status. signiﬁcantly correlated with survival (Figure 7). GRIA1, BST2, B2M, and TRIM21 were positively correlated with the overall survival. GRIA2 and MAP2 were correlated with poor prognosis. The relationship between 24 hub genes and 22 immune cells analyzed using Person’s correlation analysis is performed in Figure 8A. The remarkable relationship between inﬁltration levels of immune cell types and survival-related hub genes was validated in TIMER. The results indicated that inﬁltration levels of CD8+ T cells, neutrophils, and dendritic cells were signiﬁcantly associated with GRIA1, GRIA2, and MAP2 (Figure 8B). Furthermore, BST2 and B2M were correlated with B cells, macrophages, and dendritic cells, and TRIM21 was associated with B cells and neutrophils. genes were linked to cell adhesion molecules, cAMP signaling pathway, leukocyte transendothelial migration, protein digestion and absorption, and Toll-like receptor signaling pathway (Figure 5D). These results demonstrated that the genes were associated with the extracellular matrix of tumor microenvironment and cellular interaction. Identiﬁcation of Survival-Related Hub Genes A total of 24 hub genes with high connectivity in the modules were identiﬁed from the PPI network based on the cut-off criteria. We subsequently elevated the biological enrichment analysis of the 24 hub genes using the online tool (http://www. ncbi.nlm.nih.gov/gene) (Table 1). Six of the hub genes were In addition, gene expression data of immune cells for 134 GBM samples were downloaded from CGGA database to investigate the signiﬁcance of immune cells identiﬁed from TCGA database. The results we obtained from CCGA revealed that activated NK cells (Figure S1, p = 0.019) and monocytes (Figure S1, p = 0.023) were October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 5 The Immune Inﬁltration of Glioblastoma Huang et al. A B D C E 4 \| Screening for immune-related genes. (A–D) The volcano plot of all quantiﬁed genes in the analysis of monocytes, macrophages M0, NK cells activated, ophils. (E) Venn diagram indicates the overlap of differentially expressed genes across the four different immune cells. A B B C D C D D E E E FIGURE 4 \| Screening for immune-related genes. (A–D) The volcano plot of all quantiﬁed genes in the analysis of monocytes, macrophages M0, NK cells activated, and eosinophils. (E) Venn diagram indicates the overlap of differentially expressed genes across the four different immune cells. GURE 4 \| Screening for immune-related genes. (A–D) The volcano plot of all quantiﬁed genes in the analysis of monocytes, macro d eosinophils. (E) Venn diagram indicates the overlap of differentially expressed genes across the four different immune cells. associated with positive prognosis, which are consistent with the data we have gotten previously from the TCGA database (Figure S1). detected in any of the cells types. Notably, microglia are the vital macrophages of the brain, and they act as the primary form of immune defense in the central nervous system. A speciﬁc microglial marker in humans, TMEM 119, was used to distinguish microglia from macrophages in the brain (Figure S2). We subsequently identiﬁed the expression of hub genes in microglia and found AIF1, B2M, BST2, C3AR1, CCND1, CCT3, FCGR1A, GNG7, HMOX1, HSPA8, MNDA, TLR2, and TUBA1A were signiﬁcantly expressed (Figure S2). Validation of the Expression of Immune- Related Hub Genes by Single-Cell Sequencing The cells were classiﬁed as malignant and non-malignant cell types by combining three approaches; high expression of markers classiﬁed as non-malignant cells such as macrophages, T cells, and oligodendrocytes. The distribution of hub genes expressions in the four cell clusters is displayed in Figure 9. A1F1, C3AR1, FCGR1A, MNDA, HMOX1, and TLR2 were only expressed in macrophages. B2M, CCT3, HSPA8, and TUBA1A were signiﬁcantly expressed in all the four cell clusters. With reference to survival-related genes, BST2 was detected in macrophages, T-cells, and malignant cells. GRIA1 and GRIA2 were expressed in oligodendrocytes and malignant cells. MAP2 was only detected in malignant cells. However, TRIM21 was not DISCUSSION The present study analyzed immune cells and immune-related genes in TME of GBM to establish a potential strategy for GBM immunotherapy. The study identiﬁed immune-related genes in TME, which signiﬁcantly contributed to the survival of patients with GBM from TCGA database. Four survival-related immune cells were initially identiﬁed from GBM samples and the genes October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 6 The Immune Inﬁltration of Glioblastoma Huang et al. B A B D C FIGURE 5 \| Functional enrichment analysis of immune-related genes. (A) Biological process analysis. (B) Cellular components analysis. (C) Molecular function. (D) KEGG pathway analysis (p < 0.05). A B B C D C C D D FIGURE 5 \| Functional enrichment analysis of immune-related genes. (A) Biological process analysis. (B) Cellular components analysis. (C) Molecular function. (D) KEGG pathway analysis (p < 0 05) FIGURE 5 \| Functional enrichment analysis of immune-related genes. (A) Biological process analysis. (B) Cellular components analysis. (C) Molecular function. (D) KEGG pathway analysis (p < 0.05). FIGURE 5 \| Functional enrichment analysis of immune-related genes. (A) Biological process analysis. (B) Cellular components a (D) KEGG pathway analysis (p < 0.05). chment analysis of immune-related genes. (A) Biological process analysis. (B) Cellular components analysis. (C) Molecular function. BBB is damaged during tumor progression (21). With the accumulation of a family of monocyte chemoattractant family of proteins (MCPs), monocytes from the periphery inﬁltrate into the tumor across the BBB, and then differentiate into macrophages. Tumor-associated macrophages are often regarded as the facilitators of tumor proliferation due to their proangiogenic and immunosuppressive effects (21). M0 macrophages, which are referred to as ‘alternatively activated macrophages,’ can be polarized into M1 or M2 phenotypes by environmental signals (22). M1 macrophages can produce pro- inﬂammatory cytokines that are essential for host defense and exert tumoricidal effects in GBM (21). However, M2 macrophage phenotype is considerably involved in tumor cell proliferation and prediction of poor clinical prognosis in patients with GBM patients (23). M1 and M2 macrophages are plastic and heterogeneous immune cells, and the TME facilitates the regulation of functional polarization of TAMs (24). Currently, researchers have been working on promoting the reversal of correlating to the levels of four immune cells analyzed. Furthermore, GO and KEGG enrichment analysis were conducted to investigate the biological functions of immune- related genes. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Subsequently, all the immune-related genes were imported to construct a PPI network, and 24 hub genes obtained. Finally, the immune cell types in patients with GBM were validated using CGGA database, and hub genes validated in single-cell sequencing. Four types of survival-related immune cells associated with GBM were identiﬁed from TCGA database, including M0 macrophage, monocytes, NK cells and eosinophils. Previous research has indicated that immune cells, especially tumor- associated macrophages (TAMs) in TME interact with tumor cells through direct contact or different signaling pathways. TAMs are crucial components of inﬁltrating immune cells, accounting for 30–40% of the cellular components in GBM (20). Immune cell populations in GBM are classiﬁed into two categories: microglia and bone marrow-derived monocytes. The October 2020 \| Volume 11 \| Article 585034 7 The Immune Inﬁltration of Glioblastoma Huang et al. A B FIGURE 6 \| Protein-Protein interaction network construction and modular analysis. (A) PPI network was constructed using a total of DEGs. (B) The most signiﬁcant module was marked. The color of a node reﬂects the log(Fc) value of the gene expression, the size of a node suggests the numbers of interacting genes with others. A B FIGURE 6 \| Protein-Protein interaction network construction and modular analysis. (A) PPI network was constructed using a total of DEGs. (B) The most signiﬁcant module was marked. The color of a node reﬂects the log(Fc) value of the gene expression, the size of a node suggests the numbers of interacting genes with others. cytotoxicity (29). Moreover, GBM cells express high levels of MHC class I molecules and human leukocyte antigens (HLA)-A, HLA-B, and HLA-C ligands, which inhibit functions of NK cells via killer immunoglobulin-like receptors (KIRs) (30). Therefore, blocking KIRs could disrupt the tumor microenvironment and attenuate the activity of NK cells to kill GBM cells. Increasing the number of NK cells inﬁltrating the GMB microenvironment and modiﬁcation of NK cells could be a potential treatment intervention for GBM (31, 32). Emerging evidence has demonstrated that the activation of eosinophils induces initiation, promotion and progression of GBM (33). TAMs from M2 to M1 based on their polarization (25, 26). Therefore, the results may indicate that the macrophages in TME of GBM could be used as potential therapeutic targets for GBM immunotherapy. NK cells accounts for 2.11% of the total cellular components in GBM, which constitutes the lowest proportion of all immune cells inﬁltrating in GBM (27). Frontiers in Immunology \| www.frontiersin.org DISCUSSION 10 CXCL10 C-X-C motif chemokine ligand 10 signaling receptor binding and chemokine activity 11 GCH1 GTP cyclohydrolase 1 calcium ion binding and GTP binding 12 FCGR1A Fc fragment of IgG receptor Ia obsolete signal transducer activity, downstream of receptor and IgG binding 13 C3AR1 Complement C3a receptor 1 G protein-coupled receptor activity and complement component C3a receptor activity 14 TUBA1A Tubulin alpha 1a structural molecule activity 15 CCT3 Chaperonin containing TCP1 subunit 3 unfolded protein binding 16 HMOX1 Heme oxygenase 1 protein homodimerization activity and oxidoreductase activity 17 GNG7 G protein subunit gamma 7 obsolete signal transducer activity 18 C1R Complement C1r calcium ion binding and serine-type peptidase activity 19 BST2 Bone marrow stromal cell antigen 2 obsolete signal transducer activity 20 CYP19A1 Cytochrome P450 family 19 subfamily A member 1 iron ion binding and electron transfer activity 21 GRIA2 Glutamate ionotropic receptor AMPA type subunit 2 ionotropic glutamate receptor activity and AMPA glutamate receptor activity 22 MNDA Myeloid cell nuclear differentiation antigen Innate Immune System and Apoptosis and Autophagy 23 MAF MAF bZIP transcription factor DNA-binding transcription factor activity and DNA-binding transcription activator activity, RNA polymerase II-speciﬁc 24 TRIM21 Tripartite motif containing 21 identical protein binding and ligase activity TABLE 1 \| The function of hub genes. Innate Immune System and Apoptosis and Autophagy Innate Immune System and Apoptosis and Autophagy DNA-binding transcription factor activity and DNA-binding transcription activator activity, RNA polymerase II-speciﬁc DNA-binding transcription factor activity and DNA-binding transcription activator activity, RNA polymerase II-speciﬁc identical protein binding and ligase activity identical protein binding and ligase activity pathways are all related to the extracellular matrix components and cell’s cytoskeleton in the microenvironment. These above results further indicate the reliability of the immune differential genes and their relevance to the GBM tumor microenvironment. promotion, eosinophils are activated by GBM mediators, which in turn lead to the production of tumor promoting growth factors (35). Nevertheless, the mechanisms of immune response in GBM remain indeterminate; therefore, further studies are required to investigate the mechanism involved. Furthermore, we identiﬁed 24 hub genes, and 6 of these genes (GRIA2, GRIA1, BST2, MAP2, B2M, and TRIM21) have signiﬁcant correlation with prognosis and were considered as predictive biomarkers that could provide valuable insights into new immunotherapy strategies. Previous studies have demonstrated that glioma cells can secrete excitotoxicity glutamate that mediates neuronal death in glioma microenvironment. DISCUSSION NK cells have been reported to recognize target cells that are deﬁcient in the surface expression of major histocompatibility complex (MHC) molecules, and can directly lyse tumor cells without prior activation (28). However, TME inﬂuences the immune function of NK cells and causes immune evasion. The upregulation of growth factor signaling pathways or the loss of cell cycle regulators promotes evasion of GBM from surveillance through resistance to NK-derived Previous advances have indicated the eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) play a critical role in preventing GBM initiation (34). During GBM October 2020 \| Volume 11 \| Article 585034 8 The Immune Inﬁltration of Glioblastoma Huang et al. TABLE 1 \| The function of hub genes. Number Name Full name Function 1 FYN FYN proto-oncogene, Src family tyrosine kinase G-protein signaling_RhoA regulation pathway and Lipoprotein metabolism 2 HSPA8 Heat shock protein family A (Hsp70) member 8 ubiquitin protein ligase binding 3 CCND1 Cyclin D1 protein kinase activity and enzyme binding 4 GRIA1 Glutamate ionotropic receptor AMPA type subunit 1 PDZ domain binding and extracellularly glutamate-gated ion channel activity 5 TLR2 Toll like receptor 2 protein heterodimerization activity and transmembrane signaling receptor activity 6 B2M Beta-2-microglobulin identical protein binding 7 AIF1 Allograft inﬂammatory factor 1 calcium ion binding and actin ﬁlament binding 8 MAP2 Microtubule associated protein 2 structural molecule activity and calmodulin binding 9 OLIG2 Oligodendrocyte transcription factor 2 homodimerization activity and transcription factor activity, RNA polymerase II distal enhancer sequence-speciﬁc binding. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Genes that contribute to cell-by-cell variations cannot be detected using conventional RNA-seq data of GBM downloaded from TCGA database (47). However, single-cell RNA-seq (scRNA-seq) proﬁles for intracellular transcriptome at individual cell level can reveal potential heterogeneous tumors and the composition of glioblastoma tumor microenvironment (48). ScRNA-seq can easily identify highly variable genes in all cell types in the TME of GBM, including the two primary cell types: microglia/macrophages and oligodendrocytes, which are limited in conventional RNA-seq (49). For example, as we have mentioned above, the results of ScRNA-seq revealed that GluR1 and GluR2 were expressed in oligodendrocytes and malignant cells. The expression of Ca2+-permeable GluR confers protection against excitotoxicity and promotes progression of tumor (50). BST2 expression increases in the malignant cells of glioma during tumor progression (51). TLR2 expressed in microglia can promote glioblastoma progression by up-regulating the expression of MT1-MMP in microglia (52). The expression of CCND1 in microglia cells contributes to the differential diagnosis of oligodendrogliomas (53). The use of scRNA-seq to detect the expression of hub genes could signiﬁcantly help us to accurately understand the function of hub genes in each cell (54). In addition, scRNA-seq demonstrates transcriptional heterogeneity associated with spatial speciﬁcity in distinct TME subunit promotes permeability to Ca2+, whereas presence of GluR2 inhibits permeability to Ca2+ (42). However, GluR1 and GluR4 subunits also function as Ca2+- permeable AMPARs. Ishiuchi et, al found GluR1 proteins were substantially expressed in most tumor cells, whereas GluR2 was mainly expressed in normal tissues in human glioblastoma samples (43). Furthermore, it has been suggested that blockage of Ca2+ inﬂux through GluR2 expression suppresses migration and induces apoptosis in human glioblastoma cells (44). In addition, knocking down GluR1 inhibits glioma growth (45). Therefore, the conversion of Ca2+- permeable AMPARs to Ca2+- impermeable could be a potential therapeutic target for brain tumors (43). TRIM21 expression is correlated with prognosis, which acts as a tumor suppressor in patients with GBM (46). TRIM21 depletion in GBM enhanced cell proliferation and tumor growth. Lee et, al found that phosphofructokinase 1 (PFK1) expression promotes human glioblastoma progression, while TRIM21 exert anti-tumor effect by mediating poly ubiquitination and degradation of PFK1 (46). Therefore, TRIM21 is a novel target for glioblastoma treatment. The expression of 24 hub genes in human glioblastomas was validated using single-cell sequencing. Frontiers in Immunology \| www.frontiersin.org DISCUSSION However, single-cell RNA-seq (scRNA-seq) proﬁles for intracellular transcriptome at individual cell level can reveal potential heterogeneous tumors and the composition of glioblastoma tumor microenvironment (48). ScRNA-seq can easily identify highly variable genes in all cell types in the TME of GBM, including the two primary cell types: microglia/macrophages and oligodendrocytes, which are limited in conventional RNA-seq (49). For example, as we have mentioned above, the results of ScRNA-seq revealed that GluR1 and GluR2 were expressed in oligodendrocytes and malignant cells. The expression of Ca2+-permeable GluR confers protection against excitotoxicity and promotes progression of tumor (50). BST2 expression increases in the malignant cells of glioma during tumor progression (51). TLR2 expressed in microglia can promote glioblastoma progression by up-regulating the expression of MT1-MMP in microglia (52). The expression of CCND1 in microglia cells contributes to the differential diagnosis of oligodendrogliomas (53). The use of scRNA-seq to detect the expression of hub genes could signiﬁcantly help us to accurately understand the function of hub genes in each cell (54). In addition, scRNA-seq demonstrates transcriptional heterogeneity associated with spatial speciﬁcity in distinct TME samples. Genes that contribute to cell-by-cell variations cannot be detected using conventional RNA-seq data of GBM downloaded from TCGA database (47). However, single-cell RNA-seq (scRNA-seq) proﬁles for intracellular transcriptome at individual cell level can reveal potential heterogeneous tumors and the composition of glioblastoma tumor microenvironment (48). ScRNA-seq can easily identify highly variable genes in all cell types in the TME of GBM, including the two primary cell types: microglia/macrophages and oligodendrocytes, which are limited in conventional RNA-seq (49). For example, as we have mentioned above, the results of ScRNA-seq revealed that GluR1 and GluR2 were expressed in oligodendrocytes and malignant cells. The expression of Ca2+-permeable GluR confers protection against excitotoxicity and promotes progression of tumor (50). BST2 expression increases in the malignant cells of glioma during tumor progression (51). TLR2 expressed in microglia can promote glioblastoma progression by up-regulating the expression of MT1-MMP in microglia (52). The expression of CCND1 in microglia cells contributes to the differential diagnosis of oligodendrogliomas (53). The use of scRNA-seq to detect the expression of hub genes could signiﬁcantly help us to accurately understand the function of hub genes in each cell (54). In addition, scRNA-seq demonstrates transcriptional heterogeneity associated with spatial speciﬁcity in distinct TME samples. DISCUSSION Moreover, glutamate secretion promotes tumor expansion by inducing inﬂammatory response within the surrounding areas (39). Researchers have established that the expression of a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPARs) protects GBM cells from the glutamate-rich tumor microenvironment (40). AMPARs are complexes consisting of four subunits (GluR1, GluR2, GluR3, and GluR4). GRIA1 and GRIA2 are also referred to as GluR1and GluR2, respectively. Glutamate receptors (GluRs) are receptors that bind to glutamate, and they function as ligand-gated ion channels in the central nervous system and mediate transmission in excitatory synapses (41). The subunit composition of AMPARs depends on the conductance properties of Ca2+. Absence of GluR2 More importantly, KEGG enrichment analysis indicated that these differential immune-related genes were enriched in the classical pathway, such as cell adhesion molecules (CAMs) and cAMP signaling pathway. CAMs are glycol-proteins expressed on the cell surface and play a critical role in multiple biologic processes during tumor development (36). It has been reported that CAMs mediate the process of immune responses in the tumor microenvironment, such as immune cell recruitment, immune cell activation, and formation of immunological synapse between immune cells and tumor cells (37). The cAMP signaling pathway, which acts as universal second messengers regulates pivotal physiological processes. The increases of intracellular cAMP inhibits innate immune functions (38). At the BP level, these differential immune- related genes were signiﬁcantly enriched in cell adhesion, and extracellular matrix organization. In the CC groups, the differential immune-related genes were related to extracellular exosome, extracellular space, and extracellular region; the MF groups were enriched in protein binding, the structural constituent of cytoskeleton, and microtubule binding. Theses October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 9 The Immune Inﬁltration of Glioblastoma Huang et al. A B D E F C FIGURE 7 \| Overall survival analysis of six hub genes. (A) B2M. (B) BST2. (C) GRIA1. (D) GRIA2. (E) MAP2. (F) TRIM21 (p < 0.05). A B B A C D D C D F E E F GURE 7 \| Overall survival analysis of six hub genes. (A) B2M. (B) BST2. (C) GRIA1. (D) GRIA2. (E) MAP2. (F) TRIM21 (p < 0.05). samples. Genes that contribute to cell-by-cell variations cannot be detected using conventional RNA-seq data of GBM downloaded from TCGA database (47). DISCUSSION b genes in single- cell sequencing in GBM. t-distributed neighbor embedding(tSNE) plot of all single cells. The color represents the alignant cells (green), marcophages(magenta), oligodendrocytes (cyan), and T-cells (blue). FIGURE 9 \| Validation of hub genes in single- cell sequencing in GBM. t-distributed neighbor embedding(tSNE) plot of all single cells. The color represents the expression of markers for Malignant cells (green), marcophages(magenta), oligodendrocytes (cyan), and T-cells (blue). insights into the potential association between immune cell TME and GBM prognosis. patterns (55). ScRNA-seq has emerged as a revolutionary tool to enhance our understanding of the proﬁles of hub genes in GBM, and offers insights with implications for both targeted and immune therapies for GBM (49). DATA AVAILABILITY STATEMENT In summary, the study identiﬁed four types of survival-related immune cells from TCGA database and 24 TME-related hub genes in glioblastoma. The correlation between immune cells and hub genes in patients with GBM was validated using single-cell sequencing data. The results revealed that the hub genes are involved in the development and progression of GBM. Therefore, the candidate genes identiﬁed in the study can be used as potential prognostic biomarkers for GBM. However, further studies on the immune cells and hub genes in GBM tumor microenvironment should be conducted to investigate the underlying mechanisms. The present study provides novel The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Conventional RNA-seq is regularly performed on a bulk level and only measures the average gene expression based on mixed cell populations in October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 10 The Immune Inﬁltration of Glioblastoma Huang et al. A B FIGURE 8 \| Immune inﬁltration of survival-related genes. (A) The correlation between expression proportion of hub genes and immune cells. Red suggests the positive correlation while the blue represents negative correlation. The size of point indicates P-value, and the color reﬂects the correlation. (B) The correlation analysis between survival-related genes and tumor inﬁltrating immune cells was performed. Scatter plots were generated with partial Spearman’s correlation and A A A B nﬁltration of survival-related genes. (A) The correlation between expression proportion of hub genes and immune cells. Red suggests the le the blue represents negative correlation. The size of point indicates P-value, and the color reﬂects the correlation. (B) The correlation val-related genes and tumor inﬁltrating immune cells was performed. Scatter plots were generated with partial Spearman’s correlation and B B B tration of survival-related genes. (A) The correlation between expression proportion of hub genes and immune cells. Red s the blue represents negative correlation. The size of point indicates P-value, and the color reﬂects the correlation. (B) The c -related genes and tumor inﬁltrating immune cells was performed. Scatter plots were generated with partial Spearman’s co B FIGURE 8 \| Immune inﬁltration of survival-related genes. (A) The correlation between expression proportion of hub genes and immune cells. Red suggests the positive correlation while the blue represents negative correlation. The size of point indicates P-value, and the color reﬂects the correlation. (B) The correlation analysis between survival-related genes and tumor inﬁltrating immune cells was performed. Scatter plots were generated with partial Spearman’s correlation and statistical signiﬁcance. October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 11 The Immune Inﬁltration of Glioblastoma Huang et al. FIGURE 9 \| Validation of hub genes in single- cell sequencing in GBM. t-distributed neighbor embedding(tSNE) plot of all single cells. The color represents the expression of markers for Malignant cells (green), marcophages(magenta), oligodendrocytes (cyan), and T-cells (blue). 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Doncheva NT, Morris JH, Gorodkin J, Jensen LJ. Cytoscape StringApp: Network Analysis and Visualization of Proteomics Data. FUNDING SUPPLEMENTARY FIGURE 1 \| Overall survival analysis of 22 immune cells based on Kaplan Meier-plotter from the data of CGGA. This work was supported by the Natural Science Foundation of ZhejiangProvince(projectnumber:LZ20H090002)andtheNational Natural Science Foundational (project number: 82071285). This work was supported by the Natural Science Foundation of ZhejiangProvince(projectnumber:LZ20H090002)andtheNational Natural Science Foundational (project number: 82071285). SUPPLEMENTARY FIGURE 2 \| Hub genes expression in microglia. Microglia classiﬁed from macrophages are marked in dark red, while hub genes are in orange. AUTHOR CONTRIBUTIONS SH, ZS, JS, and JP designed the study. SH, ZS, XH, TZ, and KH collected and analyzed data. SH, ZS, XH, QZ, and JP wrote the October 2020 \| Volume 11 \| Article 585034 12 The Immune Inﬁltration of Glioblastoma Huang et al. SUPPLEMENTARY MATERIAL manuscript. All the authors approved the manuscript. All authors contributed to the article and approved the submitted version. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2020. 585034/full#supplementary-material REFERENCES Burger MC, Zhang C, Harter PN, Romanski A, Strassheimer F, Senft C, et al. CAR-Engineered NK Cells for the Treatment of Glioblastoma: Turning Innate Effectors Into Precision Tools for Cancer Immunotherapy. Front Immunol (2019) 10:2683:2683. doi: 10.3389/ﬁmmu.2019.02683 14. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. 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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. 47. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet (2009) 10(1):57–63. doi: 10.1038/nrg2484 48. Patel AP, Tirosh I, Trombetta JJ, Shalek AK, Gillespie SM, Wakimoto H, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary October 2020 \| Volume 11 \| Article 585034 Frontiers in Immunology \| www.frontiersin.org 14
https://openalex.org/W3040431287	https://europepmc.org/articles/pmc7329455?pdf=render	English	null	Access to hip and knee replacement surgery in patients with chronic diseases according to patient-reported pain and functional status	BMC health services research	2,020	cc-by	7,683	Podmore et al. BMC Health Services Research (2020) 20:602 https://doi.org/10.1186/s12913-020-05464-3 Podmore et al. BMC Health Services Research (2020) 20:602 https://doi.org/10.1186/s12913-020-05464-3 Open Access Bélène Podmore1,2* , Andrew Hutchings1,2, Sujith Konan3, John Robson4 and Jan van der Meulen1,2 Bélène Podmore1,2* , Andrew Hutchings1,2, Sujith Konan3, John Robson4 and Jan van der Me Bélène Podmore1,2* , Andrew Hutchings1,2, Sujith Konan3, John Robson4 and Jan van der Meulen1,2 * Correspondence: belene.podmore@lshtm.ac.uk Co espo de ce: be e e.pod o e@ s t .ac.u 1Department of Health Services Research & Policy, London School of Hygiene & Tropical Medicine, 15-17 Tavistock Place, London WC1H 9SH, UK 2Clinical Effectiveness Unit, The Royal College of Surgeons of England, London, UK , Full list of author information is available at the end of the article p p @ 1Department of Health Services Research & Policy, London School of Hygiene & Tropical Medicine, 15-17 Tavistock Place, London WC1H 9SH, UK 2Clinical Effectiveness Unit, The Royal College of Surgeons of England, London, UK Abstract Background: An increasing number of patients undergoing hip or knee replacement have chronic diseases. It has been suggested that the presence of chronic diseases may affect access to this type of surgery in the English National Health Service (NHS). We examined the access to hip and knee replacement surgery in patients with and without chronic diseases according to preoperative patient-reported pain, functional status and symptom duration. Methods: We analysed data of 640,832 patients who had hip or knee surgery between 2009 and 2016 in England. Multivariable regression was used to estimate the impact of 11 chronic diseases on severity of joint problems as measured on a scale from 0 to 48 by Oxford Hip (OHS) and Knee Scores (OKS) just before surgery and on likelihood of long-standing joint problems (> 5 years pre-operatively). Results: Patients with chronic diseases reported more severe joint problems than patients without (OHS differences ranged from 1.1 [95% CI 0.93, 1.2] to 2.5 [95% CI 2.3, 2.7] and OKS differences from 0.5 [95% CI 0.3, 0.7] to 2.6 [95% CI 2.4, 2.7] for the 11 chronic diseases) but the differences remain small. When analysed separately, patients with chronic diseases reported both more severe pain and poorer functional status. Six chronic diseases in hip patients and two in knee patients increased the likelihood that they had long-standing joint problems. The severity of joint problems just before surgery increased with the number of chronic diseases (OHS differences; one chronic disease (1.5 [95% CI 1.4, 1.5]) to four or more (5.8 [95% CI 5.6, 6.0])). Conclusions: Patients with chronic diseases reported more severe joint problems immediately before hip or knee replacement surgery suggesting they have hip or knee replacement later in the course of their joint disease. Keywords: Healthcare disparities, Access to surgery, Chronic disease, Surgical procedure Data sources We used data from the English national PROMs programme for elective hip and knee replacement surgery [19]. All NHS providers are required to participate and patients are asked to report their joint problems and well- being at the preoperative assessment clinic or on admis- sion to hospital and then again 6 months after surgery. Over 75% of eligible patients complete the preoperative questionnaire [20]. The PROMs data were linked at pa- tient level to data from the Hospital Episode Statistics (HES) database. HES contains administrative records of all admissions to all NHS hospitals in England. Eligibility was restricted to the first primary replacement surgery (Fig. 1). and knee replacement surgery [10]. In a recent qualitative study, we explored the views of healthcare professionals in the English NHS about refer- ring and selecting patients with chronic diseases for joint replacement surgery [11]. These professionals reported that some patients with chronic diseases are not ‘pre- pared’ for surgery because their chronic diseases are not adequately controlled. As a result, these patients are often sent back to their general practitioner in primary care, fragmenting and delaying the surgical management of their joint problems, in some cases preventing surgery altogether [12]. In addition, such delays could lead to in- creased functional deterioration and pain of the osteo- arthritic hip and knees and thereby also increased costs. Advanced osteoarthritis of the hip and knee is associated with increased health service use and opioid use [13, 14]. Previous research investigating variation in access to joint replacement surgery has used two different ap- proaches. Some papers have measured access indirectly from a population perspective by focusing on those not receiving surgery and seeking to measure unmet need [15]. Others have looked at those who did receive sur- gery, studying variation in utilisation of surgery accord- ing to factors such as geographical area [16] or socioeconomic status [17]. The Patient Reported Out- come Measures (PROMs) programme that is being car- ried out in the English National Health Service (NHS) has provided a new opportunity to explore access as it provides information on the severity and the duration of the joint problems just before surgery in a nationally representative sample [17]. © 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. Page 2 of 12 Page 2 of 12 Page 2 of 12 Podmore et al. BMC Health Services Research (2020) 20:602 Background of joint problems and in their duration according to the presence of chronic disease. A similar approach has been used previously to look at the impact of socioeconomic status [17, 18]. g Hip and knee replacement surgery is one of the most common and effective surgical treatments leading to sig- nificant improvements in quality of life [1]. Despite this in publicly funded healthcare systems such as England [2], Canada [3], and New Zealand [4] eligibility criteria restricting access to hip and knee replacement surgery, have recently been introduced to limit inappropriate use of joint replacement surgery and reduce healthcare cost. Eligibility criteria in England have included the severity of preoperative functional status [5] and pain [2], the re- quirement that a patient’s body mass index is lower than 30 kg/m2, and the optimisation of pre-existing chronic diseases [6–8]. There is no evidence, however, to suggest that limiting access according to any of these criteria is justified and these policies are not supported by clinical guidelines issued by the National Institute for Health and Care Excellence (NICE) [9]. Furthermore, in a com- prehensive systematic review of 70 studies, on outcomes of joint replacement surgery comparing patients with and without chronic diseases, the evidence does not sug- gest patients with chronic diseases benefit less from hip and knee replacement surgery [10]. In this paper, we therefore examined associations of the severity of joint problems (overall and separately in terms of pain and functional status) and the duration of the joint problems in patients with different chronic dis- eases just before hip or knee replacement surgery to get a better understanding of the impact that chronic dis- eases have on access to joint replacement surgery. Defining chronic diseases The 11 chronic diseases that were included in the ana- lysis were defined using ICD-10 codes in the linked hos- pital admissions HES data up to 1 year prior to the surgery. The 11 chronic diseases comprised heart disease, high blood pressure, problems caused by a stroke, leg pain when walking due to poor circulation, lung disease, diabetes, kidney disease, nervous system disease, liver disease, cancer, and depression. These chronic disease categories, used in the PROMs question- naire and based on the work of Bayliss et al. [21], were selected because it allowed using a combination of already existing ICD-10 diagnosis-based comorbidity in- dices (Elixhauser, Royal College of Surgeons Charlson and Quan Charlson Comorbidity Index). p Previous research investigating variation in access to joint replacement surgery has used two different ap- proaches. Some papers have measured access indirectly from a population perspective by focusing on those not receiving surgery and seeking to measure unmet need [15]. Others have looked at those who did receive sur- gery, studying variation in utilisation of surgery accord- ing to factors such as geographical area [16] or socioeconomic status [17]. The Patient Reported Out- come Measures (PROMs) programme that is being car- ried out in the English National Health Service (NHS) has provided a new opportunity to explore access as it provides information on the severity and the duration of the joint problems just before surgery in a nationally representative sample [17]. If there were differences in access, we might expect to see differences in the severity Data sources If there were differences in access, we might expect to see differences in the severity In a recent qualitative study, we explored the views of healthcare professionals in the English NHS about refer- ring and selecting patients with chronic diseases for joint replacement surgery [11]. These professionals reported that some patients with chronic diseases are not ‘pre- pared’ for surgery because their chronic diseases are not adequately controlled. As a result, these patients are often sent back to their general practitioner in primary care, fragmenting and delaying the surgical management of their joint problems, in some cases preventing surgery altogether [12]. In addition, such delays could lead to in- creased functional deterioration and pain of the osteo- arthritic hip and knees and thereby also increased costs. Advanced osteoarthritis of the hip and knee is associated with increased health service use and opioid use [13, 14]. Measures We used the Oxford Hip (OHS) and Knee Scores (OKS) as our measures of severity of joint problems just before surgery. These are derived from patient responses to 12 questions about pain and limits on physical functioning caused by the hip or the knee. Responses to each question are measured on a 5- point scale. Response values are added up to produce an overall scale from 0 (worst) to 48 (best). The OKS and OHS have been validated and found to correlate with surgeon assessment of symptoms [22]. Podmore et al. BMC Health Services Research (2020) 20:602 Page 3 of 12 Fig. 1 Data Flow Chart We also considered the questions related to pain and those related to functional status in the OHS and OKS separately. The severity of joint problems is typ- ically measured using disease-specific measures such as the OKS and OHS. The challenge of using these measures, which are designed to assess only the se- verity of the hip or knee problem, is that they may also be influenced by chronic diseases [23, 24]. To explore this further, we looked separately at the scores for pain (for example, night pain and sudden pain) and for functional status (for example, ability to go shopping on your own and climb a flight of stairs). We hypothesised that pain is more ‘joint-specific’ than functional status and that it is therefore less likely to be influenced by chronic diseases. This ap- proach has been used before to study the impact of pain and of functional status on patient satisfaction after hip and knee replacement surgery [25]. For the OKS, scores for the five questions on pain were added together as were those for the seven on func- tional status (see supplementary material). For the OHS, there were six questions each on pain and functional status. than 1 year’, ‘1–5 years’, ‘6–10 years’, and ‘More than 10 years’. We defined long-standing hip or knee problems as problems with a duration of joint problems of more than 5 years pre-operatively. Statistical analysis We estimated adjusted differences in mean preoperative pain and functional status using multivariable linear re- gression and calculated the mean scores according to the presence or absence of each chronic disease. We also estimated odds ratios (ORs) for having long-standing hip or knee problems for each chronic disease using multi- variable logistic regression. The impact of the number of chronic diseases (1, 2, 3, or ≥4) on the severity of joint problems and duration of joint problems was also inves- tigated to explore the effect of having multiple chronic diseases. We adjusted for sociodemographic factors including age, sex, ethnicity and socioeconomic status (Index of Multiple Deprivation [26]) and other chronic diseases. Hospitals were added as a random effect. Missing values for ethnicity, age, sex and socioeconomic status were im- puted with chained equations. Analyses were run on each of 10 imputed data sets and estimated parameters were combined using Rubin’s rules [27]. Statistical re- sults are presented with 95% confidence intervals and p- values. All statistical analysis were carried out using STATA V.15. A categorical measure of duration of joint problems was derived from responses to a single question asking patients how long they had experienced problems with their hip or the knee on which they were about to have surgery. The four response categories included ‘Less Page 4 of 12 Podmore et al. BMC Health Services Research (2020) 20:602 Table 1 Population characteristics Characteristic Hip replacement Knee replacement No. Patient characteristics Six hundred forty thousand eight hundred thirty-two pa- tients were eligible (Table 1). Their mean age was 68 and 41.8% were male. About 3% of patients reported a minority ethnic background with Black or Black British being the largest group but there was a high percentage of missing data. The most common chronic diseases were high blood pressure (52.8%), heart disease (17.8%), and lung disease (14.5%). The least common chronic dis- ease was liver disease (0.60%). 35.3% of patients had one chronic disease and 32.3% two or more. Severity of joint problems Patients with any of the 11 chronic diseases for both hip and knee replacement surgery reported more severe joint problems than patients without chronic diseases just before surgery (Table 2). For hip replacement sur- gery, adjusted differences in severity of joint problems ranged from 1.06 (95% CI 0.93, 1.19) for kidney disease to 2.49 (95% CI 2.31, 2.66) for diseases of the nervous system. For knee replacement surgery, adjusted differ- ences in severity of joint problems ranged from 0.46 (95% CI 0.26, 0.66) for cancer patients to 2.58 (95% CI 2.42, 2.73) for patients with diseases of the nervous sys- tem. The largest differences in severity of joint problems for both hip and knee replacement were reported by pa- tients with diseases of the nervous system, depression and liver disease and the smallest differences for high blood pressure, cancer and kidney disease. When looking at pain and functional status scores sep- arately, we found that patients with chronic diseases re- ported not only worse functional status but also more pain just before surgery than patients without chronic diseases for each of the 11 chronic diseases (Table 2). Similar to the overall OHS and OKS score, the stronger impact on both pain and functional status scores was found in patients with diseases of the nervous system and depression and the lowest in patients with kidney disease and cancer. Statistical analysis of patient 312,079 (48.7) 328,753 (51.3) Mean (SD) OHS or OKS 17.4 (8.25) 18.3 (7.87) Mean (SD) EQ-5D 0.33 (0.33) 0.39 (0.32) Long-standing problems, n (%) 57,827 (18.5) 141,559 (43.1) Age, mean (range) 68 (18–105) 69 (18–102) Gender, n (%) Male 126,925 (40.7) 140,971 (42.9) Female 184,982 (59.3) 187,525 (57.0) Missing, not stated 172 257 Socioeconomic status by quintile group, n (%) 1 (least deprived) 74,380 (23.4) 69,582 (21.2) 2 76,164 (24.4) 74,799 (22.8) 3 55,793 (17.9) 62,851 (19.1) 4 52,194 (16.7) 60,177 (18.3) 5 (most deprived) 50,408 (16.2) 58,327 (17.7) Missing 3140 3017 Ethnicity, n (%) White or White British 271,959 (98.3) 279,159 (94.5) Mixed background 546 (0.19) 836 (0.28) Asian or Asian British 1239 (0.45) 10,445 (3.53) Black or Black British 1703 (0.62) 3347 (1.13) Chinese or other ethnic 1150 (0.42) 1706 (0.58) Missing 35,482 33,260 Chronic disease, n (%) Heart disease 53,277 (17.1) 60,755 (18.5) High Blood pressure 151,163 (48.4) 187,815 (57.1) Stroke 3227 (1.03) 3530 (1.07) Leg pain due to poor circulation 5140 (1.65) 4955 (1.51) Lung Disease 43,481 (13.9) 51,176 (15.6) Diabetes 29,535 (9.46) 44,813 (13.6) Kidney Disease 16,428 (5.26) 18,000 (5.48) Diseases of the Nervous System 8483 (2.72) 9741 (2.96) Liver Disease 1888 (0.60) 1931 (0.59) Cancer 6354 (2.04) 5545 (1.69) Depression 13,367 (4.28) 14,814 (4.51) Count of chronic diseases, n (%) 0 113,479 (36.4) 94,290 (28.7) 1 107,139 (34.3) 119,012 (36.2) 2 59,976 (19.2) 75,202 (22.9) 3 22,929 (7.35) 29,761 (9.05) 4+ 8556 (2.74) 10,488 (3.19) Table 1 Population characteristics Long-standing joint problems 18.5% of patients who had hip replacements reported long-standing joint problems (> 5 years) and 43.1% of those who had knee replacements. The impact of differ- ent chronic diseases on the likelihood that patients re- ported long-standing hip or knee problems showed a mixed picture (Fig. 2). For hip replacement surgery, six chronic diseases increased the likelihood that patients reported long-standing problems whereas three others reduced it. For knee replacement, the likelihood that pa- tient reported long-standing problems was increased by two chronic diseases and decreased by two others. Page 5 of 12 Podmore et al. Long-standing joint problems BMC Health Services Research (2020) 20:602 Table 2 Preoperative severity of joint problems (OHS in hip replacement/OKS in knee replacement) separated by functional status and pain according to chronic diseases (adjusted according to age, sex, ethnicity, SES and other chronic diseases) Chronic disease Hip replacement OHS Knee replacement OKS Total (0 worse and 48 best) Functional status (0 worst 24 best) Pain (0 worst 24 best) Total (0 worse and 48 best) Functional status (0 worse and 28 best) Pain (0 worse 20 best) Mean score Adjusted difference (95% CI) Adjusted difference (95% CI) Adjusted difference (95% CI) Mean score Adjusted difference (95% CI) Adjusted difference (95% CI) Adjusted difference (95% CI) Heart disease No 17.7 – – – 18.4 – – – Yes 16.0 −1.29 (−1.37, −1.21) −0.68 (−0.72, −0.64) −0.61 (−0.65, −0.57) 17.6 −1.05 (−1.12, −0.98) −0.70 (−0.74, −0.66) −0.35 (−0.38, −0.32) High blood pressure No 18.2 – – – 18.9 – – – Yes 16.5 −1.22 (−1.29, −1.17) −0.69 (−0.73, −0.66) −0.54 (−0.57, −0.50) 17.8 −0.87 (−0.92, −0.81) −0.59 (−0.62, −0.55) −0.28 (−0.31, −0.26) Stroke No 17.4 – – – 18.3 – – – Yes 14.5 −1.39 (−1.67, −1.10) −0.81 (−0.96, −0.66) −0.57 (−0.72, −0.43) 16.2 −1.15 (−1.40, −0.89) −0.84 (−1.00, −0.68) −0.30 (−0.41, −0.19) Leg pain due to poor circulation No 17.4 – – – 18.3 – – – Yes 15.3 −1.28 (−1.50, −1.06) −0.62 (−0.74, −0.50) −0.66 (−0.78, −0.55) 17.4 −0.83 (−1.05, −0.62) −0.59 (−0.73, −0.46) −0.24 (−0.33, −0.15) Lung Disease No 17.7 – – – 18.6 – – – Yes 15.6 −1.49 (−1.57, −1.41) −0.70, (−0.75, −0.66) −0.79 (−0.83, −0.75) 16.7 −1.21 (−1.28, −1.14) −0.78 (−0.83, −0.74) −0.43 (−0.46, −0.40) Diabetes No 17.6 – – – 18.5 – – – Yes 15.7 −1.31 (−1.41, −1.21) −0.72 (−0.77, −0.67) −0.59 (−0.64, −0.53) 16.8 −1.26 (−1.34, −1.18) −0.84 (−0.89, −0.79) −0.42 (−0.45, −0.38) Kidney Disease No 17.5 – – – 18.3 – – – Yes 15.3 −1.06 (−1.19, −0.93) −0.60 (−0.67, −0.53) −0.46 (−0.53, −0.39) 17.1 −0.82 (−0.94, −0.71) −0.57 (−0.65, −0.50) −0.25 (−0.30, −0.20) Diseases of the Nervous System No 17.5 – – – 18.4 – – – Yes 14.4 −2.49 (−2.66, −2.31) −1.48 (−1.57, −1.39) −1.01 (−1.10, −0.91) 15.5 −2.58 (−2.73, −2.42) −1.87 (−1.96, −1.77) −0.71 (−0.78, −0.65) Liver Disease No 17.4 – – – 18.3 – – – Yes 14.0 −2.29 (−2.65, −1.93) −1.28 (−1.48, −1.08) −1.01 (−1.20, −0.82) 16.9 −1.30 (−1.64, −0.97) −0.90 (−1.11, −0.69) −0.41 (−0.55, −0.26) Cancer Page 6 of 12 Podmore et al. Long-standing joint problems BMC Health Services Research (2020) 20:602 Table 2 Preoperative severity of joint problems (OHS in hip replacement/OKS in knee replacement) separated by functional status and pain according to chronic diseases (adjusted according to age, sex, ethnicity, SES and other chronic diseases) (Continued) Chronic disease Hip replacement OHS Knee replacement OKS Total (0 worse and 48 best) Functional status (0 worst 24 best) Pain (0 worst 24 best) Total (0 worse and 48 best) Functional status (0 worse and 28 best) Pain (0 worse 20 best) Mean score Adjusted difference (95% CI) Adjusted difference (95% CI) Adjusted difference (95% CI) Mean score Adjusted difference (95% CI) Adjusted difference (95% CI) Adjusted difference (95% CI) No 17.4 – – – 18.3 – – – Yes 16.4 −1.22 (−1.42, −1.03) −0.72 (−0.83, −0.61) −0.50 (−0.61, −0.40) 18.6 −0.46 (−0.66, −0.26) −0.35 (−0.48, −0.22) −0.11 (−0.20, −0.03) Depression No 17.5 – – – 18.4 – – – Yes 14.6 −2.07 (−2.21, −1.93) −1.12 (−1.19, −1.04) −0.95 (−1.03, −0.88) 15.3 −1.98 (−2.10, −1.85) −1.31 (−1.38, −1.23) −0.67 (−0.73, −0.62) Table 2 Preoperative severity of joint problems (OHS in hip replacement/OKS in knee replacement) separated by functional status and pain according to chronic diseases (adjusted according to age, sex, ethnicity, SES and other chronic diseases) (Continued) Chronic disease Hip replacement OHS Knee replacement OKS Total (0 worse and 48 best) Functional status (0 worst 24 best) Pain (0 worst 24 best) Total (0 worse and 48 best) Functional status (0 worse and 28 best) Pain (0 worse 20 best) Mean score Adjusted difference (95% CI) Adjusted difference (95% CI) Adjusted difference (95% CI) Mean score Adjusted difference (95% CI) Adjusted difference (95% CI) Adjusted difference (95% CI) No 17.4 – – – 18.3 – – – Yes 16.4 −1.22 (−1.42, −1.03) −0.72 (−0.83, −0.61) −0.50 (−0.61, −0.40) 18.6 −0.46 (−0.66, −0.26) −0.35 (−0.48, −0.22) −0.11 (−0.20, −0.03) Depression No 17.5 – – – 18.4 – – – Yes 14.6 −2.07 (−2.21, −1.93) −1.12 (−1.19, −1.04) −0.95 (−1.03, −0.88) 15.3 −1.98 (−2.10, −1.85) −1.31 (−1.38, −1.23) −0.67 (−0.73, −0.62) Page 7 of 12 Podmore et al. BMC Health Services Research (2020) 20:602 Fig. 2 Impact of chronic diseases on long-standing joint problems (duration> 5 years pre-operatively) (95% CI) (adjusted according to age, sex, ethnicity, SES and other chronic disease) Fig. Long-standing joint problems 2 Impact of chronic diseases on long-standing joint problems (duration> 5 years pre-operatively) (95% CI) (adjusted according to age, sex, ethnicity, SES and other chronic disease) Multiple chronic diseases had a knee replacement, the adjusted differences in- creased from 1.06 (95% CI 0.99, 1.12) for patients with one chronic disease to 4.79 (95% CI 4.64, 4.94) for patients with four or more chronic diseases. We observed a similar gradient both in hip and in knee replacement surgery patients when looking at pain and functional status separately. In patients with four chronic diseases, irrespective of whether they had a hip or a knee replacement, the most common com- bination was high blood pressure, heart disease, dia- betes, and lung disease. The severity of joint problems just before surgery in- creased with the number of chronic diseases (Table 3). Compared with patients without chronic diseases who had a hip replacement, the adjusted differences in OHS increased from 1.45 (95% CI 1.38, 1.52) for pa- tients with one chronic disease to 2.79 (95% CI 2.70, 2.87) for patients with two chronic diseases. The ad- justed differences were largest for patients with four or more chronic diseases (5.79, 95% CI 5.61, 5.96). Compared with patients without chronic diseases who Page 8 of 12 Podmore et al. Discussion This study demonstrates that compared to patients with- out chronic diseases, patients with chronic diseases, es- pecially those with liver disease, depression, and diseases of the nervous system, reported more severe joint prob- lems immediately before undergoing a hip or knee re- placement. Patients with chronic diseases reported not only worse functional status but also more pain just be- fore surgery which suggests that patients with chronic diseases have truly worse joint problems regardless of any direct impact of chronic diseases on the disease- specific measure. When looking at the number of chronic diseases, the severity of the joint problems in- creased with the number of chronic diseases. Patients with chronic diseases however reported little to no dif- ference in duration of their joint problems compared to patients without chronic diseases although the difference was larger in patients undergoing hip replacement com- pared to patients undergoing knee replacement. We acknowledge that our finding of an association be- tween the presence of chronic conditions and more se- vere joint problems does not prove a causal relationship. However, our interpretation is supported by the findings from our previous qualitative study which explored the views of healthcare professionals on the referral and se- lection of patients with chronic diseases for hip or knee replacement surgery in the English NHS [11]. This study showed that chronic diseases may create subtle barriers, for example when patients who are considered to be un- prepared for surgery are referred back to their general practitioner in primary care. The observed differences in severity of joint problems were small but statistically significant for all of the 11 different chronic diseases. To interpret the size of the difference, they can be compared with defined ‘minim- ally important differences’ (MID), the smallest important differences in scores that patients report as beneficial. Suggested MID values are five points for both the OHS and OKS [28]. Only hip and knee patients with four or more chronic diseases reported differences in OHS and OKS scores more than five points larger than patients without chronic diseases. Furthermore, delays in access in patients with chronic conditions may be linked to patients’ reluctance to undergo surgery [31] or to clinicians’ uncertainty about the indications for replacement surgery [32–34]. Multiple chronic diseases BMC Health Services Research (2020) 20:602 Table 3 Preoperative severity of joint problems (OHS in hip replacement and OKS in knee replacement) and likelihood of long-standing problems by the number of chronic diseases (95% CI, P-value for trend) (adjusted according to age, sex, ethnicity and SES) Count of chronic diseases Severity of joint problems (0 worse and 48 best) Functional status (0 worst 24 best) Pain (0 worst 24 best) Long-standing joint problems (duration > 5 years pre-operatively) Mean score Adjusted difference (95% CI) P- value Mean score Adjusted difference (95% CI) P- value Mean score Adjusted difference (95% CI) P- value % Adjusted OR (95% CI) P- value Hip replacement:OHS 0 18.8 Reference 10.6 Reference 8.21 Reference 21.3 Reference 1 17.4 −1.45 (−1.52, −1.38) < 0.001 9.73 −0.79 (−0.83, −0.76) < 0.001 7.67 −0.66 (−0.69, −0.62) < 0.001 17.3 0.93 (0.91, 0.95) 0.076 2 16.2 −2.79 (−2.87, −2.70) 9.03 −1.52 (−1.56, −1.47) 7.17 −1.28 (−1.32, −1.23) 16.5 0.98 (0.95, 1.00) 3 14.9 −4.15 (−4.27, −4.04) 8.31 −2.23 (−2.30, −2.17) 6.58 −1.92 (−1.98, −1.86) 16.1 1.02 (0.98, 1.06) 4+ 13.3 −5.79 (−5.96, −5.61) 7.42 −3.13 (−3.23, −3.03) 5.89 −2.66 (−2.75, −2.56) 17.5 1.15 (1.09, 1.23) Knee replacement:OKS 0 19.5 Reference 12.5 Reference 7.01 Reference 45.7 Reference 1 18.5 −1.06 (−1.12, −0.99) < 0.001 11.7 −0.72 (−0.76, −0.68) < 0.001 6.77 −0.34 (−0.37, −0.31) < 0.001 42.2 0.95 (0.93, 0.97) 0.663 2 17.6 −2.16 (−2.24, −2.09) 11.0 −1.50 (−1.51, −1.42) 6.51 −0.70 (−0.73, −0.67) 41.9 0.97 (0.95, 0.99) 3 16.5 −3.38 (−3.48, −3.28) 10.3 −2.28 (−2.34, −2.22) 6.17 −1.10 (−1.15, −1.06) 41.8 0.98 (0.96, 1.01) 4+ 15.1 −4.79 (−4.94, −4.64) 9.39 −3.21 (−3.31, −3.11) 5.75 −1.58 (−1.64, −1.51) 41.9 0.99 (0.95, 1.04) Page 9 of 12 Page 9 of 12 Podmore et al. BMC Health Services Research (2020) 20:602 Podmore et al. BMC Health Services Research (2020) 20:602 been reported to be a significant predictor of poorer out- come after surgery [30]. The impact of the number of chronic diseases on the likelihood of reporting long-standing joint problems was inconsistent. In hip patients, only patients with four or more chronic diseases were more likely to report long- standing problems (OR 1.15, 95% CI 1.09, 1.23). In knee patients, an increasing number of chronic diseases had no impact on the likelihood of patients reporting long- standing problems. Multiple chronic diseases This study is the first to examine the relationship be- tween chronic diseases and patient-reported pain, func- tional status and duration of joint problems immediately before surgery in a large representative sample of pa- tients. One possible interpretation of our findings is that the severity of joint problems at the time of surgery rep- resents delays in access to surgery, although our findings do not give an indication about the length of these de- lays. It can also be argued that our observation that pa- tients with chronic disease report more severe joint problems than patients without may also be explained by information bias (e.g. patients with more severe joint problems are more likely to have other comorbidities re- corded) or that pre-operative conservative treatment may be more effective in patients without than in pa- tients with comorbidities. Discussion In pre- vious studies with different groups of healthcare profes- sionals, the presence of chronic diseases was found reported to be a reason that surgery should not be rec- ommended because of the increased risks of surgery [35]. This is also one of the arguments to justify the re- stricted eligibility criteria for hip and knee replacement imposed by some regional commissioners of healthcare in England who require that chronic diseases are opti- mised before surgery [6–8]. Our findings about the duration of joint problems were not in alignment with the findings related to the severity of joint problems just before surgery. These in- consistent results may be due to recall bias, because pa- tients may find it difficult to remember the actual onset of their hip or knee problems. Patients were asked, ‘How long they had experienced problems with the hip or the knee on which they were about to have surgery?’ In re- sponse to this question, patients may have reported the duration of problems of their most recent episode with a specific level of severity rather than their overall dur- ation [29]. Previous studies have reported that this may be due to the lack of clarity of the question that was used to elicit information about symptom duration [29]. Nevertheless, increasing symptom duration has recently Delays to surgery have also been linked to patient health-seeking behaviour and reluctance to undergo sur- gery [36]. Firstly, differences in thresholds for pain may explain variation in seeking clinical advice or having sur- gery. There is evidence that people from more socio- economically deprived backgrounds – who are also more likely to have chronic diseases [37] – tend to accept a higher threshold of chronic pain and functional limita- tion before having surgery [38]. Secondly, a number of studies have reported differences in patient preferences and expectations for joint replacement surgery. For Page 10 of 12 Podmore et al. BMC Health Services Research (2020) 20:602 surgery [10]. This suggests that patients with chronic diseases do not benefit significantly less from hip and knee replacement surgery. example, it was shown that elderly people may prefer to delay surgery and manage the pain and the limited mo- bility rather than undergo a surgery with risks of compli- cations [39]. This may be similar for patients with chronic diseases given their higher risks of short-term complications after surgery [10]. Funding This work was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care North Thames at Barts Health NHS Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. The funding body did not have any role in the design of the study and collection, analysis and interpretation of the data and in writing the manuscript. Ethics approval and consent to participate The study was approved by the Health Research Authority NHS Research Ethics Committee (Reference: 16/WA/0241) as well as the London School of Hygiene & Tropical Medicine ethics committee (Reference: 11628). Acknowledgements The authors acknowledge NHS Digital for the Patient Reported Outcome Measures data and the Hospital Episode Statistics Inpatient Admissions data linkage. Discussion Thirdly, patients with chronic diseases may also prioritise the treatment of their chronic diseases rather than starting surgical treat- ment of their joint problems [40]. Abbreviations HES: Hospital Episode Statistics; ICD-10: International Classification of Diseases, 10th Revision; MID: Minimally Important Difference; NICE: National Institute for Health and Care Excellence; OHS: Oxford Hip Score; OKS: Oxford Knee Score; PROMs: Patient Reported Outcome Measures programme. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05464-3. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05464-3. Additional file 1. Breakdown of OHS and OKS by pain and functional status questions (P = pain, F = functional status). Furthermore, it is likely that there is a ‘healthy-surgical patient effect’. This effect may have contributed to an underestimation of the effect of chronic diseases as a consequence of the selection of patients for surgery be- ing influenced by ‘unmeasured’ or ‘unobserved’ con- founders that are not accounted for. As a result, patients with chronic diseases who had surgery may be less frail and less severe than patients with a similar chronic dis- ease profile in the general population [41]. In addition, due to the lack of a control group (patients with comor- bidities who have not had joint replacement surgery) it is not possible to fully account for this selection bias. Authors’ contributions BP designed the study, conducted the main study, analysis and wrote the manuscript. AH helped design the study design, analysis and with the drafting of the manuscript. SK helped with the analysis of the manuscript and revised the manuscript. JR helped with helped with the analysis of the manuscript and revised the manuscript. JvM helped design the study design, with the analysis and the drafting of the manuscript. All authors have read and approved the manuscript. Also, our sample of patients represents 71% of all pa- tients who had a hip or knee replacement in the English NHS between 2009 and 2016. While the response rate to the PROMs survey is high, non-recruitment may have led to confounders being unevenly distributed between different groups of patients and hospitals [42]. This was especially apparent with only 3% of patients reported to be from a minority ethnic group, although this may be explained in part by the high percentage of missing data (> 10%). To account for this, we adjusted for clustering of outcomes within hospitals and for socioeconomic fac- tors. This adjustment had a minimal impact on the findings. Availability of data and materials The data that support the findings of this study are available from NHS Digital but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. The negative consequences of the presence of comor- bidities before surgery also need to be interpreted in the context of the outcomes after joint replacement surgery. In our previous systematic review of 70 studies looking at 10 outcomes comparing patients with and without pa- tients chronic diseases, we found that chronic diseases predominantly had an impact on the safety e.g. compli- cations) but little impact on the effectiveness (e.g. func- tional and pain outcomes) after joint replacement Conclusions In conclusion, patients with chronic diseases undergoing hip or knee replacement surgery reported more severe pain and a poorer functional status immediately before surgery than patients without chronic diseases. These findings suggest that on average patients with chronic diseases have hip or knee replacement later in the course of their joint disease, likely as a result of delays in access to surgery. There were several study limitations. First, it is import- ant to mention in this context is that we could not ad- just for the severity of the chronic diseases because the administrative data could capture the presence of chronic conditions but not their severity. This is espe- cially important for the interpretation of the observed association between of the number of chronic conditions and the severity of the joint problems at the time of sur- gery. It is likely that we would have found an even stron- ger association if we had been able to take severity of the chronic conditions into account. References 23. Wylde V, Learmonth ID, Cavendish VJ. The Oxford hip score: the patient’s perspective. Health Qual Life Outcomes. 2005;3(1):66. 23. Wylde V, Learmonth ID, Cavendish VJ. The Oxford hip score: the patient’s perspective. Health Qual Life Outcomes. 2005;3(1):66. 1. Ethgen O, Bruyere O, Richy F, Dardennes C, Reginster JY. Health-related quality of life in total hip and total knee arthroplasty. 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BMJ. 2010;341:c4092. 39. Hudak PL, Clark JP, Hawker GA, Coyte PC, Mahomed NN, Kreder HJ, et al. “You’re perfect for the procedure! Why don’t you want it?” elderly arthritis patients’ unwillingness to consider total joint arthroplasty surgery: a qualitative study. Med Decis Mak. 2002;22(3):272–8. 16. Martin CT, Callaghan JJ, Liu SS, Gao YB, Warth LC, Johnston RC. Disparity in Total joint Arthroplasty patient comorbidities, demographics, and postoperative outcomes based on insurance payer type. J Arthroplasty. 2012;27(10):1761–5. 17. Neuburger J, Hutchings A, Allwood D, Black N, van der Meulen JH. Sociodemographic differences in the severity and duration of disease amongst patients undergoing hip or knee replacement surgery. J Public Health (Oxf). 2012;34(3):421–9. 40. Pugh JA. Priority setting for patients with multiple comorbidities: diabetes may not end up number one. J Gen Intern Med. 2007;22(12):1783–4. 17. Neuburger J, Hutchings A, Allwood D, Black N, van der Meulen JH. Sociodemographic differences in the severity and duration of disease amongst patients undergoing hip or knee replacement surgery. J Public Health (Oxf). 2012;34(3):421–9. 41. Dreinhofer KE, Dieppe P, Sturmer T, Grober-Gratz D, Floren M, Gunther KP, et al. Indications for total hip replacement: comparison of assessments of Page 12 of 12 Podmore et al. BMC Health Services Research (2020) 20:602 Podmore et al. BMC Health Services Research (2020) 20:602 orthopaedic surgeons and referring physicians. Ann Rheum Dis. 2006;65(10): 1346–50. 42. Hutchings A, Neuburger J, van der Meulen J, Black N. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2621590165	https://dergipark.org.tr/en/download/article-file/343039	Turkish	null	A case with MTHFR gene mutation after secondary cirrhosis to portal vein thrombosis	Ortadoğu tıp dergisi	2,017	cc-by	2,265	Öz Portal ven trombozu; portal hipertansiyonun sebeplerinden bir tanesidir. Portal venin tıkanması, yavaş ve iyi tolere edilebilen bir durum olabileceği gibi, şiddetli ve potansiyel olarak ölümcül bir durumda olabilir. Trombofili, trom boza eğilim oluşturan durumları tanımlayan bir terimdir. Trombofili çok yaygın bir fenomen değildir ve edinsel ve kalıtsal sebepleri vardır. Trombofilik anormallikler, portal ven trombusu gelişiminde rol oynarlar. Kalıtsal faktörler arasında Faktör V Leiden mutasyonu, Protrombin 20210A mutasyonu, protein S ve C eksiklikleri ve antitrombin 3 eksikliği en sık bilinen ve trombofilik hastalarda en sık etiyolojik faktör olarak sorgulanan faktörlerdir. Hiperhomo sisteinemi ise hem kalıtsal ve hemde edinsel nedenler ile gelişebilen bir risk faktörüdür. MTHFR enzimi homosis teinin metionine dönüşmesinde rol oynar. MTHFR mutasyonu olduğunda homosistein metionine dönüştürülemez ve hiperhomosisteinemi meydana gelir. Homosisteinin kanda yükselmesi kan damarlarında irritasyona neden olur ve sonuçta kan damarlarında pıhtı oluşması için artmış bir risk faktörüdür. Bu olgu sunumu ile portal ven trombozu bulunan ve karaciğer sirozu ile kliniğimize başvuran, etiyolojik araştırmalar neticesinde MTHFR geni homozigot mutasyonu tespit edilen 37 yaşındaki bayan hastayı sunmayı amaçladık. Anahtar Kelimeler: Portal ven, kalıtsal trombofili, tromboz, metilentetrahidrofolat redüktaz A case with MTHFR gene mutation after secondary cirrhosis to portal vein thrombosis Kenan Çadırcı1, Ahmet Yalçın2 ¹Bölge Eğitim ve Araştırma Hastanesi, İç Hastalıkları Kliniği, Erzurum, Türkiye ²Bölge Eğitim ve Araştırma Hastanesi, Radyoloji Kliniği, Erzurum, Türkiye Geliş Tarihi: 15.12.2016 Kabul Tarihi: 01.02.2017 Doi: 10.21601/ortadogutipdergisi.277896 Kenan Çadırcı1, Ahmet Yalçın2 ¹Bölge Eğitim ve Araştırma Hastanesi, İç Hastalıkları Kliniği, Erzurum, Türkiye ²Bölge Eğitim ve Araştırma Hastanesi, Radyoloji Kliniği, Erzurum, Türkiye ¹Bölge Eğitim ve Araştırma Hastanesi, İç Hastalıkları Kliniği, Erzurum, Türkiye ²Bölge Eğitim ve Araştırma Hastanesi, Radyoloji Kliniği, Erzurum, Türkiye 5.12.2016 Kabul Tarihi: 01.02.2017 Doi: 10.21601/ortadogutipdergisi.277896 eliş Tarihi: 15.12.2016 Kabul Tarihi: 01.02.2017 Doi: 10.21601/ortadogutipdergi aka sunumu v aka sunumu v Giriş Portal ven trombozu (PVT), portal venin herhangi bir ye rinde trombüs gelişmesi ile karakterize bir hastalıktır. Por tal ven trombozu portal hipertansiyonun sebeplerinden bir tanesidir. Portal venin tıkanması, yavaş ve iyi tolere edi lebilen bir durum olabileceği gibi, şiddetli ve potansiyel olarak ölümcül bir durumda olabilir [1,2]. Abstract Portal vein thrombosis is one of the causes of portal hypertension. Portal vein thrombosis can be a cause slowly and well tolerance, and severely and mortal. Thrombophilia, forming tendency of thrombosis is a term that describes situations. Thrombophilia is not a very common phenomenon and has acquired or inherited causes. Thrombophilic abnormalities may have a role in the development of portal vein thrombosis. Inherited factors include the Factor V Leiden and prothrombin 20210A mutation, protein S, C, and antithrombin 3 deficiency is the most common and known thrombophilic patients are questioned as to why the most common etiological factor. Hyperhomocysteinemia is a risk factor that can develop with both hereditary and acquired causes. The MTHFR enzyme plays a role in the conversion of homocysteine to methionine. When the MTHFR mutation is present, homocysteine can not be con verted to methionine and hyperhomocytheinemia occurs. Elevated homocysteine levels may cause irritation of the blood vessels and its show an increased risk for eventually result in a blood clots in the veins. In this case presenta tion, we report a 37 year old woman patient with portal vein thrombosis and to apply our clinic with liver cirrhosis that outcome MTHFR homozygous gene mutation. words: Portal vein, Hereditary thrombophilia, thrombosis, methylenetetrahydrofolate reductase 146 ORTADOGU MEDICAL JOURNAL 9 (3): 146-149, 2017 Portal ven trombozuna sekonder siroz gelişen MTHFR gen mutasyon olgusu kilen batın ultrasonografisinde ‘Karaciğer normalden kü çük olup, karaciğer sol ve sağ lob medial seğmentleri at rofik olarak değerlendirildi ve dalak uzun aksı 200 mm ve splenik ven tortioze olarak izlenmektedir’ şeklinde yorum lanmıştı. Hastaya bunun üzerine dinamik batın bilgisayarlı tomografi (BT) çekildi. Portal fazda çekilen kontrastlı ak siyel ve koronal görüntülerde portal venin ekstra-hepatik seğmentinde trombüs izlendi (Resim 1A). Dalak hilusunda yaygın kollateral vasküler yapılar mevcuttu (Resim 1B). Yapılan endoskopisinde özafagus varisi (F2) tespit edilen hastaya bunun üzerine Nadroparine calcium 0,6 ml 2x1 tedavisi başlanıldı. Portal ven trombüsü etiyolojisine yö nelik olarak yapılan tetkiklerinde Homosistein değeri 20,6 µmol/L (5-15) ile yüksek olarak tespit edildi. Protein C ve protein S eksikliği, Faktör V Leiden (1691G/A), Faktör ΙΙ (20210 G/A) mutasyonları tespit edilmezken MTHFR 677 C/T mutasyonu homozigot olarak tespit edildi. Hastada özafagus varisleri olması nedeniyle Nadroparine calcium tedavisi önerilerek kontrollere çağrıldı. Olgu Sunumu Otuzyedi yaşında bayan hasta kliniğimize yaklaşık olarak bir yıldır süren karın ağrısı ve halsizlik şikayeti ile baş vurdu. Hastanın hikayesinden yaklaşık olarak bir yıl önce karın ağrısı şikayeti ile başvurduğu dış merkezli bir sağlık kuruluşu tarafından yapılan tetkikleri neticesinde dalağın da büyüme olduğu şeklinde bilgi verilerek ilaç önerildiği öğrenildi. Mevcut şikayetlerinde rahatlama olmayan has tanın kliniğimizde yapılan muayenesinde cilt ve konjonk tivalar soluk, dalak palpabl olarak tespit edildi. Hastanın eskiye ait tetkikleri incelendiğinde yaklaşık 1 yıl önce çe Resim 1A. Portal fazda elde olunan koronal kontrastlı BT’de portal venin ekstra-hepatik segmentinde trombus (oklar) Resim 1B. Portal fazda elde edilen aksiyel kontrastlı BT’de dalak hilusunda yaygın kollateral vasküler yapılar (asterisk) Resim 1A. Portal fazda elde olunan koronal kontrastlı BT’de portal venin ekstra-hepatik segmentinde trombus (oklar) Resim 1A. Portal fazda elde olunan koronal kontrastlı BT’de portal venin ekstra-hepatik segmentinde trombus (oklar) Resim 1B. Portal fazda elde edilen aksiyel kontrastlı BT’de dalak hilusunda yaygın kollateral vasküler yapılar (asterisk) Resim 1B. Portal fazda elde edilen aksiyel kontrastlı BT’de dalak hilusunda yaygın kollateral vasküler yapılar (asterisk) B. Portal fazda elde edilen aksiyel kontrastlı BT’de dalak hilusunda yaygın kollateral vasküler yapılar (asterisk) Tartışma MTHFR, lipit, protein, membran ve DNA’nın metilasyo nunda ve nükleotidlerin sentezi için önemlidir ve tek kar bon döngüsü içinde yer almaktadır. En yaygın MTHFR polimorfizmleri C677T ve A1298C’dir. Bu gen mutasyon ları sonucu enzim aktivitesi azalmakta ve orta dereceli bir hiperhomosisteinemi meydana gelmektedir [7]. Venöz tromboemboli (VTE), genellikle hemostazda rol alan düzenleyici proteinlerdeki kalıtsal veya edinsel deği şiklikler sonucu meydana gelir [1]. Venöz tromboz oluşu munda iki ana mekanizma, antikoagülan proteinlerin azal ması veya fonksiyon kaybı ile prokoagülan proteinlerdeki fonksiyon artması olarak sayılabilir [2]. Hiperhomosisteinemi tanısında ilk aşamada, serumda açlık ve metionin yüklenmesinden sonra homosistein düzeyle rinin tayini önerilmektedir. Normalde açlık plazma homo sistein konsantrasyonu 5-15 μmol/L dir. Homosisteinemiler hafif (15-30 μmol/L), orta ( 30-100 μmol/L) ve ciddi (>100 μmol/L) homosisteinemiler olmak üzere 3 gruba ayrılır [8]. Bizde hastamızın açlık serum homosistein düzeyini 20,6 ile orta derecede yükselmiş olarak tespit ettik. VTE için kabul edilmiş risk faktörleri; kalıtımsal veya ka zanılmış olarak sınıflanabilir. İleri yaş, uzun süreli immo bilizasyon, cerrahi travma, kırıklar, oral kontraseptif (OK) kullanımı, hormon replasman tedavisi, gebelik, puerperi um, kanser, antifosfolipit sendromu, kazanılmış risk fak törlerini oluşturur [3]. Bizim hastamız mevcut kazanılmış risk faktörlerine sahip değildi. Kalıtsal trombofili nedenleri arasında Faktör V Leiden mutasyonu, Protrombin G20210 A mutasyonu, Protein S ve C eksikliği, Antitrombin eksikliği, disfibrinojenemi ve Faktör XIII 34 val sayılabilir [4]. Literatürde MTHFR mutasyonlarına bağlı meydana gelmiş venöz tromboz olguları bildirilmiştir. Gürsoy ve arkadaşları tarafından karın ağrısı ile başvuran inaktif ülseratif kolitli bir erkek hastada heterozigot MTHFR mutasyonu sonucu meydana gelen kombine portal, splenik ve mezenterik ven trombozu olgusu sunulmuştur [7]. Bizim vakamızda da MTHFR mutasyonu homozigot olarak tespit edildi. Hiperhomosisteinemi, hem arteriyel hem de venöz trom boza neden olabildiği gösterilmiş bir trombofili nedenidir. Kalıtsal ve edinsel nedenler ile gelişebilmektedir. Hiper homosisteinemi, metabolizması ile ilişkili olan enzimle rin (sistatyonin-β-sentetaz, methionin sentetaz ve metilen tetrahidrofolat redüktaz (MTHFR)), konjenital eksiklikle rine bağlı olabileceği gibi folik asit ve vitamin B12 gibi kofaktör olarak rol oynayan vitaminlerin diyette yetersiz alımıyla ilişkili de olabilir [5]. Hiperhomosisteinemi, hem arteriyel hem de venöz trom boza neden olabildiği gösterilmiş bir trombofili nedenidir. 147 ORTADOĞU TIP DERGİSİ 9 (3): 146-149, 2017 147 ORTADOĞU TIP DERGİSİ 9 (3): 146-149, 2017 A case with MTHFR gene mutation after secondary cirrhosis to portal vein thrombosis zukluklar ve bazı kanser türlerinde rol oynamaktadır. zukluklar ve bazı kanser türlerinde rol oynamaktadır. Tartışma Circulation 2005; 111: 289-93 Zhou ve arkadaşları tarafından yapılan bir çalışmada, si rozlu hastalarda MTHFR mutasyonunun kontrol grubuna göre anlamlı derecede yüksek olarak tespit edildiği, benzer olarak homosistein düzeylerinin de yüksek olduğu ve hi perhomosisteineminin karaciğer sirozu için bağımsız bir risk faktörü olabileceği bildirilmiştir [12]. 7. Gürsoy G, Çimbek A, Acar Y, et al. Combined portal, splenic and mesenteric venous thrombosis in inactive ulcerative colitis with heterozygous mutation in MTHFR gene: A rare case of thrombop hilia. J Res Med Sci 2011;16: 1500–6. Kalıtımsal trombofilik anormallikler, portal ven trombüsü gelişiminde rol oynarlar. Bunlar arasında Faktör V Leiden mutasyonu, Protrombin 20210A mutasyonu, protein S ve C eksiklikleri ve antitrombin 3 eksikliği en sık bilinen ve trombofilik hastalarda en sık etiyolojik faktör olarak sor gulanan faktörlerdir. 8. Küçükkaya RD, Aydın M. Trombofili genetiği. Türk Hematoloji Derneği. Moleküler Hematoloji Kursu.www.thd.org.tr/thdData/ userfiles/file/molhem_13.pdf 01.08.2016. 9. 9. 9. Bayan K, Tüzün Y, Yılmaz S, Canoruç N, Dursun M. Analysis of inherited thrombophilic mutations and natural anticoagulant de ficiency in patients with idiopatic portal hypertension. J Thromb Thrombolysis 2009;28:57-62. Biz de portal ven trombüsü tespit ettiğimiz ve yaptığımız araştırma neticesinde Faktör V Leiden mutasyonu tespit etti ğimiz 28 yaşında genç erkek bir olgu sunumu yapmıştık [13]. Sonuç olarak; karın ağrısı ve/veya karaciğer fonksiyon test lerinde anormallikle başvuran bir hastada etiyolojik amaçlı olarak veya herhangi bir sebeple yapılan görüntüleme tet kikleri neticesinde portal vende trombüs tespit edilen, özel likle genç olgularda trombofilik mutasyonların araştırılma sı hem tedavi açısından farklı bir bakış açısı kazandıracak ve hem de hastanın daha sonraki bir embolik/ trombotik olaya maruz kalmasının önüne geçilmiş olacaktır. Biz de portal ven trombüsü tespit ettiğimiz ve yaptığımız araştırma neticesinde Faktör V Leiden mutasyonu tespit etti ğimiz 28 yaşında genç erkek bir olgu sunumu yapmıştık [13]. 10. Elhajj II, Salem ZM, Birjawi GA, Taher AT, Soweid AM. Hete rozygous prothrombin 20210G/A mutation, associated with hyper homocysteinemia and homozygous methylenetetrahydrofolate re ductase 677C/T mutation in a patient with portal and mesenteric venous thrombosis. Hematol J 2004;5:540-2. 11. Gabr MA, Bessa SS, El-Zamarani EA. Portal vein thrombosis in Egyptian patients with liver cirrhosis: role of methylenetetrahy drofolate reductase C677T gene mutation. Hepatol Res 2010; 40:486-93. Maddi Destek ve Çıkar İlişkisi 12. Zhou XM, Lin JS, Sun XM, et al. The relationship between the plasma homocysteine level and the polymorphism of MHFR gene C677T in liver cirhosis. Zhonghua Gan Zang Bing Za Zhi 2005;:908-10. Çalışmayı maddi olarak destekleyen kişi/kuruluş yoktur ve yazarların çıkara dayalı bir ilişkisi yoktur. Çalışmayı maddi olarak destekleyen kişi/kuruluş yoktur ve yazarların çıkara dayalı bir ilişkisi yoktur. Tartışma Bayan ve arkadaşları tarafından idiopatik portal ven trom büsü olan hastalarda, MTHFR C677T ve MTHFR A1298C mutasyonlarının da içinde olduğu FaktörV R506Q, Fak törV H1299R, protrombin 20210 A ve Faktör XIII V34L gibi kalıtsal trombofilik mutasyonların analiz edildiği bir çalışmada, ortalama Protein C ve Antitrombin 3 düzeyle rinin kontrol grubuna göre düşük olduğu, ayrıca MTHFR C677T , MTHFR A1298C, Faktör XIII V34L, PAI-1 ( Plazminojen aktivatör inhibitör-1) ve GP IIIa L33P gibi genetik polimorfizm frekanslarının ise kontrol grubuna göre daha yüksek olarak bulunduğu bildirilmiştir [9]. Yüksek homosistein düzeylerine ayrıca böbrek yetmezli ğinde, tiroid hormon düzeylerinin düşük olduğu durumlar da, psöriazis hastalığında, bazı ilaçların kullanımına bağlı olarak (antiepileptik ve metotreksat) ve MTHFR gen mu tasyonuna bağlı olarak da rastlanılabilir [6]. MTHFR enzimi 5,10-metilen tetrahidrofolatı 5-metil tet rahidrofolata dönüştürür. Bu bileşik homosisteinin metio nine dönüşmesinde rol oynar. Bu enzimin mutasyonların da homosistein metionine dönüştürülemediğinden dolayı kanda homosistein birikir ve hiperhomosisteinemi olarak adlandırılan durum ortaya çıkar. MTHFR geni ile ilişkili çeşitli polimorfizmler nöral tüp defektleri ile ilişkili bu lunmuştur. MTHFR genindeki polimorfizm kalp hastalığı, inme, hipertansiyon, preeklampsi, glokom, psikiyatrik bo Elhajj ve arkadaşları tarafından heterozigot faktör II 20210 G/A ve homozigot MTHFR 677C/T mutasyonu olan ve hi perhomosisteinemisi tespit edilen, karın ağrısı olması üze rine yapılan tetkiklerinde portal ve mezenterik ven trom büsü tespit edilen bir erkek hasta bildirmişlerdir [10]. Sirozlu hasta gruplarında da trombofilik mutasyonların varlığı birçok çalışma ile araştırılmıştır [11,12]. Gabr ve arkadaşları tarafından Mısır’da yapılan bir çalışma 148 ORTADOGU MEDICAL JOURNAL 9 (3): 146-149, 2017 Portal ven trombozuna sekonder siroz gelişen MTHFR gen mutasyon olgusu 5. Beyan C. Trombofilili hastada tanısal yaklaşım. Türk Hematoloji Derneği - Temel Hemostaz Tromboz Kursu 68-75. da MTHFR homozigot mutasyonunun PVT’ lu sirozlu has talarda artmış risk ile ilişkili olduğunu bildirmişlerdir [11]. da MTHFR homozigot mutasyonunun PVT’ lu sirozlu has talarda artmış risk ile ilişkili olduğunu bildirmişlerdir [11]. Zhou ve arkadaşları tarafından yapılan bir çalışmada, si rozlu hastalarda MTHFR mutasyonunun kontrol grubuna göre anlamlı derecede yüksek olarak tespit edildiği, benzer olarak homosistein düzeylerinin de yüksek olduğu ve hi perhomosisteineminin karaciğer sirozu için bağımsız bir risk faktörü olabileceği bildirilmiştir [12]. 6. Varga EA, Sturm AC, Misita CP, Moll S. Homocysteine and MTHFR mutations relation to thrombosis and coronary artery di sease. Kaynaklar 13. Çayır K, Çadırcı K, Bilici M, Tekin SB, Keleş M, Emre H. Faktör V Leiden mutasyonuna bağlı gelişen portal ven trombüsü. Haseki Tıp Bülteni 2009;47:181-2. 1. Hooper WC, Evatt BL. The role of activated protein C resis tance in the pathogenesis of venous thrombosis. Am J Med Sci 1998;316:120-8. 1. Hooper WC, Evatt BL. The role of activated protein C resis tance in the pathogenesis of venous thrombosis. Am J Med Sci 1998;316:120-8. 2. Martinelli I, De Stefano V, Mannucci PM. Inherited risk factors for venous thromboembolism. Nat Rev Cardiol 2014; 11:140-56. 2. Martinelli I, De Stefano V, Mannucci PM. Inherited risk factors for venous thromboembolism. Nat Rev Cardiol 2014; 11:140-56. Sorumlu Yazar: Kenan Çadırcı, Bölge Eğitim ve Araştır ma Hastanesi, İç Hastalıkları Kliniği, Erzurum, Türkiye, E-posta: doktorcadirci@hotmail.com 3. Franco RF, Reitsma PH. Genetic risk factors of venous thrombo sis. Hum Genet 2001;109:369-84. 3. Franco RF, Reitsma PH. Genetic risk factors of venous thrombo sis. Hum Genet 2001;109:369-84. 4. Rosendaal FR. Venous thrombosis: the role of genes, environ ment, and behavior. Hematology Am Soc Hematol Educ Program 2005:1-12. 4. Rosendaal FR. Venous thrombosis: the role of genes, environ ment, and behavior. Hematology Am Soc Hematol Educ Program 2005:1-12. 149 ORTADOĞU TIP DERGİSİ 9 (3): 146-149, 2017
https://openalex.org/W4298296218	https://cdr.lib.unc.edu/downloads/1c18dr645	English	null	Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis	Carolina Digital Repository (University of North Carolina at Chapel Hill)	2,002	cc-by	7,635	Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis* Received for publication, March 25, 2002, and in revised form, May 22, 2002 Published, JBC Papers in Press, May 28, 2002, DOI 10.1074/jbc.M202856200 Kevin P. Troyer and R. Mark Wightman‡ From the Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Kevin P. Troyer and R. Mark Wightman‡ From the Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To deter- mine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules re- leased and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber micro- electrodes. In external solutions of high osmolarity, re- lease events following entry of divalent ions (Ba2 or Ca2) were less frequent. However, the vesicles ap- peared to be fused to the membrane without extruding their contents, since the maximal observed concentra- tions of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term “kiss-and-hold,” was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demon- strate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation. molecular associations lead to a state (7, 8) that is iso-osmotic with the cytoplasm (6). These close associations have been documented by nuclear magnetic resonance in vesicles from both cell types (9, 10). Upon cell stimulation, the vesicle contacts the plasma mem- brane and forms a fusion pore, the dynamics and control of which have been reviewed recently (11, 12). Capacitance and amperometry techniques have indicated that a portion of the vesicular contents can be released through this fusion pore (13, 14). Furthermore, at both mast and chromaffin cells, patch- amperometry measurements reveal that the fusion pore can exist for several milliseconds prior to full extrusion of vesicle contents (15, 16). Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis* Following secretory vesicle-cell membrane fusion in mast cells vesicular swelling occurs (17), which has been attributed to the displacement of associated cations in the granule with hydrated cations such as Na from the external solution (18). This ion exchange mechanism, with its associated hydration effects (19), has been hypothesized and experimen- tally supported at several cell types as reviewed by Artalejo et al. (20). Theoretical considerations of this process have likened the extrusion and swelling to a controlled explosion that drives full release of the vesicular contents and dissociation of the vesic- ular matrix (18). Amperometric recordings revealed that the time course of release is longer than that expected for free diffusion (1), having a duration of several milliseconds at chro- maffin cells (21, 22), and even longer at mast cells (23), con- sistent with a rate-limiting dissociation. The time course of extrusion can be decreased by perturbing any of the physical and chemical gradients that exist between the vesicle interior and the extracellular environment. These include osmolarity, pH, and cation concentration (24–26). For example, increasing the osmotic gradient by lowering the extracellular solution osmolarity decreases the release time course and increases the amount released, whereas high extracellular osmolarity has the opposite effect (27). In the present work, the effects of high osmolarity (using elevated NaCl) on secretory cells were eval- uated in an attempt to freeze exocytotic events at the step between fusion pore formation and extrusion of vesicle con- tents. The results reported are consistent with such a state and reveal that extrusion of the vesicular contents is a distinct step in the sequence of events termed exocytosis. Vesicles in secretory cells perform two different functions: they are a storage depot for small molecules and they release these molecules during exocytosis. Storage in dense core vesi- cles requires stable packaging for an extended period of time. This is accomplished by association of the vesicular contents. On the other hand, release requires dissociation of this storage matrix on a relatively fast time scale so the contents can be extruded (1, 2). In mast cells, histamine and 5-HT are associated with a negatively charged heparin matrix in the vesicles through a cation exchange type of interaction (3). These vesicles contain 150 mM histamine, which alone with its associated anion would lead to a free osmolality of 550 mosM (4). Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis* However, the vesi- cles are iso-osmotic with the 300-mosM cytoplasm because of the association of the molecules within the granule matrix (5). Similarly, a single chromaffin cell vesicle contains 550 mM catecholamine along with 122 mM ATP, 17–30 mM Ca2, 5 mM Mg2, 22 mM ascorbate, and the acidic protein chromogranin A, leading to a total soluble concentration of 750 mM (6). Again, Vol. 277, No. 32, Issue of August 9, pp. 29101–29107, 2002 Printed in U.S.A. Vol. 277, No. 32, Issue of August 9, pp. 29101–29107, 2002 Printed in U.S.A. Vol. 277, No. 32, Issue of August 9, pp. 29101–29107, 2002 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Tel.: 919-962-1472; Fax: 919-962-2388; E-mail: rmw@unc.edu. This is an Open Access article under the CC BY license. This paper is available on line at http://www.jbc.org * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Tel.: 919-962-1472; Fax: 919-962-2388; E-mail: rmw@unc.edu. This paper is available on line at http://www.jbc.org 29 This is an Open Access article under the CC BY license. RESULTS After the exposure to isotonic buffer, the spikes continued for as long as 5 min at a frequency similar to that in isotonic buffer (Table I). Confocal Microscopy—DH labeling was used to visualize events associated with exocytosis at chromaffin cells. To measure surface- bound DH, a measure of cell membrane-vesicle fusion, cells were exposed to 5 mM Ba2 for 2 min in Tris-HCl buffer (no added Ca2) in iso- or hypertonic buffer. Control cells were incubated without Ba2. The cells were then fixed with a non-permeant protocol by quickly removing the bathing solutions and replacing them with identical buff- ers containing 4% p-formaldehyde at 4 °C for 20 min (33). The cells were then rinsed twice with buffer, incubated with 0.2% bovine serum albu- min for 10 min, rinsed two more times, and incubated with mouse anti-DH monoclonal antibody (1:300; Chemicon, Temecula, CA) for 45 min at 4 °C. To monitor DH retention on the surface, an identical procedure was followed except the cells were incubated in iso- or hy- pertonic buffer for 20 min following Ba2 but before fixation. To monitor vesicle recycling, the procedure was again identical except the anti- DH antibody was present during the exposure to Ba2. These cells were fixed and permeabilized with ethanol to allow visualization of endocytosed DH. Since the average diameter of mast cell vesicles is 700 nm (29), the degranulation that occurs during exocytosis can be observed with light microscopy. In hypertonic medium, the mast cells shrank and degranulation was not apparent upon application of A23187 (Fig. 2b). This was the case despite the fact that the voltammetric results showed spikes of histamine and 5-HT (Fig. 1), albeit small ones. However, degranulation was readily observed immediately after transient application of isotonic buffer, and it continued for the time scale of the release detected with voltammetry (Fig. 2, c and d). After rinsing, DH was visualized by incubating the cells with Alexa- Fluor 568 goat anti-mouse IgG conjugate (1:1000, Molecular Probes, Eugene, OR). The coverslips containing the cells were washed and mounted on microscope slides with Vectashield (Vector Labs, Burl- ingame, CA). Images were acquired using a 100 oil immersion objec- tive on a Leica-LCS confocal microscope (Leica Instruments, Exton, PA). Analysis of images was performed using Scion Image software (Frederick, MD). Mast Cell Optical Imaging—Visualization of release from mast cells was enhanced with 50 g/ml ruthenium red (29). 1 The abbreviations used are: FSCV, fast scan cyclic voltammetry; DH, dopamine--hydroxylase; 5-HT, 5-hydroxytryptamine. Separating Vesicle Release from Fusion 29102 TABLE I Effects of elevated osmolarity buffer on release from peritoneal mast cells. Cmax is the maximal concentration of histamine measured from each current spike. ISO, ISO buffer contained 150 mM NaCl. Secretagogue [NaCl] Cmax % histamine spikes 0.4 M Spike frequency mM M Hz A23187 150 (ISO) 12.04 0.45 87.5 0.64 A23187 350 0.14 0.02 0 0.17 ISO after A23187 350 5.06 0.28 89.1 0.61 p g f TABLE I were prepared using doubly distilled deionized water and adjusted to pH 7.4 by addition of NaOH. backfilled with a 4 M potassium acetate, 150 mM KCl solution and calibrated using 4 M epinephrine, norepinephrine, and histamine and 1 M serotonin. FSCV1 was performed using locally written Labview software (National Instruments, Austin, TX) with an EI-400 poten- tiostat (Cypress Systems, Lawrence, KS) (31). Potentials were scanned from 200 mV to 1600 mV and back for catecholamines and from 100 to 1400 mV and back for histamine and serotonin (potentials versus a Ag/AgCl reference electrode, Bioanalytical Systems, Lafayette, IN). A scan rate of 2000 V/s was used with a 60-Hz repetition rate. Spike detection and analysis at the peak oxidation potentials for the mole- cules of interest were performed (32), and the resulting current spike amplitudes were converted to concentration based on electrode calibrations. RESULTS Mast Cells—Following transient exposure (5 s) to the Ca2 ionophore A23187 in isotonic buffer (150 mM NaCl), mast cells exhibited frequent exocytotic release events that were detected by cyclic voltammetry (Table I), consistent with previous re- ports. Every exocytotic event resulted in the simultaneous re- lease of 5-HT and histamine (Fig. 1) that each gave a distinct voltammetric peak (29). Each event was characterized by a simultaneous increase in the concentrations of 5-HT and his- tamine followed by a more gradual returned to base line. Oc- casionally the exocytotic spikes were preceded by a foot-like event (29) in which a transient, steady-state, low concentration of the amine was present (data not shown). Such events have been shown to be due to secretion through the fusion pore (13). Cell Experiments—Individual cells were stimulated to release by pressure ejection (Picospritzer; General Valve Corp., Fairfield, NJ) of 5 mM BaCl2 or 60 mM KCl (chromaffin cells), 0.5 M A23187 (mast cells), or transient isotonic buffer solution (chromaffin and mast cells) from a micropipette placed 30 m from the cell. The carbon fiber microelec- trode was placed 1 m from the cell membrane using a piezoelectric manipulator (PCS-1000, Burleigh Instruments, Fishers, NY) on the stage of an inverted microscope (Axiovert 35; Zeiss, Thornwood, NY). Cells were rinsed with isotonic Tris-HCl buffer at room temperature prior to experiment. For experiments in which bathing buffer osmolar- ity was changed, glass coverslips containing the cells were placed in a perfusion chamber positioned on the microscope. Mast cells displayed greater sensitivity to elevations in os- molarity than chromaffin cells. Application of external solution with NaCl concentrations of 520 mM, as used with chromaffin cells (see below), caused either complete inhibition of Ca2- evoked release events or rapid, spontaneous degranulation. At a lower NaCl concentration (350 mM NaCl), only a few cells degranulated spontaneously. However, for intact cells, release was quite dramatically altered. Exposure to A23187 in hyper- tonic medium containing Ca2 still evoked spikes, but their maximal concentration was 1% of that evoked in isotonic solution. Indeed, they resemble feet unaccompanied by spikes. The frequency of spikes was also reduced (Table I). Spikes with large concentrations could be obtained if the cells were subse- quently exposed to an isotonic puff for a transient (5 s) period 3 min after the initial A23187 exposure (an example is shown in Fig. 1). EXPERIMENTAL PROCEDURES Cell Culture—Bovine adrenal medullary cells, enriched in either epinephrine or norepinephrine using a Renografin gradient, were cul- tured as previously described and plated at a density of 3 105 cells/ plate (28). Cells were used on days 3–7 after culture. Murine peritoneal mast cells were cultured as described previously (29). Cells were plated on 25-mm glass coverslips (Carolina Biological Supply, Burlington, NC) contained in 35-mm diameter tissue culture plates (Falcon; Fisher Scientific). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Electrochemistry—Carbon fiber microelectrodes for the detection of released molecules were prepared as described (30). Electrodes were ‡ To whom correspondence should be addressed. Tel.: 919-962-1472; Fax: 919-962-2388; E-mail: rmw@unc.edu. 29101 29101 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Separating Vesicle Release from Fusion RESULTS For sero- tonin, the oxidation is maximal at 0.7 V, and for histamine oxidation is maximal at 1.3 V. Middle trace, current arising from oxidation of histamine. Upper trace, fura-2 monitoring of intracellular diva- lent ion concentration at a different cell. Separating Vesicle Release from Fusion 29103 eparating Vesicle Release from Fusion FIG. 1. Cyclic voltammetric moni- toring of exocytosis at a mast cell in isotonic solution (150 mM NaCl) and hypertonic solution (350 mM NaCl). Left panel, typical release from a mast cell exposed to A23187 in isotonic solution. A different cell, in hypertonic solution, was exposed to A23187 for 5 s during the first dashed box (middle panel) and then ex- posed to isotonic solution for 10 s during the second dashed box (right panel). Color plots: release events were monitored with background subtracted, FSCV that allows a simultaneous view of serotonin and his- tamine (29). The potential applied to the carbon fiber microelectrode is plotted on the y axis (from 0.1 to 1.4 and back to 0.1 V versus a Ag/AgCl reference), while the x axis is time. The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). For sero- tonin, the oxidation is maximal at 0.7 V, and for histamine oxidation is maximal at 1.3 V. Middle trace, current arising from oxidation of histamine. Upper trace, fura-2 monitoring of intracellular diva- lent ion concentration at a different cell. FIG. 2. Optical micrographs of a mast cell during degranulation. a, ini- tial view of the cell in hypertonic solution (350 mM NaCl). b, cell following exposure to A23187. c, same cell following subse- quent exposure (10 s) to isotonic solution (150 mM NaCl). d, five minutes after c. Chromaffin Cells—In isotonic solution without Ca2, tran- sient exposure of chromaffin cells to Ba2 (5 mM, 5 s) causes exocytotic release of catecholamines (Fig. 3) (24). In isotonic solution containing Ca2, transient exposure of chromaffin cells to elevated KCl (60 mM, 5 s) also causes exocytotic release of catecholamines (Fig. 4) (35). Cyclic voltammetry can be used to monitor the release and can distinguish cells that secrete norepinephrine from those that secrete epinephrine (36). Cyclic voltammograms (37) also reveal that the foot present for many exocytotic events is comprised of catecholamines (data not shown). Release evoked by the two secretagogues is quite sim- ilar (24) (Table II). RESULTS hypertonic medium induced massive release (Table II, Figs. 3 and 4). In 27% of the chromaffin cells pretreated with 60 mM KCl and 36% of the cells pretreated with Ba2, this release consisted of one to two spikes with maximal concentrations greater than 40 M that appeared to be compound exocytotic events. In the remainder of cells, spikes were observed shortly after exposure to isotonic buffer with a delay time of 4 s to the first spike. The isotonic buffer caused cell swelling on a similar time scale. The large spikes diminished immediately following cessation of transient exposure to isotonic buffer. When cells were completely restored to isotonic conditions, exposure to either secretagogue evoked spikes with a normal frequency and maximal concentrations (data not shown). In hyperosmotic solutions (520 mM NaCl) without Ca2, transient exposure to 5 mM Ba2 also results in exocytotic spikes from chromaffin cells (Fig. 3), but they are less frequent and much smaller in amplitude (Table II). Similar results are obtained for K-induced release in hypertonic solutions (Fig. 4, Table II). The maximal concentration of spikes in hypertonic medium with Ba2 is only 7% and with K is only 10% of that seen in isotonic medium. In all cases the cyclic voltammograms revealed that the small, foot-like events were the same cate- cholamine that the cell normally secreted. Thus, the results at chromaffin cells are quite similar to those at mast cells: in hyperosmotic solutions, release events of small amplitude can be evoked, but their concentration and frequency is lower than in isotonic solution. Fura-2 was used to monitor either intracellular Ba2 or Ca2 in these experiments. It is well established in isotonic solutions that both secretagogues cause an immediate increase in intra- cellular divalent ions (34), and this was replicated in this work (data not shown). In hypertonic solutions, pressure ejection of Ba2 or K similarly caused an elevation in intracellular diva- lent ion concentrations. In the case of Ba2, the signal re- mained elevated for greater than 3 min (Fig. 3) because intra- cellular Ca2 stores do not effectively sequester it (38). Following K in hypertonic solution, the divalent ion signal returned to base-line levels 10 s after its application. Thus, the observed, divalent ion entry accompanies the small release events obtained in hypertonic solutions. RESULTS Individual cells in both iso-osmotic and elevated osmolarity buffers were stimulated to release and monitored using a CCD camera (Sensys, Photometrics, Tucson, AZ) through the microscope. The lack of visual degranulation is not due to failure of Ca2 to enter the cell. Ca2 entry was immediate upon exposure to A23187 in hypertonic buffer as revealed by fluorescence from fura-2, the ratiometric divalent ion indicator (34). Further- more, upon exposure to isotonic buffer, the larger spikes, with their accompanying visual degranulation, were not accompa- nied by a further influx of Ca2 (Fig. 1, upper trace). Thus, in hypertonic solution, it appears that Ca2 entry evokes vesicle- cell membrane fusion, but extrusion of vesicular contents is suppressed unless isotonic conditions are restored for a tran- sient period. Reagents and Solutions—All chemicals were obtained from Sigma and used as received. A23187 was prepared by 1000-fold dilution of a stock solution in Me2SO. Iso-osmotic buffer contained 12.5 mM Tris- HCl, 150 mM NaCl, 4 mM KCl, 1.2 mM MgCl2, and 5 mM glucose). Hypertonic buffers were prepared by elevating the NaCl concentration to 520 mM for chromaffin cells and 350 mM for mast cells. All solutions Separating Vesicle Release from Fusion FIG. 2. Optical micrographs of a mast cell during degranulation. a, ini- tial view of the cell in hypertonic solution (350 mM NaCl). b, cell following exposure to A23187. c, same cell following subse- quent exposure (10 s) to isotonic solution (150 mM NaCl). d, five minutes after c. FIG. 1. Cyclic voltammetric moni- toring of exocytosis at a mast cell in isotonic solution (150 mM NaCl) and hypertonic solution (350 mM NaCl). Left panel, typical release from a mast cell exposed to A23187 in isotonic solution. A different cell, in hypertonic solution, was exposed to A23187 for 5 s during the first dashed box (middle panel) and then ex- posed to isotonic solution for 10 s during the second dashed box (right panel). Color plots: release events were monitored with background subtracted, FSCV that allows a simultaneous view of serotonin and his- tamine (29). The potential applied to the carbon fiber microelectrode is plotted on the y axis (from 0.1 to 1.4 and back to 0.1 V versus a Ag/AgCl reference), while the x axis is time. The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). RESULTS Transient application of an isotonic solution to chromaffin cells for 15 s at 2 min after exposure to either secretagogue in Fura-2 measurements were also made during reintroduction of isotonic solution after exposure to secretagogues. The accom- panying cell swelling and dye dilution complicates interpreta- tion of the changes in divalent ions There was no observable Confocal Microscopy—To provide evidence that vesicles fused with the plasma membrane in hypertonic solution a FIG. 3. Cyclic voltammetric moni- toring of exocytosis at a chromaffin cell in isotonic (150 mM NaCl) and hy- pertonic solution (520 mM NaCl). Left panel, release from a cell in isotonic solu- tion upon exposure to Ba2. The cell in hypertonic solution (middle panel) was exposed to Ba2 for 5 s during the first dashed box and then exposed to isotonic solution for 15 s during the second dashed box (right panel). Color plots: release events were monitored with FSCV. The potential applied to the carbon fiber mi- croelectrode is plotted on the y axis, while the x axis is time. The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). For epi- nephrine, oxidation of the catechol to its o-quinone occurs at 0.75 V with a second peak at 1.5 V from the oxidation of the secondary amine as described previously (36). Middle trace, current arising from oxidation of epinephrine at 0.75 V. Up- per trace, fura-2 monitoring of intracellu- lar divalent ion concentration at a differ- ent cell. FIG. 4. Cyclic voltammetric monitoring of exocytosis at a chromaffin cell in isotonic (150 mM NaCl) and hypertonic solution (520 mM NaCl). Left panel, release from a cell exposed to 60 mM K in isotonic solution. The cell in hypertonic solution was exposed to K for 5 s during the first dashed box (middle panel) and then exposed to isotonic solution for 15 s during the second dashed box (right panel). Color plots: release events were monitored with FSCV. The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). Middle trace, current arising from oxidation of epinephrine at 0.75 V. The experimental parameters are the same as those in Fig. 3. Upper trace, fura-2 monitoring of intracellular divalent ion concentration at a different cell. Separating Vesicle Release from Fusion 29104 Separating Vesicle Release from Fusion 29104 Separating Vesicle Release from Fusion FIG. RESULTS 3. Cyclic voltammetric moni- toring of exocytosis at a chromaffin cell in isotonic (150 mM NaCl) and hy- pertonic solution (520 mM NaCl). Left panel, release from a cell in isotonic solu- tion upon exposure to Ba2. The cell in hypertonic solution (middle panel) was exposed to Ba2 for 5 s during the first dashed box and then exposed to isotonic solution for 15 s during the second dashed box (right panel). Color plots: release events were monitored with FSCV. The potential applied to the carbon fiber mi- croelectrode is plotted on the y axis, while the x axis is time. The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). For epi- nephrine, oxidation of the catechol to its o-quinone occurs at 0.75 V with a second peak at 1.5 V from the oxidation of the secondary amine as described previously (36). Middle trace, current arising from oxidation of epinephrine at 0.75 V. Up- per trace, fura-2 monitoring of intracellu- lar divalent ion concentration at a differ- ent cell. FIG. 4. Cyclic voltammetric monitoring of exocytosis at a chromaffin cell in isotonic (150 mM NaCl) and hypertonic solution (520 mM NaCl). Left panel, release from a cell exposed to 60 mM K in isotonic solution. The cell in hypertonic solution was exposed to K for 5 s during the first dashed box (middle panel) and then exposed to isotonic solution for 15 s during the second dashed box (right panel). Color plots: release events were monitored with FSCV. The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). Middle trace, current arising from oxidation of epinephrine at 0.75 V. The experimental parameters are the same as those in Fig. 3. Upper trace, fura-2 monitoring of intracellular divalent ion concentration at a different cell. FIG. 4. Cyclic voltammetric monitoring of exocytosis at a chromaffin cell in isotonic (150 mM NaCl) and hypertonic solution (520 mM NaCl). Left panel, release from a cell exposed to 60 mM K in isotonic solution. The cell in hypertonic solution was exposed to K for 5 s during the first dashed box (middle panel) and then exposed to isotonic solution for 15 s during the second dashed box (right panel). Color plots: release events were monitored with FSCV. DISCUSSION In high osmolarity solutions, secretion from both mast and chromaffin cells is dramatically altered. The results presented here show that vesicle-cell membrane fusion can still occur under these conditions, but that full extrusion of the vesicle contents does not occur. Indeed, the fused vesicles in hyper- tonic solution appear to be frozen at the cell surface, and only release after swelling of the vesicle matrix is allowed to occur. Previous studies have demonstrated that both chromaffin and mast cell vesicles contract in response to hypertonic stress without disruption of their stored contents (5). Indeed, the chromaffin vesicle has been termed an ideal osmometer be- tween 300 and 1000 mosM (6). The new finding of this work is that high osmolarity solutions prevent full extrusion of vesicle contents and allow temporal isolation of an intermediate state in the sequence of events that occur during exocytosis, a state we refer to as “kiss-and-hold.” either isotonic (Fig. 5a) or hypertonic solutions (Fig. 5b) causes DH localization on the cell perimeter. The fluorescence at the cell surface, which is eight times more intense in cells stimu- lated in isotonic solution, indicates vesicle-cell membrane fu- sion has occurred under both conditions, exposing the vesicle interior to the external solution. In both solutions, unstimu- lated cells showed no fluorescence (data not shown). A similar experiment was used to test vesicle lifetime at the cell surface. With the same treatment as in Fig. 5a, but with a 20-min incubation in buffer before the addition of DH anti- body, DH was not found on cell surfaces when stimulated in isotonic medium (Fig. 5c). This is consistent with vesicle endo- cytosis that occurs on a time scale much shorter than 20 min in isotonic solutions (40). Supporting this conclusion, fluorescence was found within cells that were stimulated to release in iso- tonic solutions in the presence of DH antibody (Fig. 5e). In contrast, DH antibody was present on cell surfaces stimulated in hypertonic medium under all conditions, but was not seen to incorporate within the cell (Fig. 5, d and f). The rate of endo- cytosis is known to be lower in hypertonic solutions, consistent with this finding (41). Hypertonic solutions clearly lower the rate of exocytosis at both mast and chromaffin cells. RESULTS Cmax values are measured from catecholamine oxidative peak. ISO, ISO buffer contained 150 mM NaCl. Secretagogue [NaCl] (mM) Cmax % spikes 2 M Total spike frequency M Hz Ba2 150 (ISO) 13.86 1.77 89.5 1.18 Ba2 520 1.36 0.14 19.6 0.15 ISO after Ba2 520 7.08 1.41 72.9 0.23 K 150 (ISO) 12.22 1.23 93.2 1.66 K 520 0.82 0.06 2.6 0.12 ISO after K 520 3.68 0.52 47.3 0.22 Separating Vesicle Release from Fusion 29105 FIG. 5. Confocal images of chromaffin cells stimulated to re- lease under different conditions and stained with DH anti- body. a, c, and e, results obtained in isotonic solution. b, d, and f, results obtained in hypertonic (520 mM NaCl) solution. a and b, cell was fixed after 2-min exposure to 5 mM Ba2 followed by DH antibody staining. c and d, cells were exposed to Ba2 as in a and rinsed for 20 min without Ba2 before fixation and staining. e and f, cells were stimulated as in a but in the presence of anti-DH antibody to reveal endocytosis. TAB Effects of elevated osmolarity buffer on release from ch Cmax values are measured from catecholamine oxidative peak. ISO, I Secretagogue [NaCl] (mM) Cmax M Ba2 150 (ISO) 13.86 1.7 Ba2 520 1.36 0.1 ISO after Ba2 520 7.08 1.4 K 150 (ISO) 12.22 1.2 K 520 0.82 0.0 ISO after K 520 3.68 0.5 p g FIG. 6. Model for separation of vesicle fusion and subsequent extrusion of contents. When stimulated in hypertonic solution, ves- icles dock with the plasma membrane and unassociated contents can diffuse through the fusion pore (upper). Subsequent exposure to isotonic solution causes expansion of the vesicle matrix and extrusion of con- tents (lower). LE II omaffin cells stimulated with 5 mM Ba2 or 60 mM K. SO buffer contained 150 mM NaCl. % spikes 2 M Total spike frequency Hz 7 89.5 1.18 4 19.6 0.15 1 72.9 0.23 3 93.2 1.66 6 2.6 0.12 2 47.3 0.22 f TABLE II h ff FIG. 6. Model for separation of vesicle fusion and subsequent extrusion of contents. When stimulated in hypertonic solution, ves- icles dock with the plasma membrane and unassociated contents can diffuse through the fusion pore (upper). RESULTS Subsequent exposure to isotonic solution causes expansion of the vesicle matrix and extrusion of con- tents (lower). FIG. 5. Confocal images of chromaffin cells stimulated to re- lease under different conditions and stained with DH anti- body. a, c, and e, results obtained in isotonic solution. b, d, and f, results obtained in hypertonic (520 mM NaCl) solution. a and b, cell was fixed after 2-min exposure to 5 mM Ba2 followed by DH antibody staining. c and d, cells were exposed to Ba2 as in a and rinsed for 20 min without Ba2 before fixation and staining. e and f, cells were stimulated as in a but in the presence of anti-DH antibody to reveal endocytosis. RESULTS The current intensity is encoded in color as indicated by the scale bar (note change in scale bars). Middle trace, current arising from oxidation of epinephrine at 0.75 V. The experimental parameters are the same as those in Fig. 3. Upper trace, fura-2 monitoring of intracellular divalent ion concentration at a different cell. Confocal Microscopy—To provide evidence that vesicles fused with the plasma membrane in hypertonic solution, a fluorescently labeled antibody to DH was employed. DH is a vesicular enzyme that appears on the surface of chromaffin cells following exocytosis (39). Exposure to Ba2 for 2 min in panying cell swelling and dye dilution complicates interpreta- tion of the changes in divalent ions. There was no observable change in the bound to free ratio at cells that had been exposed to Ba2; however, results in cells that had been exposed to K were equivocal. Separating Vesicle Release from Fusion Separating Vesicle Release from Fusion 29105 DISCUSSION In high osmolarity solutions, secretion from both mast and chromaffin cells is dramatically altered. The results presented here show that vesicle-cell membrane fusion can still occur under these conditions, but that full extrusion of the vesicle contents does not occur. Indeed, the fused vesicles in hyper- tonic solution appear to be frozen at the cell surface, and only release after swelling of the vesicle matrix is allowed to occur FIG. 5. Confocal images of chromaffin cells stimulated to re- lease under different conditions and stained with DH anti- body. a, c, and e, results obtained in isotonic solution. b, d, and f, results obtained in hypertonic (520 mM NaCl) solution. a and b, cell was fixed after 2-min exposure to 5 mM Ba2 followed by DH antibody staining. c and d, cells were exposed to Ba2 as in a and rinsed for 20 min without Ba2 before fixation and staining. e and f, cells were stimulated as in a but in the presence of anti-DH antibody to reveal endocytosis. FIG. 6. Model for separation of vesicle fusion and subsequent extrusion of contents. When stimulated in hypertonic solution, ves- icles dock with the plasma membrane and unassociated contents can diffuse through the fusion pore (upper). Subsequent exposure to isotonic solution causes expansion of the vesicle matrix and extrusion of con- tents (lower). TABLE II Effects of elevated osmolarity buffer on release from chromaffin cells stimulated with 5 mM Ba2 or 60 mM K. DISCUSSION relating well with the decreased frequency of electrochemically detected spikes. At mast cells, visual evidence for vesicle-cell membrane fusion was not obtained in hypertonic medium, al- though secretion was clearly evident by the cyclic voltammetry. The secretory events that are seen are so small that they resemble the feet that precede some exocytotic events in iso- osmolar solutions at both types of cells in hypertonic media. This is the expected outcome if vesicle fusion occurred in hy- pertonic solutions with only minor extrusion of the contents. Despite their small size, the cyclic voltammograms establish that the substances detected are those stored in the vesicles. All of the evidence from both cell types indicates that the small size of the spikes obtained in hypertonic medium is a consequence of inhibition of vesicle-matrix dissociation. The association of the vesicle contents in chromaffin cells has been characterized as a “dynamic viscous solution that is stabilized by ternary complex formation” (10) and is also well documented in mast cells (47). Upon exposure to the extracellular solution through the fusion pore, at the initial “kiss,” exchange between the vesicular interior and the extracellular fluid begins. Nor- mally the matrix dissociation and extrusion of its contents relies on hydration, with its accompanying matrix swelling, that is driven by a physical or chemical gradient across the fusion pore. By removing the osmotic gradient in hypertonic solutions, one of the driving forces for dissociation and extru- sion is dissipated. The small amount of secretion that does occur could arise from components originally in an unassoci- ated state or a small amount of matrix dissociation that occurs at the fusion pore, but which is unable to propagate into the remainder of the vesicle. Because rates of endocytosis are low- ered in hypertonic solutions, the fused, intact vesicle “holds” at the cell surface for an extended period of time. The hypertonically constructed kiss-and-hold configuration inhibits the dissociation of matrix constituents following vesi- cle fusion with the cell membrane. This provides access to the vesicle interior while maintaining the viability of the cell, which may allow for a more complete understanding of the complex interaction of molecules within the vesicle matrix in their natural environment as well as a glimpse of their disso- ciation during exocytosis. REFERENCES p The frozen, fused vesicle state established in hypertonic me- dium is destroyed once the osmotic gradient is re-established. This occurs with the transient isotonic exposure that evokes multiple spikes on the time scale of cellular swelling. In mast cells, the maximal concentration of these spikes is consistent with extrusion of contents from previously fused vesicles. At chromaffin cells, the maximal concentration and time course of some observed spikes are larger than under normal exocytotic conditions, consistent with multiple, simultaneous secretory events. For cells stimulated with K, the original stimulus is no longer present upon isotonic solution exposure, and yet secre- tion is still observed. This strongly suggests that the observed spikes arise from previously fused vesicles. With Ba2 at chro- maffin cells and with mast cells, the isotonic-evoked spikes are unaccompanied by an increase in intracellular divalent ions. 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DH labeling is lowered in hypertonic solutions, cor- Separating Vesicle Release from Fusion 29106 removing the driving force for swelling, we were able to isolate the intermediate kiss-and-hold state. The mathematical mod- els predict that “kiss-and-run” events, events where fusion pore formation allows escape of some transmitter followed by vesic- ular retreat from the cellular membrane without fully fusing, are unlikely under normal conditions at cells with large vesi- cles. Consistent with these models, all fusion pore events in mast cells are followed by complete fusion of the vesicle with the cellular membrane and subsequent release (15). Patch- amperometry studies on chromaffin cells reveal only 10% of the observed fusion events are kiss-and-run (16), but these occur- rences are increased with elevated external Ca2 concentra- tions (49). In the absence of endocytosis caused by elevated osmolarity, one would expect these events to be restricted to the cell membrane, as shown in this work, until acted upon by external forces. For small vesicles, the force of matrix expan- sion is insufficient to destabilize the fusion pore, and kiss-and- run is more likely. This has also been observed for small clear vesicles at hippocampal synapses (50). relating well with the decreased frequency of electrochemically detected spikes. At mast cells, visual evidence for vesicle-cell membrane fusion was not obtained in hypertonic medium, al- though secretion was clearly evident by the cyclic voltammetry. The secretory events that are seen are so small that they resemble the feet that precede some exocytotic events in iso- osmolar solutions at both types of cells in hypertonic media. This is the expected outcome if vesicle fusion occurred in hy- pertonic solutions with only minor extrusion of the contents. 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https://openalex.org/W2806191867	https://europepmc.org/articles/pmc6010545?pdf=render	English	null	Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens	Frontiers in microbiology	2,018	cc-by	14,892	Citation: Fleeman RM, Debevec G, Antonen K, Adams JL, Santos RG, Welmaker GS, Houghten RA, Giulianotti MA and Shaw LN (2018) Identiﬁcation of a Novel Polyamine Scaffold With Potent Efﬂux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens. Front. Microbiol. 9:1301. doi: 10.3389/fmicb.2018.01301 Identiﬁcation of a Novel Polyamine Scaffold With Potent Efﬂux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens 1 Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, United States, 2 Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, United States We have previously reported the use of combinatorial chemistry to identify broad- spectrum antibacterial agents. Herein, we extend our analysis of this technology toward the discovery of anti-resistance molecules, focusing on efﬂux pump inhibitors. Using high-throughput screening against multi-drug resistant Pseudomonas aeruginosa, we identiﬁed a polyamine scaffold that demonstrated strong efﬂux pump inhibition without possessing antibacterial effects. We determined that these molecules were most effective with an amine functionality at R1 and benzene functionalities at R2 and R3. From a library of 188 compounds, we studied the properties of 5 lead agents in detail, observing a ﬁvefold to eightfold decrease in the 90% effective concentration of tetracycline, chloramphenicol, and aztreonam toward P. aeruginosa isolates. Additionally, we determined that our molecules were not only active toward P. aeruginosa, but toward Acinetobacter baumannii and Staphylococcus aureus as well. The speciﬁcity of our molecules to efﬂux pump inhibition was conﬁrmed using ethidium bromide accumulation assays, and in studies with strains that displayed varying abilities in their efﬂux potential. When assessing off target effects we observed no disruption of bacterial membrane polarity, no general toxicity toward mammalian cells, and no inhibition of calcium channel activity in human kidney cells. Finally, combination treatment with our lead agents engendered a marked increase in the bactericidal capacity of tetracycline, and signiﬁcantly decreased viability within P. aeruginosa bioﬁlms. As such, we report a unique polyamine scaffold that has strong potential for the future development of novel and broadly active efﬂux pump inhibitors targeting multi-drug resistant bacterial infections. Keywords: efﬂux pumps, efﬂux pump inhibitors, polyamines, bacterial resistance, potentiation Edited by: Xian-Zhi Li, Health Canada, Canada Reviewed by: Katy Jeannot, UMR6249 Chrono Environnement, France Paola Sperandeo, Università degli Studi di Milano, Italy Reviewed by: Katy Jeannot, UMR6249 Chrono Environnement, France Paola Sperandeo, Università degli Studi di Milano, Italy *Correspondence: Lindsey N. Shaw shaw@usf.edu Specialty section: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology Received: 07 February 2018 Accepted: 28 May 2018 Published: 14 June 2018 Keywords: efﬂux pumps, efﬂux pump inhibitors, polyamines, bacterial resistance, potentiation ORIGINAL RESEARCH published: 14 June 2018 doi: 10.3389/fmicb.2018.01301 INTRODUCTION This presents the scenario of a much talked about post-antibiotic era, where conventional antibiotics will no longer be eﬀective and common infections may once again become fatal (Reardon, 2014). A primary issue is that typical drug discovery eﬀorts often result in the development of therapeutics with known mechanisms of action, thus allowing bacteria to rapidly evolve resistance to these new agents (Falagas and Bliziotis, 2007). Consequently, new strategies are urgently needed for the discovery of novel therapeutics targeting multi-drug resistant organisms (Spellberg et al., 2008; Reardon, 2014). The selective pressure antibiotics place on heterogeneous bacterial communities often directly leads to resistant clones becoming dominant within infectious populations (Spellberg et al., 2008). Novel therapeutics targeting resistant bacterial strains would be therapeutically advantageous, speciﬁcally focusing on those isolates that prove the most diﬃcult to eradicate. A unique way to do this is to interfere with bacterial resistance mechanisms, rather than focusing on bacterial viability. Such treatment options could restore the eﬀectiveness of numerous obsolete clinical antibiotics, reclaiming many important therapeutics. Co-administration of such anti- resistance agents alongside existing antibiotics may also lead to decreased resistance, as multiple targets within the cell are impacted simultaneously (Yoneyama and Katsumata, 2014). Hence, anti-resistance approaches may exponentially increase the number of available therapeutic options, whereas conventional antibiotic development commonly yield only a single new drug (Renau et al., 1999; Lomovskaya et al., 2001). A primary method by which bacteria resist the action of antibacterial agents is via eﬄux pump extrusion. Eﬄux pumps are complexes within the bacterial cell envelope used to export toxic substances such as antibiotics from the intracellular environment before damage to the cell occurs (Sun et al., 2014). Eﬄux pumps are found in most multi-drug resistant nosocomial pathogens, with many having similar and overlapping substrate speciﬁcities. As such, targeting bacterial eﬄux pumps via therapeutic intervention could eﬀectively re-sensitize cells to a broad spectrum of antibacterial agents (Renau et al., 1999; Lomovskaya et al., 2001; Weinstein and Hooper, 2005). Indeed, recent studies have shown that strains overexpressing eﬄux pumps commonly display an average >twofold increased minimal inhibitory concentrations (MIC) toward multiple antibiotics (Weinstein and Hooper, 2005; Adabi et al., 2015). In previous work by our group we used combinatorial chemistry to identify broad spectrum antibacterial agents (Fleeman et al., 2015; Sandhaus et al., 2016). MATERIALS AND METHODS Design and Synthesis of the Combinatorial Libraries INTRODUCTION The continued increase of antimicrobial resistant bacterial infections is an ongoing public health crisis in the United States, with mortality rates increasing yearly (Boucher et al., 2009). This problem can be directly linked to ever growing demands for antibiotics, coupled with a diminishing therapeutic arsenal that has been exacerbated by a continual decline in antibiotic discovery over June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org Polyamines as EPIs Fleeman et al. transporters as well (Cornwell et al., 1987; Neyrolles et al., 2016). Speciﬁcally, molecules such as verapamil when administered at higher doses have been shown to cause cardiac arrest due to calcium channel inhibition (Chevalier et al., 1999). More recent agents, such as MC-207,110 (phenylalanine arginine beta naphthalimide, or PaβN), have been shown to have increased speciﬁcity toward bacterial eﬄux systems, (Renau et al., 1999; Lomovskaya et al., 2001; Misra and Bavro, 2009; Opperman and Nguyen, 2015); however, the advancement of this scaﬀold has been abandoned as it has been shown to non-speciﬁcally depolarize prokaryotic membranes and cause signiﬁcant nephrotoxicity (Watkins et al., 2003; Webber et al., 2013; Opperman and Nguyen, 2015). Although a number of eﬄux pump inhibitors with improved activity have been identiﬁed in recent years (Chamberland et al., 1996, 1999, 2000, 2001; Zelle and Harding, 1996; Levy, 1998; Zelle, 1998; Markham et al., 2000; De Souza et al., 2002; Nakayama et al., 2002; Pages et al., 2003; Nelson and Alekhsun, 2004; Lemaire et al., 2006), the only advancement into clinical trials to date has been for the proton pump inhibitor omeprazole, used in combination with amoxicillin and clarithromycin to treat Helicobacter pylori infections (Handzlik et al., 2013; ClinicalTrials.gov, 2014). Therefore, there is a clear need to identify new eﬄux pump inhibitors with enhanced properties, and limited toxicity. This is particularly true for Gram negative species, such as Pseudomonas aeruginosa, which have impermeable outer membranes and commonly overexpress eﬄux systems (Palmer and Whiteley, 2015; Tommasi et al., 2015). Indeed, this organism has 10 resistance nodulation division (RND) eﬄux pumps that collectively extrude β-lactams, ﬂuoroquinolones, sodium dodecyl sulfate, tetracycline, erythromycin, ethidium bromide, crystal violet, and homoserine lactones (Askoura et al., 2011). Moreover, given the broad substrate range of P. aeruginosa eﬄux pumps, the inhibition of one pump can be alleviated by the upregulation of additional eﬄux pumps with parallel targets (Lister et al., 2009). the past 30 years (Spellberg et al., 2008). INTRODUCTION In the present study, we extend our analysis of this technology toward the discovery of anti-resistance agents, speciﬁcally focusing on eﬄux pump inhibitors. Using high throughput combinatorial scaﬀold library screening against multi-drug resistant P. aeruginosa isolates we identiﬁed a polyamine scaﬀold derived from a reduced acyl peptide that demonstrated strong eﬄux pump inhibition and limited cytotoxicity toward eukaryotic cells. We suggest that these molecules possess excellent potential for future development as anti-resistance agents targeting bacterial eﬄux pumps. Eﬄux mediated resistance was ﬁrst described almost 40 years ago in a study demonstrating that tetracycline insensitivity could result from plasmid-encoded transport systems (McMurry et al., 1980). Following this, Nelson et al. (1993, 1994) and Nelson and Levy (1999) observed that polyamine tetracycline derivatives could increase the eﬀectiveness of tetracycline when administered concomitantly. Early inhibitors targeting eﬄux pumps, such as reserpine, were discovered from existing drugs, however their use was limited by the need for administration at very high doses in order to be eﬀective (Van Bambeke et al., 2006). They also suﬀered from oﬀ-target eﬀects, with compounds such as verapamil, reserpine, and thioridazine not only inhibiting bacterial eﬄux pumps, but eukaryotic Frontiers in Microbiology \| www.frontiersin.org Checkerboard Potentiation Assays C ec e boa d o e a o ssays Scaﬀold ranking library samples and individual polyamines were screened using checkerboard inhibitory assays to assess the potentiation of tetracycline and chloramphenicol. The test utilized a 96-well plate microtiter assay where the concentration of the scaﬀold or individual polyamine was decreased from 25 to 0.8 µg mL−1 (average molarity of mixture samples 65 to 4 µM) along the rows, and the concentration of tetracycline or chloramphenicol was increased from 0.4 to 100 µM across the columns. The EC90 values for all polyamines are reported in µg mL−1 which more accurately displays the eﬀective concentrations for mixture samples. However, with the known antibiotics, we report EC90 values in µM, which more accurately describes puriﬁed compounds. Plates were incubated statically at 37◦C for 20 h, and the optical density (OD600) was determined using a Synergy 2 plate reader (BioTek). Potentiation modeling (detailed below) was performed to determine fold change in the 50 and 90% eﬀective concentration of tetracycline or chloramphenicol. Bacterial Strains and Growth Conditions The bacterial strains used in this study are multi-drug resistant clinical isolates that have previously been described (Supplementary Table S1; Fleeman et al., 2015). Organisms were grown in tryptic soy broth (TSB) for overnight cultures and cation adjusted Mueller Hinton broth (CA-MH II) was used for experimental procedures. All incubations were performed at 37◦C. The minimum inhibitory concentrations (MICS) for all antibiotics tested against these organisms are shown in Supplementary Table S1. Eﬀ%Antibiotic(x1) = %Antibiotic & Compound(x1, x2) −%Compound(x2) 1 −%Compound(x2) Thus, the model-adjusted checkerboards show the antibiotic activity post-potentiation, and from that one can determine the true change in Mic. Ethidium Bromide Efﬂux Inhibition Assay d u o de u b o ssay Ethidium bromide eﬄux assessment was performed by following the ﬂuorescence of ethidium bromide in a 96-well plate assay, as described previously (Renau et al., 1999; Lomovskaya et al., 2001; Webber et al., 2013; Blanchard et al., 2014; Vasudevan et al., 2014). This varies from ethidium bromide accumulation assays as it includes a pre-incubation step to allow accumulation before assessment of eﬄux (Raherison et al., 2002; Paixao et al., 2009). Bacterial cells were grown overnight at 37◦C in TSB, before being synchronized for 3 h in fresh media to exponential phase. Cultures were pelleted at 900 × g for 20 min and the supernatant removed. The resulting pellet was thrice washed and resuspended in 20 mM sodium phosphate buﬀer to an OD600 of 0.2. Ethidium bromide was next added at a sub inhibitory concentration of 25 µM and incubated at room temperature for 15 min to equilibrate. After equilibration, cells were inoculated into a black walled 96-well plate to a density of 1 × 106 CFU mL−1. Using a Biotek plate reader, the ﬂuorescence of cells was monitored for 2 min with 530 nm excitation and 600 nm emission. When baseline readings were complete, polyamines 247, 250, 266, 271, and 314 were added at 25 µg mL−1 alongside the positive control PaβN at the same concentration [all eﬄux pumps inhibitor concentrations were maximum potentiating concentrations (MPC)]. The solvent dimethylformamide (DMF) was used as a no treatment control alongside tetracycline alone. After addition of all compounds, ﬂuorescence was monitored every 5 min for a total of 90 min. After this time, cells were serially diluted and plated to ensure that treatment with ethidium bromide did not aﬀect viability. There was an initial spike in ﬂuorescence upon addition of the compound, with minimal changes observed thereafter, therefore for graphical representation, the ﬁnal maximum relative ﬂuorescence at 90 min was used for comparison of lead agents to controls. 1987): %Antibiotic & Comp (x1, x2) =%Antibiotic (x1) + %Comp (x2) −%Antibiotic (x1) · %Comp (x2) Here, x1 and x2 are the concentrations of antibiotic (tetracycline or chloramphenicol) and library/compound (Comp) tested, respectively. This equation can be rearranged to model the eﬀective percent activity (EC) of the antibiotic alone, after accounting for compound activity: Frontiers in Microbiology \| www.frontiersin.org Design and Synthesis of the Combinatorial Libraries The design and synthesis of the Torrey Pines scaﬀold ranking library has previously been described (Houghten et al., 1999; Reilley et al., 2010; Santos et al., 2013; Wu et al., June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 2 Polyamines as EPIs Fleeman et al. 2013). The library is comprised of 84 diﬀerent scaﬀolds, each with 10,000–750,000 compounds, in approximately equal molar amounts. The polyamine library chosen for analysis contains 399,766 analogs; from this, 188 individual compounds were chosen for analysis. Detailed chemical characterization for scaﬀold libraries and individual compounds can be found in the general chemistry method section in Supplementary Figure S1. Individual compounds were synthesized as described in Scheme a in Supplementary Figure S1. Statistical Analysis of Checkerboard Assays Potentiation was quantiﬁed using a mathematical model developed by our group to assess the ability of library samples and individual compounds to eﬀectively enhance the activity of tetracycline or chloramphenicol (Hoel, 1987). This was used to diﬀerentiate libraries or compounds that possessed only antibacterial activity from those that had synergistic activity with tetracycline or chloramphenicol. In this way, only libraries and compounds that potentiated tetracycline or chloramphenicol activity were pursued. The model is based on the following equation for modeling the percentage activity of a mixture of two agents with independent action (Hoel, Bacterial Membrane Depolarization To determine the level of membrane depolarization by polyamine compounds a 3,3′-dipropylthiadicarbocyanine Iodide (DiSC3) Bacterial Membrane Depolarization To determine the level of membrane depolarization by polyamine compounds a 3,3′-dipropylthiadicarbocyanine Iodide (DiSC3) June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 3 Polyamines as EPIs Fleeman et al. ﬂuorescence dye was used. Exponentially growing cultures were prepared as described above, before being harvested by centrifugation. Cells were next washed in buﬀer a [5 mM Hepes (pH 7.2), 5 mM glucose] and resuspended to an OD600 of 0.2 in the same buﬀer containing 100 mM KCL and 2 µM DiSC3. Samples were allowed to equilibrate for 15 min at room temperature to ensure uptake and quenching of the dye in bacterial membranes. Cells were aliquoted into 96-well plates and polyamines were added alongside the known eﬄux inhibitor PAβN (all at 25 µg mL−1). Nisin (25 µg mL−1) was used as a positive control to display depolarization eﬀects. A Biotek plate reader was used to monitor ﬂuorescence of wells, with a 622 nm excitation and 670 nm emission. For graphical representation, the maximum relative ﬂuorescence at 2 min was used for comparison of lead agents to controls. tetracycline (0, 12, 25, and 50 µM) were used in combination with the MPC (25 µM) of lead agents 247, 250, 266, 271, and 314 against P. aeruginosa. Data is shown as percent recovery by dividing the CFU mL−1 of treatment groups by the CFU mL−1 recovered from a no treatment control that did not have tetracycline or eﬄux inhibitors. Bioﬁlm experiments were performed similar to those described by us previously (Fleeman et al., 2015; von Salm et al., 2016), with the following modiﬁcations. Polyamine agents 247, 250, 266, 271, and 314 were added at MPC into bioﬁlm containing wells alongside varying concentrations of tetracycline (0, 12, 25, and 50 µM). Cellular viability was determined by serial dilution after a 20-h incubation at 37◦C. Values were converted to percent recovery using no treatment controls. All data was generated from three biological replicates and two technical replicates. Eukaryotic Calcium Channel Activity Assay y The eﬀects of polyamine eﬄux inhibitors on eukaryotic ion channels was performed using a calcium channel assay kit (Life Technologies) and the Hek293T kidney cell line. Cells (5 × 104) were inoculated into a black walled 96-well plate and allowed to attach overnight at 37◦C with 5% CO2. After this, the Fluo-4 dye supplemented with Probenecid (5 mM) was added and allowed to equilibrate for 1 h at 37◦C with 5% CO2. After this time, ﬂuorescence was measured for 120 s using a Biotek plate reader with a 495 nm excitation and 516 nm emission. Cells were then treated with solvent only controls (10% DMF), as well as polyamine compounds 250, 266, 271, and the known calcium channel inhibitor verapamil (all at 25 µg mL−1). Fluorescence was monitored for 120 s, before calcium channels were stimulated with carbamylcholine chloride (137 µM). Readings were then taken at 6 s intervals, with peak relative ﬂuorescence at 18 s used graphically for comparison of lead agents to controls. RESULTS To assess toxicity of polyamine compounds we used HepG2 human liver carcinoma cells and Hek293T human embryonic kidney epithelial cells. The viability of cell lines was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) molecular probe as previously described by our group (Fleeman et al., 2015). Brieﬂy, 247, 266, 271 (Hek293T), or 250, 271, 314 (HepG2), alongside control eﬄux pump inhibitor PaβN, were diluted in 10% DMF from 25 µg mL−1 to 3 µg mL−1 using twofold dilutions, before being added to cells in DMEM with 10% FBS and 1% penicillin/streptomycin. Cells were then incubated for 48 h at 37◦C with 5% CO2. Following this, viability was assessed by the addition of MTT and measuring the OD570 in a Biotek plate reader. Percent recovery was determined compared to no drug controls. The Use of a Scaffold Ranking Library to Identify Potential Efﬂux Pump Inhibitors The Use of a Scaffold Ranking Library to Identify Potential Efﬂux Pump Inhibitors Identify Potential Efﬂux Pump Inhibitors We have previously described the synthesis and antibacterial activity of the Torrey Pines scaﬀold ranking library toward the ESKAPE pathogens. With the success of this screening, we decided to expand our approaches toward the development of anti-resistance agents, speciﬁcally targeting eﬄux mechanisms (Fleeman et al., 2015). As such, the 81 Torrey Pines scaﬀold samples were screened for their ability to decrease the 90% eﬀective concentration (EC90) of the known eﬄux pump substrate, tetracycline, toward a tetracycline resistant strain of P. aeruginosa (tetracycline alone EC90 = 82.5 µM). EC90s were deﬁned as the concentration required to produce 90% inhibition of the test organism. The capacity for potentiation of all scaﬀolds was determined by mathematical modeling to identify (and thus exclude) those molecules that also possessed antibacterial activity themselves (see Materials and Methods section “Statistical Analysis of Checkerboard Assays” for details). Upon analysis, 17 libraries resulted in a potentiated tetracycline EC90 of ≥twofold, whilst 6 resulted in a fold potentiation of ≥4 (Figure 1 and Supplementary Table S2). A consideration with these studies is that we wish to identify eﬄux pump inhibitors, rather than compounds that have bacterial killing activity themselves. Upon testing the top 17 libraries we determined that 7 of them, including the 5 most active scaﬀolds, had individual antibacterial activity. Of the remaining 10 libraries, 2229 (polyamines derived from reduced acyl peptides) had the best potentiating eﬀects (>fourfold, tetracycline EC90 lowered to 21.03 µM), without itself having antibacterial activity. For this reason, we prioritized the 2229 polyamine scaﬀold for further assessment. Bactericidal and Bioﬁlm Assessment In each assay, the TPL concentration used was 25 µg mL−1. The libraries represented by a red bar displayed inhibition of bacterial growth themselves, in the absence of tetracycline, whilst blue bars represent libraries that display no inhibition of bacterial growth. Positive (PAβN) and negative (10% DMF) control compounds were used, and are denoted by black bars. Note that only libraries displaying twofold or greater potentiation are shown. FIGURE 1 \| Screening of combinatorial libraries to identify scaffolds that inhibit bacterial efﬂux pumps. The Torrey Pines scaffold libraries (TPL) were screened for potentiation of tetracycline activity against a clinical tetracycline-resistant P. aeruginosa isolate 1419. Data is represented as fold potentiation, which is deﬁned as the EC90 tetracycline concentration (no TPL)/EC90 tetracycline concentration with TPL. In each assay, the TPL concentration used was 25 µg mL−1. The libraries represented by a red bar displayed inhibition of bacterial growth themselves, in the absence of tetracycline, whilst blue bars represent libraries that display no inhibition of bacterial growth. Positive (PAβN) and negative (10% DMF) control compounds were used, and are denoted by black bars. Note that only libraries displaying twofold or greater potentiation are shown. of tetracycline toward P. aeruginosa (Supplementary Table S3). These studies were expanded to include EC50 determinations as well as EC90 to provide depth to our structure activity relationship analysis. 271), as this was the most promising orientation for the positive charge. In addition, while the 10 most potentiating polyamines were shown to decrease the EC50 of tetracycline from 47.8 µM to ≤9 µM, there were a subset of four agents (247, 250, 271, 314) that were more eﬀective at decreasing the EC90 than the EC50 revealing their activity does not plateau before 90% bacterial inhibition is achieved. Therefore, we chose polyamines 247, 250, 266, 271, 314 from the 10 most potentiating polyamines to undergo secondary validation assessment to explore their eﬄux pump inhibitor-like properties (Figure 2). Upon analysis, 37 individual polyamines were found to inhibit bacterial growth alone at or below the maximum concentration tested 25 µg mL−1. Of the 151 remaining polyamines, 72 reduced the tetracycline EC50 by <twofold, 30 decreased the tetracycline EC50 between twofold and ﬁvefold, and 49 decreased the tetracycline EC50 by ≥ﬁvefold. From this latter group, 10 were also successful at decreasing the 90% eﬀective concentration by ≥ﬁvefold. Bactericidal and Bioﬁlm Assessment Four of the 10 most eﬀective polyamines (247, 250, 266, 271) had an amine functionality at the R1 position, S-methylbenzene at the R2 position, and ethylbenzene at the R3 position. Interestingly, both stereoisomers of methylbutylamine (247 = S-N-methylbutylamine: 266 = R-N-methylbutylamine) were found to create strong potentiation at the R1 position. From the remaining six polyamines, three (314, 338, and 348) had S-methylbenzene at the R1 position, an amine functionality at the R2 position, and ethylbenzene at the R3 position; while three (393, 414, and 453) had S-methylbenzene at the R1 and R2, and varied aromatic groups at the R3 position; thus, lacking an amine functionality at the R1 or R2 position. Although polyamines 393, 414, and 453 displayed promising fold-potentiation values, these agents were not selected as lead agents when considering that a large portion (24%) of polyamines with the R1 and R2 functionality deﬁned by S-methylbenzene displayed antibacterial activity themselves. In contrast, however, the majority (52%) of polyamines with S-methylbenzene at the R2 and R3 positions displayed ≥twofold potentiation of tetracycline activity without displaying inhibition alone. Therefore, we prioritized polyamines with amine functionalities at the R1 position (247, 250, 266, Using dose response studies (Figure 3A and Supplementary Figure S2A), we determined that the most eﬀective lead was compound 271, potentiating the tetracycline EC90 by 8.5-fold and its EC50 by 8.2-fold (Table 1). With regards to the remaining four compounds, we determined that 247 resulted in an 8.3-fold decrease of the EC90 and a ﬁvefold decrease of the EC50. Of note, these two compounds are similar with S-methylbenzene at the R2 position, and ethylbenzene at the R3 position, however, they diﬀer slightly at the R1 position (247 = S-N-methylbutylamine; 271 = R-N-propylamine). Additionally, compounds 250 and 266 both display a 7.8- and 5.8-fold potentiation of the tetracycline EC90, respectively, with strong EC50 values of 7.0- and 6.8-fold potentiation. Interestingly, 266 displayed more promising EC50 fold-potentiation than EC90, however this is a common feature of competitive eﬄux pump inhibitor (Askoura et al., 2011); indeed, our studies reveal a similar eﬀect for the well described molecules reserpine and PAβN (Table 1). Both 250 and 266 also have S-methylbenzene at the R2 position and ethylbenzene at the R3 position similar to compounds 247 and 271, but again vary at the R1 position (250 = S-ethylamine; 266 = R-N- methylbutylamine). Bactericidal and Bioﬁlm Assessment Bactericidal and Bioﬁlm Assessment Lead agents were screened for bactericidal activity as described by us previously (Van Horn et al., 2014; Fleeman et al., 2015), with the following modiﬁcations. Varying concentrations of To explore suitability of polyamine derivatives as eﬄux pump inhibitors, a library of 188 individual compounds contained within the Torrey Pines chemical collection were screened for their ability to decrease the 50 and 90% eﬀective concentration June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 4 Polyamines as EPIs Fleeman et al. FIGURE 1 \| Screening of combinatorial libraries to identify scaffolds that inhibit bacterial efﬂux pumps. The Torrey Pines scaffold libraries (TPL) were screened for potentiation of tetracycline activity against a clinical tetracycline-resistant P. aeruginosa isolate 1419. Data is represented as fold potentiation, which is deﬁned as the EC90 tetracycline concentration (no TPL)/EC90 tetracycline concentration with TPL. In each assay, the TPL concentration used was 25 µg mL−1. The libraries represented by a red bar displayed inhibition of bacterial growth themselves, in the absence of tetracycline, whilst blue bars represent libraries that display no inhibition of bacterial growth. Positive (PAβN) and negative (10% DMF) control compounds were used, and are denoted by black bars. Note that only libraries displaying twofold or greater potentiation are shown. FIGURE 1 \| Screening of combinatorial libraries to identify scaffolds that inhibit bacterial efﬂux pumps. The Torrey Pines scaffold libraries (TPL) were screened for potentiation of tetracycline activity against a clinical tetracycline-resistant P. aeruginosa isolate 1419. Data is represented as fold potentiation, which is deﬁned as the EC90 tetracycline concentration (no TPL)/EC90 tetracycline concentration with TPL. In each assay, the TPL concentration used was 25 µg mL−1. The libraries represented by a red bar displayed inhibition of bacterial growth themselves, in the absence of tetracycline, whilst blue bars represent libraries that display no inhibition of bacterial growth. Positive (PAβN) and negative (10% DMF) control compounds were used, and are denoted by black bars. Note that only libraries displaying twofold or greater potentiation are shown. FIGURE 1 \| Screening of combinatorial libraries to identify scaffolds that inhibit bacterial efﬂux pumps. The Torrey Pines scaffold libraries (TPL) were screened for potentiation of tetracycline activity against a clinical tetracycline-resistant P. aeruginosa isolate 1419. Data is represented as fold potentiation, which is deﬁned as the EC90 tetracycline concentration (no TPL)/EC90 tetracycline concentration with TPL. Frontiers in Microbiology \| www.frontiersin.org Bactericidal and Bioﬁlm Assessment Compound 314 was found to have EC50 and June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 5 Fleeman et al. Polyamines as EPIs FIGURE 2 \| Structure of core polyamine scaffold (2229) and individual polyamine lead molecules. FIGURE 3 \| Polyamine lead agents potentiate the activity of unrelated antibiotic efﬂux substrates. P. aeruginosa (1419) cells were treated with polyamine molecules at increasing concentrations, alongside tetracycline (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic (EC90 values) as the efﬂux pump inhibitor concentration was increased. EC f ld t ti ti l f 5 d 7 5 f ld ti l (Fi 3B d S l t Fi S2B) E h f th Polyamines as EPIs Fleeman et al. FIGURE 2 \| Structure of core polyamine scaffold (2229) and individual polyamine lead molecules. FIGURE 2 \| Structure of core polyamine scaffold (2229) and individual polyamine lead molecules. FIGURE 3 \| Polyamine lead agents potentiate the activity of unrelated antibiotic efﬂux substrates. P. aeruginosa (1419) cells were treated with polyamine molecules at increasing concentrations, alongside tetracycline (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic (EC90 values) as the efﬂux pump inhibitor concentration was increased. FIGURE 3 \| Polyamine lead agents potentiate the activity of unrelated antibiotic efﬂux substrates. P. aeruginosa (1419) cells were treated with polyamine molecules at increasing concentrations, alongside tetracycline (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic (EC90 values) as the efﬂux pump inhibitor concentration was increased. EC90, fold potentiation values of 5- and 7.5-fold, respectively. Of note, 314 has an S-methylbenzene at the R1 position and an amine functionality (this time propylamine) at the R2. (Figure 3B and Supplementary Figure S2B). Each of the polyamine agents again displayed an increase in the potentiation of chloramphenicol EC90 in a dose responsive manner. Agent 266 displayed the highest potentiation values, although all compounds performed in a markedly similar, and eﬀective manner. To determine if P. aeruginosa was the only organism targeted by our polyamine lead molecules, we next performed similar experiments with two other leading, and unrelated, ESKAPE pathogens: A. baumannii and Methicillin Resistant S. aureus (MRSA). It should be noted that in these studies, we only used a single compound for each, for proof of principle, due Polyamines Inhibit Efﬂux Pump Activity for a Wide Range of Organisms and Antibiotic Substrates To conﬁrm that the activity of our lead agents was not merely conﬁned to tetracycline, we next explored the potentiation of an unrelated antibiotic eﬄux substrate, chloramphenicol June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 6 Polyamines as EPIs Fleeman et al. TABLE 1 \| Potentiation assessment of lead polyamine compounds. MW EPI∗a TET+EPI∗b FP∗c TET+EPI#b FP#c 247 368.56 >25 9 5.0 12 8.3 250 340.51 >25 9 7.0 12 7.8 266 368.56 >25 9 6.8 16 5.8 271 354.53 >25 5 8.2 10 8.5 314 354.53 >25 9 5.0 12 7.5 Reserpine 608.68 >25 4 11.2 >50 1 PAβN 519.47 >25 0.7 65.5 11.63 8 All assays were performed in triplicate alongside no drug controls, known efﬂux pump inhibitors (EPIs) reserpine and PAβN, and tetracycline alone controls (data not shown due to repetitive values, average concentration for EC50 across all replicates = 47.8 µM; EC90 = 82.5 µM). Molecular weight (MW) of all compounds is displayed as grams mole−1. ∗50% effective concentrations (EC50). #90% effective concentrations (EC90). aEPI is the inhibitory concentration of the individual Efﬂux Pump Inhibitor (EPI) alone (µg mL−1). bTET+EPI is the respective EC tetracycline (µM) in the presence of each compound. cFP is Fold Potentiation = EC tetracycline + EPI/EC tetracycline (no EPI). TABLE 1 \| Potentiation assessment of lead polyamine compounds. similar results (Figure 5B), indicating the broad-spectrum nature of these agents. Furthermore, when we assayed the gram- positive pathogen S. aureus, we again derived similar ﬁndings (Supplementary Figure S3A). To provide further direct evidence of eﬄux inhibition by our lead compounds, we next tested their activity using strains with varying eﬄux potential. As such, alongside our test P. aeruginosa strain (1419) which has strong eﬄux capacity, we also tested an eﬄux impaired strain of this same organism, 1418. The eﬄux capacity of these two strains was determined using an ethidium bromide agar assay that has been previously documented as a measure of eﬄux capacity for bacterial strains (Supplementary Figure S4; Martins et al., 2013). Here, we tested all 5 leads against the two strains and the clinically relevant eﬄux antibiotic substrate aztreonam (Figure 6A). Upon analysis we found that all of our lead molecules were capable of eﬀectively potentiating the activity of aztreonam toward our test strain 1419, however no such inhibition was seen for isolate 1418. Polyamines Function via the Inhibition of Bacterial Efﬂux Mechanisms Following these promising results, we sought to validate our ﬁndings using more direct means. Accordingly, the polyamines were assessed using an ethidium bromide ﬂuorescence assay that has been widely used to identify eﬄux inhibitors (Renau et al., 1999; Lomovskaya et al., 2001; Webber et al., 2013; Blanchard et al., 2014; Vasudevan et al., 2014). This assay is a well documented variation on ethidium bromide accumulation assays, as it allows for a pre-incubation step to facilitate accumulation, before compound treatment to measure eﬄux (Raherison et al., 2002; Paixao et al., 2009). Fluorescence of ethidium bromide occurs during intercalation with DNA; thus, active eﬄux mechanisms decrease such ﬂuorescence by extruding ethidium bromide before it can interact with its target. Thus, disruption of eﬄux pump activity leads to the accumulation of intracellular ethidium bromide and a subsequent increase in ﬂuorescence over time compared to eﬄux proﬁcient cells. Importantly, when we treated P. aeruginosa with lead polyamines, followed by a sub-lethal concentration of ethidium bromide, we observed an increase in ﬂuorescence (Figure 5A) compared to no drug controls; indicating inhibition of eﬄux systems. To determine if these eﬀects are solely limited to P. aeruginosa, we next tested other Gram-negative pathogens. When these assays were repeated with A. baumannii, we observed Polyamines Inhibit Efﬂux Pump Activity for a Wide Range of Organisms and Antibiotic Substrates Interestingly, we observed strong activity correlation with these studies, as our two best potentiators of tetracycline (271 and 250) were also the best potentiators of aztreonam. to limited available material. For the former organism we used lead molecule 271 and the antibiotic eﬄux substrate levoﬂoxacin, again revealing a dose response curve of antibiotic potentiation as seen with P. aeruginosa (Figure 4A). When MRSA was tested in a similar manner using the substrate chloramphenicol and lead 247, we also noted marked potentiation of antibiotic activity in a dose responsive manner (Figure 4B). As such, it would appear that our polyamines are capable of potentiating the activity of multiple antibiotic substrates, from a variety of diﬀerent classes, and toward both Gram-negative and Gram- positive pathogens. To ensure that these ﬁndings were not speciﬁc only to P. aeruginosa, we next tested one of our lead polyamines, 247 (only 1 used due to limited available compound), against an eﬄux deﬁcient strain of MRSA. For this we used a mutant of the major eﬄux pump NorA in the United States 300 background. When tested, we observed that whilst our lead polyamine was able to strongly potentiate chloramphenicol activity in the wild-type strain, there was little potentiation observed in the norA knockout (Figure 6B). The small amount of potentiation seen in the mutant strain is likely a result of the fact that, although NorA is indeed the major eﬄux pump in S. aureus, others also exist in this organism (Costa et al., 2013). Importantly, the MRSA strain used in these studies was diﬀerent to those for our ethidium bromide analysis (USA 300 vs. United States 100), presenting further evidence that our molecules are not strain (or species) speciﬁc in their eﬀects. Collectively, these data suggest that our polyamine molecules are not only eﬀective inhibitors of bacterial eﬄux mechanisms, but that these eﬀects appear to be broad-spectrum in range. Frontiers in Microbiology \| www.frontiersin.org Polyamines Act Competitively With PAβN to Potentiate the Tetracycline Effective Concentration baumannii (1643) or MRSA (USA300) cells were treated with the noted polyamine molecules at increasing concentrations, alongside levoﬂoxacin (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic FIGURE 4 \| Lead polyamines potentiate antibiotic activity toward other ESKAPE pathogen species. A. baumannii (1643) or MRSA (USA300) cells were treated with the noted polyamine molecules at increasing concentrations, alongside levoﬂoxacin (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic (EC90 values, blue bars) as the efﬂux pump inhibitor concentration was increased, and the concomitant decline in antibiotic MIC (EC90 values, red bars). Error bars are shown ± SEM FIGURE 5 \| Polyamine molecules have broad spectrum efﬂux pump inhibitor activity. P. aeruginosa (1419) (A) or A baumannii (1643) (B) cells were treated with a sub-lethal concentration of ethidium bromide (25 µM) in combination with tetracycline, the known efﬂux inhibitor PAβN, lead polyamine agents at 25 µg mL−1, or vehicle (10% DMF) (ND). Graphs demonstrate ﬂuorescence after 90-min exposure displayed as relative ﬂuorescent units. Error bars are shown ± SEM. FIGURE 5 \| Polyamine molecules have broad spectrum efﬂux pump inhibitor activity. P. aeruginosa (1419) (A) or A baumannii (1643) (B) cells were treated with a sub-lethal concentration of ethidium bromide (25 µM) in combination with tetracycline, the known efﬂux inhibitor PAβN, lead polyamine agents at 25 µg mL−1, or vehicle (10% DMF) (ND). Graphs demonstrate ﬂuorescence after 90-min exposure displayed as relative ﬂuorescent units. Error bars are shown ± SEM. FIGURE 5 \| Polyamine molecules have broad spectrum efﬂux pump inhibitor activity. P. aeruginosa (1419) (A) or A baumannii (1643) (B) cells were treated with a sub-lethal concentration of ethidium bromide (25 µM) in combination with tetracycline, the known efﬂux inhibitor PAβN, lead polyamine agents at 25 µg mL−1, or vehicle (10% DMF) (ND). Graphs demonstrate ﬂuorescence after 90-min exposure displayed as relative ﬂuorescent units. Error bars are shown ± SEM. June 2018 \| Volume 9 \| Article 1301 Polyamines Act Competitively With PAβN to Potentiate the Tetracycline Effective Concentration To further conﬁrm the eﬄux pump inhibitor activity of polyamine agents, we performed a checkerboard assessment to determine the relationship between our front runner molecules and the control compound PAβN. If our polyamine agents inhibit a target other than eﬄux pumps, then combination treatment would produce a synergistic action. However, if the agents are both inhibiting eﬄux pumps, the result of combination treatment would be antagonistic (Auerbach et al., 2010; Goodman et al., 2011). Upon analysis we observed a clear competitive interaction between PAβN and polyamine agents in the presence of tetracycline (Supplementary Figure S5). This further conﬁrms that the polyamine agents are inhibiting eﬄux pumps of bacterial species. June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 7 Polyamines as EPIs Fleeman et al. FIGURE 4 \| Lead polyamines potentiate antibiotic activity toward other ESKAPE pathogen species. A. baumannii (1643) or MRSA (USA300) cells were treated with the noted polyamine molecules at increasing concentrations, alongside levoﬂoxacin (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic (EC90 values, blue bars) as the efﬂux pump inhibitor concentration was increased, and the concomitant decline in antibiotic MIC (EC90 values, red bars). Error bars are shown ± SEM. FIGURE 5 \| Polyamine molecules have broad spectrum efﬂux pump inhibitor activity. P. aeruginosa (1419) (A) or A baumannii (1643) (B) cells were treated with a sub-lethal concentration of ethidium bromide (25 µM) in combination with tetracycline, the known efﬂux inhibitor PAβN, lead polyamine agents at 25 µg mL−1, or vehicle (10% DMF) (ND). Graphs demonstrate ﬂuorescence after 90-min exposure displayed as relative ﬂuorescent units. Error bars are shown ± SEM. Polyamine Molecules Do Not Randomly on bacterial membranes, as they allowed for stabilization of FIGURE 4 \| Lead polyamines potentiate antibiotic activity toward other ESKAPE pathogen species. A. baumannii (1643) or MRSA (USA300) cells were treated with the noted polyamine molecules at increasing concentrations, alongside levoﬂoxacin (A) or chloramphenicol (B). Shown is the fold potentiation of each antibiotic (EC90 values, blue bars) as the efﬂux pump inhibitor concentration was increased, and the concomitant decline in antibiotic MIC (EC90 values, red bars). Error bars are shown ± SEM. FIGURE 4 \| Lead polyamines potentiate antibiotic activity toward other ESKAPE pathogen species. A. Polyamine Molecules Do Not Randomly Depolarize Prokaryotic or Eukaryotic Membranes (B) Lead polyamine 247 was assessed for its ability to potentiate the activity of chloramphenicol toward S. aureus strain United States 300 and its norA mutant. Shown is the potentiated chloramphenicol EC90 calculated as described in A. Error bars are shown ± SEM. FIGURE 7 \| Polyamines do not destabilize prokaryotic or eukaryotic membranes. (A) Shown is the change in ﬂuorescence of P. aeruginosa (1419) cells using a Disc3 dye assay. Data is presented as change in ﬂuorescence before and after addition of leads compounds, PAβN, and nisin at 25 µg mL−1. (B) Calcium channel activity assays to assess inhibition of calcium channel pumps in HEK 293 cells after the addition of polyamines 250, 266, 271 or the positive control verapamil at 25 µg mL−1. Data is presented as change in ﬂuorescence of cells before and after addition of the Fluo-4 dye. No drug (ND) and/or chloramphenicol (CM) were used as negative controls. Error bars are shown ± SEM. FIGURE 7 \| Polyamines do not destabilize prokaryotic or eukaryotic membranes. (A) Shown is the change in ﬂuorescence of P. aeruginosa (1419) cells using a Disc3 dye assay. Data is presented as change in ﬂuorescence before and after addition of leads compounds, PAβN, and nisin at 25 µg mL−1. (B) Calcium channel activity assays to assess inhibition of calcium channel pumps in HEK 293 cells after the addition of polyamines 250, 266, 271 or the positive control verapamil at 25 µg mL−1. Data is presented as change in ﬂuorescence of cells before and after addition of the Fluo-4 dye. No drug (ND) and/or chloramphenicol (CM) were used as negative controls. Error bars are shown ± SEM. other, unrelated polyamines have been shown to have the capacity to destabilize membranes by others (Katsu et al., 2002). lead compound treated cells exhibited increased ﬂuorescence in the presence of the Fluoro-4 dye, whilst verapamil decreased ﬂuorescence, representing the inhibition of calcium channel activity. Thus, it would appear that our polyamines are not only speciﬁc eﬄux pump inhibitors, but that their eﬀects are selective for prokaryotic membrane pumps, over their eukaryotic counterparts. Another key consideration when developing eﬄux pump inhibitors is their impact on eukaryotic eﬄux systems, as many such molecules identiﬁed to date have non-speciﬁc eﬀects on mammalian ion transport systems as well (Van Bambeke et al., 2006). Polyamine Molecules Do Not Randomly Depolarize Prokaryotic or Eukaryotic Membranes This is of particular importance when testing polyamines, as other, unrelated molecules of this class have been shown to disrupt calcium release in bacterial cells as well as eukaryotic cells (Katsu et al., 2002). As such, we tested the eﬀects of the polyamines in this regard against human embryonic kidney epithelial cells (Hek293T), alongside the known, and toxic eﬄux pump inhibitor, verapamil. In these studies, we determined that our polyamine eﬄux inhibitors mirrored no drug controls when assessed for their ability to interfere with eukaryotic calcium channel activity (Figure 7B). Speciﬁcally, Polyamine Molecules Do Not Randomly Depolarize Prokaryotic or Eukaryotic Membranes on bacterial membranes, as they allowed for stabilization of the quenched dye (Figure 7A). Whilst the positive control nisin and PAβN treated cell membranes displayed a strong increase in ﬂuorescence, indicating membrane destabilization, cells treated with chloramphenicol or solvent only (10% DMF) decreased in ﬂorescence indicating a continued quenching of the membrane dye. Polyamines treated cells displayed essentially no change in ﬂuorescence, revealing little to no depolarization when compared to the known eﬄux inhibitor PAβN. To ensure there is no depolarization of Gram-positive bacterial membranes we repeated this experiment with S. aureus and observed a lack of depolarization toward this species as well (Supplementary Figure S3B). As such, these results are considered promising given that A number of eﬄux pump inhibitors discovered to date have been shown to non-speciﬁcally inhibit eﬄux mechanisms through the non-speciﬁc depolarization of charge across bacterial membranes (Askoura et al., 2011; Webber et al., 2013). To determine if such eﬀects were true of our polyamines, we assessed membrane depolarization using the molecular probe DiSC3. In cells with normal membrane polarity, the bacterial membrane will quench ﬂuorescence of the DiSC3 dye. However, if the membrane is depolarized, the dye is released, and ﬂuorescence increases over time. Our results reveal that the polyamines had minimal eﬀect June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 8 Polyamines as EPIs Fleeman et al. FIGURE 6 \| Conﬁrming efﬂux pump inhibition by polyamines using efﬂux deﬁcient strains. (A) A P. aeruginosa proﬁcient (1419) and deﬁcient (1418) strain were subjected to combination testing of the polyamines at 12.5 µg mL−1 and 1/2 X MIC of aztreonam. Fold potentiation was calculated by dividing the CFU mL−1 for aztreonam only by the CFU mL−1 of the combined treatment groups. (B) Lead polyamine 247 was assessed for its ability to potentiate the activity of chloramphenicol toward S. aureus strain United States 300 and its norA mutant. Shown is the potentiated chloramphenicol EC90 calculated as described in A. Error bars are shown ± SEM. FIGURE 7 \| Polyamines do not destabilize prokaryotic or eukaryotic membranes. (A) Shown is the change in ﬂuorescence of P. aeruginosa (1419) cells using a Disc3 dye assay. Data is presented as change in ﬂuorescence before and after addition of leads compounds, PAβN, and nisin at 25 µg mL−1. Polyamine Molecules Do Not Randomly Depolarize Prokaryotic or Eukaryotic Membranes (B) Calcium channel activity assays to assess inhibition of calcium channel pumps in HEK 293 cells after the addition of polyamines 250, 266, 271 or the positive control verapamil at 25 µg mL−1. Data is presented as change in ﬂuorescence of cells before and after addition of the Fluo-4 dye. No drug (ND) and/or chloramphenicol (CM) were used as negative controls. Error bars are shown ± SEM. FIGURE 6 \| Conﬁrming efﬂux pump inhibition by polyamines using efﬂux deﬁcient strains. (A) A P. aeruginosa proﬁcient (1419) and deﬁcient (1418) strain were subjected to combination testing of the polyamines at 12.5 µg mL−1 and 1/2 X MIC of aztreonam. Fold potentiation was calculated by dividing the CFU mL−1 for aztreonam only by the CFU mL−1 of the combined treatment groups. (B) Lead polyamine 247 was assessed for its ability to potentiate the activity of chloramphenicol toward S. aureus strain United States 300 and its norA mutant. Shown is the potentiated chloramphenicol EC90 calculated as described in A. Error bars are shown ± SEM. FIGURE 6 \| Conﬁrming efﬂux pump inhibition by polyamines using efﬂux deﬁcient strains. (A) A P. aeruginosa proﬁcient (1419) and deﬁcient (1418) strain were subjected to combination testing of the polyamines at 12.5 µg mL−1 and 1/2 X MIC of aztreonam. Fold potentiation was calculated by dividing the CFU mL−1 for 1 FIGURE 6 \| Conﬁrming efﬂux pump inhibition by polyamines using efﬂux deﬁcient strains. (A) A P. aeruginosa proﬁcient (1419) and deﬁcient (1418) strain were subjected to combination testing of the polyamines at 12.5 µg mL−1 and 1/2 X MIC of aztreonam. Fold potentiation was calculated by dividing the CFU mL−1 for aztreonam only by the CFU mL−1 of the combined treatment groups. (B) Lead polyamine 247 was assessed for its ability to potentiate the activity of chloramphenicol toward S. aureus strain United States 300 and its norA mutant. Shown is the potentiated chloramphenicol EC90 calculated as described in A. Error bars are shown ± SEM. FIGURE 6 \| Conﬁrming efﬂux pump inhibition by polyamines using efﬂux deﬁcient strains. (A) A P. aeruginosa proﬁcient (1419) and deﬁcient (1418) strain were subjected to combination testing of the polyamines at 12.5 µg mL−1 and 1/2 X MIC of aztreonam. Fold potentiation was calculated by dividing the CFU mL−1 for aztreonam only by the CFU mL−1 of the combined treatment groups. Frontiers in Microbiology \| www.frontiersin.org Lead Polyamine Efﬂux Pump Inhibitors Lack General Toxicity Toward Eukaryotic Cells Furthermore, 50 µM treatment with tetracycline decreased bacterial viability to 1% alone, however this was drastically decreased with combination eﬄux pump inhibitor treatment. Speciﬁcally, polyamines 250, 266, and 314 resulted in the greatest decrease in bacterial recovery, allowing for 0.01% recovery. This was marginally less recovery than that of the control PAβN and polyamine 271, which allowed for 0.02% recovery. Polyamine 247 displayed the least decrease in viability with combination treatment, although it still decreased bacterial recovery to 0.04%. Given that bactericidal activity is often preferred to bacteriostatic eﬀects, particularly (Figure 8). In so doing, we determined that front runners 247, 266, and 271 had extremely low toxicity toward Hek293T cells. Speciﬁcally, when treated with 25 µg mL−1 of these compounds, cells displayed 84, 72, and 75% recovery compared to solvent only controls, whilst the known eﬄux pump inhibitor PAβN returned only 63% cell viability. In support of this, HepG2 cell recovery after treatment with 250, 314, or 271 generated similar results; even at the highest concentration tested (again 25 µg mL−1) we observed 80, 77, and 74% cell viability. In comparison, the known eﬄux inhibitor PAβN tested at the same concentration allowed for 68% recovery of HepG2 cells. The higher toxicity of PAβN was perhaps unsurprising considering that this agent has been shown to depolarize membranes at higher concentrations (Webber et al., 2013). Lead Polyamine Efﬂux Pump Inhibitors Lack General Toxicity Toward Eukaryotic Cells Given the lack of eﬀect of polyamines toward eukaryotic ion channels, we next assessed general cytotoxicity toward human cells. As such, polyamine lead compounds were tested against both HepG2 and Hek293T cell lines using MTT assays June 2018 \| Volume 9 \| Article 1301 9 Polyamines as EPIs Fleeman et al. FIGURE 8 \| Lead polyamines lack general toxicity toward eukaryotic cells. Shown is the percent recovery of Hek293T cells (A) and HepG2 cells (B) when tested using a MTT cytotoxicity assay following treatment with polyamine leads. Conversion of MTT to formazan was assessed and converted to percent recovery using 0.01% Triton 100× as 100% death, and no treatment (DMF) as 100% survival. These controls were used to calculate percent recovery, and to determine LD50s (dotted line). Data is from at least three biological replicates, with error bars shown ± SEM. FIGURE 8 \| Lead polyamines lack general toxicity toward eukaryotic cells. Shown is the percent recovery of Hek293T cells (A) and HepG2 cells (B) when tested using a MTT cytotoxicity assay following treatment with polyamine leads. Conversion of MTT to formazan was assessed and converted to percent recovery using 0.01% Triton 100× as 100% death, and no treatment (DMF) as 100% survival. These controls were used to calculate percent recovery, and to determine LD50s (dotted line). Data is from at least three biological replicates, with error bars shown ± SEM. Tetracycline treatment alone at 12, 25, and 50 µM allowed for 53, 35, and 1% respective bacterial recovery. However, combination treatment with tetracycline and the MPC of all lead agents resulted in decreased bacterial viability. For example, combination treatment with 12 µM of tetracycline and polyamine 266 displayed the greatest decrease in bacterial viability, similar to the control PAβN. Speciﬁcally, the percent recovery decreased to 0.76 and 0.79% when treated with 266 or polyamine PaβN, respectively (Figure 9). Although not as impressive as 266 and PAβN, combination treatment with polyamines 247, 250, 271, and 314 resulted in 2.9, 2.2, 2, and 5.3% recovery, respectively. Interestingly, we found that increasing tetracycline alone from 12 to 25 µM resulted in 17.8% less recovery, however combination treatment revealed a signiﬁcant decrease in bacterial viability. In combination with 25 µM tetracycline, our polyamines appeared to outperform PAβN as they allowed for ≤0.08% recovery, whilst combination treatment with PAβN allowed for 0.2% recovery. June 2018 \| Volume 9 \| Article 1301 DISCUSSION Antibiotic resistance is a developing crisis in clinical settings, with an increasing number of bacterial isolates discovered each year that are resistant to our therapeutic arsenal (Rossolini et al., 2014; Ventola, 2015). Eﬄux pumps are a major contributor to multi-drug resistance because they help circumvent the action of a broad range of substrates that includes multiple antibiotics classes (Askoura et al., 2011; Handzlik et al., 2013; Soto, 2013). This is compounded by the observation that most bacterial species utilize multiple eﬄux pumps with an overlapping range of substrates (Weinstein and Hooper, 2005; Van Bambeke et al., 2006; Sun et al., 2014). With this considered, the development of broadly active eﬄux pump inhibitors is considered desirable, so as to focus on the reclamation or reactivation of a wide swath of existing therapeutics (Masuda et al., 2000; Lomovskaya et al., 2001; Kumar and Varela, 2012; Blanchard et al., 2014). FIGURE 10 \| Polyamine potentiation of tetracycline activity limits bioﬁlm FIGURE 10 \| Polyamine potentiation of tetracycline activity limits bioﬁlm recovery by P. aeruginosa. The lead polyamine agents were tested for their ability to impact viability of a pre-formed bioﬁlm. P. aeruginosa (1419) cells were treated with tetracycline at 0, 12, 25, and 50 µM in combination with no drug (ND), or 247, 250, 266, 271, 314 and PAβN at 25 µg mL−1. The dotted line on the graph denotes 90% reduction in cell viability within bioﬁlms. Data is from at least three biological replicates, with error bars shown ± SEM. for immunocompromised patients, these ﬁndings are considered encouraging. The potentiating modeling utilized in this study allowed for the identiﬁcation of polyamines that increased the eﬀectiveness of tetracycline without displaying any toxic eﬀects themselves. This highlights the importance of potentiation modeling for the identiﬁcation of anti-resistance agents, as opposed to synergistic agents that display antimicrobial properties as well. Potentiation modeling is a more advantageous approach to identifying adjuvant agents because synergy assessment is reliant on the therapeutic agent having antibacterial activity (Hoel, 1987). Eﬄux pump inhibitors identiﬁed using synergistic activities, i.e., PaβN, have been unsuccessful due to oﬀ-target eﬀects, causing bacterial growth inhibition (Renau et al., 1999; Lomovskaya et al., 2001; Webber et al., 2013). DISCUSSION Importantly, in support of our approach, it has been shown that the concomitant treatment with an antibiotic and adjuvant agent that blocks the mechanism of resistance toward that antibiotic, but that has no antimicrobial properties itself, can lead to decreased resistance development Polyamine Efﬂux Pump Inhibitors Strongly Enhance the Bactericidal Activity of Tetracycline FIGURE 9 \| Polyamine efﬂux pump inhibitors strongly enhance the bactericidal activity of tetracycline. P. aeruginosa (1419) cells were treated with tetracycline at 0, 12, 25, and 50 µM in combination with no drug (ND), or 247, 250, 266, 271, 314 and PAβN at 25 µg mL−1. The dotted line on the graph denotes 90% bactericidal activity. Data is from at least three biological replicates, with error bars shown ± SEM. Polyamine Efﬂux Pump Inhibitors Strongly Enhance the Bactericidal Activity of Tetracycline We next set out to explore the impact of polyamines on the bactericidal eﬀects of tetracycline. The rationale for this was that, although tetracycline is a bacteriostatic antibiotic, it is known to be bactericidal at high concentrations (Pankey and Sabath, 2004). Treatment with our polyamines alone at 25 µg mL−1 resulted in minimal impact to bacterial viability, with ≥95% of cells recovered for all compounds, in contrast to PAβN which returned only 76% viability at the same concentration. June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 10 Polyamines as EPIs Fleeman et al. Combination treatment with 12 µM tetracycline and the MPC of all lead agents resulted in a signiﬁcant decrease in bioﬁlm recovery, however, with viability of 9.1–11% observed for 271, 247, 266, and 314, respectively. Combination treatment with lead agent 250 resulted in a slightly higher bioﬁlm recovery of 21%, however still improving tetracycline alone bioﬁlm eradication by 67%. Increasing tetracycline concentration by itself from 12 to 25 µM only resulted in 4% more eradication, however in combination with our eﬄux pump inhibitors, recovery decreased to 0.5 and 0.6% for 271 and 247, respectively. Similarly, treatment with agents 266, 250, and 314 resulted in 1% bioﬁlm recovery. This bioﬁlm eradication was particularly impressive when compared to the activity of the positive control PAβN (7% recovery at 25 µM tetracycline). Furthermore, at the highest tetracycline concentration (50 µM) combination treatment with PaβN produced a 1% bioﬁlm recovery while agents 271 and 247 allowed for only 0.2% recovery. Treatment with agent 250, 266, and 314 resulted in similar recovery of 0.5, 0.6, and 0.6%, respectively. These results suggest a potential beneﬁt of combination treatment with our polyamine molecules and known eﬄux antibiotics to reduce bioﬁlms. Combination treatment with 12 µM tetracycline and the MPC of all lead agents resulted in a signiﬁcant decrease in bioﬁlm recovery, however, with viability of 9.1–11% observed for 271, 247, 266, and 314, respectively. Combination treatment with lead agent 250 resulted in a slightly higher bioﬁlm recovery of 21%, however still improving tetracycline alone bioﬁlm eradication by 67%. Increasing tetracycline concentration by itself from 12 to 25 µM only resulted in 4% more eradication, however in combination with our eﬄux pump inhibitors, recovery decreased to 0.5 and 0.6% for 271 and 247, respectively. Similarly, treatment with agents 266, 250, and 314 resulted in 1% bioﬁlm recovery. Polyamine Efﬂux Pump Inhibitors Strongly Enhance the Bactericidal Activity of Tetracycline This bioﬁlm eradication was particularly impressive when compared to the activity of the positive control PAβN (7% recovery at 25 µM tetracycline). Furthermore, at the highest tetracycline concentration (50 µM) combination treatment with PaβN produced a 1% bioﬁlm recovery while agents 271 and 247 allowed for only 0.2% recovery. Treatment with agent 250, 266, and 314 resulted in similar recovery of 0.5, 0.6, and 0.6%, respectively. These results suggest a potential beneﬁt of combination treatment with our polyamine molecules and known eﬄux antibiotics to reduce bioﬁlms. FIGURE 9 \| Polyamine efﬂux pump inhibitors strongly enhance the bactericidal activity of tetracycline. P. aeruginosa (1419) cells were treated with tetracycline at 0, 12, 25, and 50 µM in combination with no drug (ND), or 247, 250, 266, 271, 314 and PAβN at 25 µg mL−1. The dotted line on the graph denotes 90% bactericidal activity. Data is from at least three biological replicates, with error bars shown ± SEM. FIGURE 10 \| Polyamine potentiation of tetracycline activity limits bioﬁlm recovery by P. aeruginosa. The lead polyamine agents were tested for their ability to impact viability of a pre-formed bioﬁlm. P. aeruginosa (1419) cells were treated with tetracycline at 0, 12, 25, and 50 µM in combination with no drug (ND), or 247, 250, 266, 271, 314 and PAβN at 25 µg mL−1. The dotted line on the graph denotes 90% reduction in cell viability within bioﬁlms. Data is from at least three biological replicates, with error bars shown ± SEM. FIGURE 9 \| Polyamine efﬂux pump inhibitors strongly enhance the bactericidal activity of tetracycline. P. aeruginosa (1419) cells were treated with tetracycline at 0, 12, 25, and 50 µM in combination with no drug (ND), or 247, 250, 266, 271, 314 and PAβN at 25 µg mL−1. The dotted line on the graph denotes 90% bactericidal activity. Data is from at least three biological replicates, with error bars shown ± SEM. FIGURE 9 \| Polyamine efﬂux pump inhibitors strongly enhance the bactericidal activity of tetracycline. P. aeruginosa (1419) cells were treated with tetracycline at 0, 12, 25, and 50 µM in combination with no drug (ND), or 247, 250, 266, 271, 314 and PAβN at 25 µg mL−1. The dotted line on the graph denotes 90% bactericidal activity. Data is from at least three biological replicates, with error bars shown ± SEM. June 2018 \| Volume 9 \| Article 1301 Polyamine Potentiation of Tetracycline Activity Reduces Bioﬁlms by P. aeruginosa Kwon and Lu (2007) revealed the endogenous polyamines spermidine and spermine, found within all living cells, when administered exogenously were eﬀective at increasing the therapeutic potential of β-lactams toward Gram-negative organisms by blocking the outer membrane porin OprD. They also found these natural polyamines were shown to potentiate activity of chloramphenicol and β-lactams toward Escherichia coli and S. aureus (Kwon and Lu, 2007). Similarly, in Bacillus subtilis, the eﬄux pump Blt is dedicated to the extrusion of spermidine, however it has also been shown to opportunistically bind to other polyamine molecules (Vazquez-Laslop, 1997). Furthermore, in Mycobacterium tuberculosis, there is a signiﬁcant increase in the eﬀectiveness of ﬂuoroquinolones administered alongside polyamine treatment (Rockey et al., 2013). Conversely, it has been shown that natural polyamines and PaβN have toxicity associated with their amine substituents, therefore care must be taken when developing therapeutics of this nature (Opperman et al., 2013; Pegg, 2013). Even though this toxicity is known, amine molecules continue to be pursued to target multiple disease states (Goldberg and Rajfer, 1982; Thomas and Thomas, 2001; Bogatcheva et al., 2006; de Jonge et al., 2015; Li et al., 2015a). Moreover, there are many therapeutics in use in clinics today that contain amines, such as aminoglycosides; thus, despite their potential for toxicity, approaches have been used successful to decrease these eﬀects (Poulikakos and Falagas, 2013). Indeed, the FDA recently approved Zerbaxa (ceftolozane/tazobactam) for treatment of β-lactamase producing enteric species. Importantly, the ceftolozane component of this therapeutic combination contains multiple amine substituents (Fernandes and Martens, 2017). These studies collectively reveal the therapeutic potential of polyamine molecules despite the potential for compounds of this class to display unwanted side-eﬀects. Notably, the direct measurement of ethidium bromide ﬂuorescence revealed our polyamines inhibit the eﬄux pumps of Gram-negative species P. aeruginosa and A. baumannii, as well as the Gram-positive organism S. aureus. There are ﬁve families of eﬄux pumps: MFS, ABC, SMR, MATE, and RND. The ﬁrst four are used by all bacterial species, whilst RND eﬄux pumps are largely used by Gram-negative species (Sun et al., 2014). The RND family is the main Gram-negative eﬄux system suggesting that, as our polyamine agents are active in both Gram-positive and -negative organisms, they are likely inhibiting more than one family of eﬄux pumps (although it is acknowledged that some Gram-positive organisms do harbor them). Polyamine Potentiation of Tetracycline Activity Reduces Bioﬁlms by P. aeruginosa Bioﬁlm formation is responsible for chronic, drug-resistant bacterial infections by a number of pathogens, and particularly P. aeruginosa. Considering the strong potentiation of tetracycline activity engendered by our lead agents, we next chose to determine if they had signiﬁcant impact on the viability of cells within bioﬁlms. Treatment with eﬄux pump inhibitors 247, 250, 266, 271, and 314 alone at 25 µg mL−1 (MPC), respectively, resulted in negligible impact on bioﬁlm viability, with 99.99% of cells recovered for all compounds other than 266 which allowed for 92% recovery. Similarly, tetracycline treatment alone at 12 and 25 µM had little impact, allowing for 88 and 84% bioﬁlm recovery, respectively (Figure 10); only at 50 µM were more pronounced eﬀects observed, with only 9% cells recovered. June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 11 Polyamines as EPIs Fleeman et al. (Olofsson and Cars, 2007; Worthington and Melander, 2013). Although a subset of polyamines was discovered during our screening with antimicrobial eﬀect themselves, their structure activity relationship was taken into consideration during lead polyamine selection as discussed in detail below, and these molecules were eliminated from further consideration. (Olofsson and Cars, 2007; Worthington and Melander, 2013). Although a subset of polyamines was discovered during our screening with antimicrobial eﬀect themselves, their structure activity relationship was taken into consideration during lead polyamine selection as discussed in detail below, and these molecules were eliminated from further consideration. the substrate binding extrusion protomer on the distal binding site (Nikaido and Pages, 2012; Opperman and Nguyen, 2015). Interestingly, tetracycline binds to the “groove” region of the binding pocket in the binding protomer, while chloramphenicol binds to the deeper “cave” region. Covalent binding to this “cave” region is the ultimate in eﬄux inhibition as it causes the binding pocket to collapse and become non-functional, therefore inhibiting multiple substrates from binding (Nikaido and Pages, 2012). Eﬄux pump discovery began with the ﬁnding that eﬄux of host antimicrobial polyamines by the S. aureus Tet38 eﬄux pump facilitated skin colonization, and the ability to survive within an abscess environment (Truong-Bolduc et al., 2013). Beginning with the initial development of polyamine tetracycline derivatives to increase the eﬀectiveness of tetracycline, (Nelson et al., 1993, 1994; Nelson and Levy, 1999), polyamine molecules have been shown to increase the therapeutic potential of common antibiotics (Vazquez-Laslop, 1997; Kwon and Lu, 2007; Rockey et al., 2013). Polyamine Potentiation of Tetracycline Activity Reduces Bioﬁlms by P. aeruginosa This can be attributed to the competitive inhibition nature of eﬄux pumps inhibitors that harnesses the broad substrate recognition of eﬄux pumps for more eﬀective eﬄux inhibition in multiple organisms. For example, the eﬄux pump families of RND, ABC, SMR, and MFS all recognize substrates with polycationic properties (Kumar and Pooja Patial, 2016). Further to this, RND pumps found in P. aeruginosa and E. coli recognize and extrude tetracycline, while ABC and SMR pumps in S. aureus also expel this same antibiotic (Kumar and Pooja Patial, 2016). This would explain the activity of the previously identiﬁed eﬄux pump inhibitor baicalein, which is derived from the plant Thymus vulgaris, and has been found to potentiate tetracycline activity by blocking the MFS family TetK eﬄux pumps of E. coli and S. aureus (Wax et al., 2001; Fujita et al., 2005; Kumar and Pooja Patial, 2016). Moreover, our polyamine eﬄux pump inhibitors appear to be acting in a competitive manner with the positive control PAβN, potentially competing for the same substrate binding pocket (Webber et al., 2013; Venter et al., 2015). In fact, our polyamines resemble the known eﬄux pump inhibitor PAβN more so than the other well-known eﬄux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP), which is shown not to potentiate antibiotic activity toward P. aeruginosa (Coban et al., 2009). This would explain the activity of the recent identiﬁcation of a pyranopyridine inhibitor, MBX2319 that was designed based on NMP and found to have potent activity toward Enterobacteriaceae but little activity toward P. aeruginosa (Sjuts et al., 2016). However, a pyridopyrimidine scaﬀold discovered by Nakashima et al. (2013), with more similarities to PAβN was found to potentiate chloramphenicol and tetracycline and further revealed to bind the distal pocket of both P. aeruginosa MexAB and E. coli AcrB, whereas MBX2319 was speciﬁc to AcrB (Venter et al., 2015). However, even with the apparent binding diﬀerences observed in recent studies, MexAB of P. aeruginosa is still a close homolog of AcrB and has the same dependence on outer membrane stability as all RND eﬄux pumps do (Li et al., 2015b). Therefore, this dependence on membrane stability The polyamine agents discovered in this study were successful in not only returning the eﬀectiveness of tetracycline but also an unrelated antibiotic eﬄux substrate, chloramphenicol. Frontiers in Microbiology \| www.frontiersin.org June 2018 \| Volume 9 \| Article 1301 Polyamine Potentiation of Tetracycline Activity Reduces Bioﬁlms by P. aeruginosa Importantly, this ﬁnding reveals that our polyamines are not just allowing for the increased eﬀectiveness of one, but multiple commercial antibiotics from a broad range of diﬀerent classes. This ﬁnding suggests that the polyamines discovered in our study inhibit through direct competitive inhibition (Kourtesi, 2013). This mechanism of eﬄux inhibition capitalizes on the broad-spectrum binding aﬃnity of eﬄux pumps by blocking June 2018 \| Volume 9 \| Article 1301 12 Polyamines as EPIs Fleeman et al. have been shown to have non-speciﬁc inhibition of eukaryotic transporters, and therefore create unwanted side eﬀects as therapeutic agents (Lomovskaya et al., 2001; Ughachukwu and Unekwe, 2012; Amin, 2013; Lebeaux et al., 2013). Verapamil is a non-speciﬁc inhibitor of calcium channels, found to also inhibit the function of P-glycoprotein ABC transporters in mammalian cells (Andersen et al., 2006). This inhibitor was found to have in vitro function as a bacterial eﬄux inhibitor, however due to its general toxicity, the use of this compound is limited to angina, hypertension, and cardiac arrhythmia (Andersen et al., 2006; Gupta et al., 2013). This assessment is extremely important for our polyamines as calcium release has also been observed from polyamines on bacterial cells (Katsu et al., 2002). With limited toxicity and secondary eﬀects, the polyamine eﬄux pump inhibitors discovered in our study appear to have more favorable characteristics than others previously discovered, suggesting promising potential as adjuvant agents. necessitates a rigorous analysis of membrane disruption for all agents (polyamine or otherwise) being developed as eﬄux pump inhibitors. Importantly, we observed our polyamines did not have deleterious eﬀects on bacterial cell membranes, as is seen for the known eﬄux pump inhibitor PAβN (Webber et al., 2013). Many eﬄux pump inhibitors discovered to date have been found to cause disruption of the bacterial cell membrane leading to their ineﬀectiveness as therapeutic agents (Misra and Bavro, 2009; Machado et al., 2017). Disruption of the bacterial cell membrane causes inhibition of eﬄux through a secondary eﬀect of membrane depolarization, leading to inhibitory activity alone, and the common identiﬁcation of false positive, non-speciﬁc eﬄux pump inhibitors (Cornwell et al., 1987; Webber et al., 2013; Li et al., 2015b). Considering the fact that polyamines have been shown to disrupt bacterial membranes it is, perhaps surprising that our polyamines do not disturb bacterial membranes, however the potentiation modeling used herein speciﬁcally sought to eliminate the selection of false positive eﬄux pump inhibitors. Amin, M. L. (2013). P-glycoprotein inhibition for optimal drug delivery. Drug Target Insights 7, 27–34. doi: 10.4137/DTI.S12519 Andersen, C. L., Holland, I. B., and Jacq, A. (2006). Verapamil, a Ca2+ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli. Biochim. Biophys. Acta 1758, 1587–1595. doi: 10.1016/j.bbamem.2006. 05.022 SUPPLEMENTARY MATERIAL To increase our conﬁdence in the speciﬁcity of the polyamines discovered herein, we demonstrate that they have limited toxicity toward two human cell lines and no inhibitory eﬀects on the eukaryotic Ca2+ channel activity of human kidney cells. To date, many eﬄux pump inhibitors (such as verapamil) The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.01301/full#supplementary-material FUNDING This work was funded in part by grants AI103715 and AI124458 (both LS) from the National Institutes of Health and the Florida Drug Discovery Acceleration Program by the State of Florida, Department of Health (RH). AUTHOR CONTRIBUTIONS Project was designed by RF, MG, and LS. Experiments were performed by RF, JA, GD, and KA. Data was analyzed by RF, RS, GW, RH, MG, and LS. Manuscript was written by RF, JA, RH, MG, and LS. Funding was provided by RH and LS. Polyamine Potentiation of Tetracycline Activity Reduces Bioﬁlms by P. aeruginosa In our analysis we did identify polyamines, derived from our scaﬀold, with antimicrobial activity that was eliminated from study due to unwanted, oﬀ-target eﬀects. Regarding our lead agents, at least at the maximum concentrations tested, we observed little to no membrane destabilization for MRSA and P. aeruginosa. We note, however, that other polyamine molecules do have the potential for this activity, thus these types of studies will be an important part of future development for our frontrunners. The identiﬁcation of 37 polyamines that inhibited bacterial viability themselves, although less than ideal, lead to knowledge of a structure activity relationship for eﬄux pump speciﬁcity. Those polyamines found to inhibit bacterial viability themselves may be disturbing the cellular membrane based on the position of positive charges within their structure. Our analysis revealed 24% of the polyamines (26 out of 103) with R1 and R2 both deﬁned by S-methylbenzene lead to antibacterial activity, while only 7% (4 out of 60) that have S-methylbenzene at R2 and R3, and 12% (7 out of 60) that have S-methylbenzene at R1 and R3, had antibacterial activity. With this knowledge, we focused our attention on compounds that had no antibacterial activity themselves to avoid selecting membrane depolarizing agents. In the future development of our molecules, iterative structure activity relationship studies will ensure bias away from such unwanted activity, and focus on harnessing only the positive, eﬄux pump inhibiting eﬀects observed for our lead molecules. Taken together, the polyamines discovered in this study have potential as therapeutic adjuvants to rescue the eﬀects of multiple antibiotics toward both Gram-positive and Gram-negative species. They appear to be acting in a speciﬁc manner and have none of the undesirable membrane targeting characteristics of previously developed eﬄux pump inhibitors. As such, we suggest that the polyamines developed herein are a promising scaﬀold for further development of anti-resistance agents to help alleviate the burden of multi-drug resistant bacterial pathogens. 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L., Witowski, C. G., Fleeman, R. M., McClintock, J. B., Amsler, C. D., Shaw, L. N., et al. (2016). Darwinolide, a new diterpene scaﬀold that inhibits methicillin-resistant Staphylococcus aureus bioﬁlm from the antarctic sponge Dendrilla membranosa. Org. Lett. 18, 2596–2599. doi: 10.1021/acs. orglett.6b00979 Renau, T. E., Léger, R., Flamme, E. M., Sangalang, J., She, M. W., Yen, R., et al. (1999). Inhibitors of eﬄux pumps in Pseudomonas aeruginosa potentiate the activity of the ﬂuoroquinolone antibacterial levoﬂoxacin. J. Med. Chem. 42, 4928–4931. doi: 10.1021/jm9904598 June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 15 Fleeman et al. Polyamines as EPIs Watkins, W. J., Landaverry, Y., Léger, R., Litman, R., Renau, T. E., Williams, N., et al. (2003). The relationship between physicochemical properties, In vitro activity and pharmacokinetic proﬁles of analogues of diamine-Containing eﬄux pump inhibitors. Bioorgan. Med. Chem. Lett. 13, 4241–4244. doi: 10.1016/ j.bmcl.2003.07.030 nAChR-selective compounds suitable for optimization studies. J. Med. Chem. 56, 10103–10117. doi: 10.1021/jm401543h nAChR-selective compounds suitable for optimization studies. J. Med. Chem. 56, 10103–10117. doi: 10.1021/jm401543h Yoneyama, H., and Katsumata, R. (2014). Antibiotic resistance in bacteria and its future for novel antibiotic development. Biosci. Biotechnol. Biochem. 70, 1060–1075. doi: 10.1271/bbb.70.1060 Wax, R. G., Lewis, K., Salyers, A. A., and Taber, H. (2001). Bacterial Resistance to Antimicrobials. June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org REFERENCES Boca Raton, FL: CRC Press. Zelle, R. E. (1998). US5726184. Zelle, R. E., and Harding, M. W. (1996). US5543423. Webber, M. A., Lamers, R. P., Cavallari, J. F., and Burrows, L. L. (2013). The eﬄux inhibitor phenylalanine-arginine beta-naphthylamide (PAβN) permeabilizes the outer membrane of gram-negative bacteria. PLoS One 8:e60666. doi: 10. 1371/journal.pone.0060666 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. Weinstein, R. A., and Hooper, D. C. (2005). Eﬄux pumps and nosocomial antibiotic resistance: a primer for hospital epidemiologists. Clin. Infect. Dis. 40, 1811–1817. doi: 10.1086/430381 Copyright © 2018 Fleeman, Debevec, Antonen, Adams, Santos, Welmaker, Houghten, Giulianotti and Shaw. 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 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. Worthington, R. J., and Melander, C. (2013). Combination approaches to combat multidrug-resistant bacteria. Trends Biotechnol. 31, 177–184. doi: 10.1016/j. tibtech.2012.12.006 Wu, J., Zhang, Y., Maida, L. E., Santos, R. G., Welmaker, G. S., LaVoi, T. M., et al. (2013). Scaﬀold ranking and positional scanning utilized in the discovery of June 2018 \| Volume 9 \| Article 1301 Frontiers in Microbiology \| www.frontiersin.org 16
https://openalex.org/W4394621067	https://ujmm.org.ua/index.php/journal/article/download/431/335	English	null	Disseminated HIV-associated venous thrombosis (a case report)	Ukraïnsʹkij žurnal vìjsʹkovoï medicini	2,024	cc-by	6,646	1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine Introduction. In 1983, Robert Gallo (USA) and Luc Montagnier (France) discovered the human immunodeficiency virus (HIV). Today, about 39 million people are living with HIV worldwide. Thanks to antiretroviral therapy (ART), HIV has become a manageable chronic condition for most patients. However, the number of associated complications, including cardiovascular disease and thrombosis, is increasing. HIV-associated venous thrombosis, such as deep vein thrombosis and pulmonary embolism, requires attention and research due to their serious consequences. The purpose was to summarize, analyze, and demonstrate the clinical sequence, diagnostic difficulties and treatment of a rare case of disseminated venous thrombosis in the setting of HIV infection. f f g f f Materials and methods. Periodical medical publications, patient records, and materials of scientific and practical conferences were used. Research methods: historical, bibliographic, systematic approach, analytical, generalization. Results. The article analyses in detail the data on a severe complication of HIV infection - disseminated venous thrombosis. To establish the final diagnosis, differential diagnosis with infectious, rheumatological, and myeloproliferative diseases was performed. As a result of the differential diagnosis, a multidisciplinary team of doctors established homozygous carriage of the MTHFR 1298 gene, which determines an increased risk of thrombosis. The addition of HIV infection could be a trigger for the development of severe thrombosis with impaired internal organ function. f Conclusions. All countries of the world continue to face the problem of HIV infection, which requires constant detection among the population, including military personnel. In the case of thrombosis under consideration, both HIV infection itself (reduced CD4 T-cell counts, late HIV detection) and genetic factors could be the cause. The patient's multidisciplinary management proved to be effective, which allowed him to achieve a positive result, and his fight against the disease will continue. Key words: human immunodeficiency virus, thrombosis, MTHFR 1298, differential diagnosis. Introduction. In 1983, two research teams led by Robert Gallo (USA) and Luc Montagnier (France) isolated the human immunodeficiency virus (HIV) [1]. About 39 million people are living with HIV (PLHIV) worldwide, 1.3 million were infected in 2022, and 630 thousand died [2]. Human immunodeficiency virus (HIV), once considered a fatal disease, has become a manageable chronic condition for PLHIV who have access to antiretroviral therapy (ART) (76% of the total number of PLHIV) [2]. is a topic of great interest. 148 ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ «УКРАЇНСЬКИЙ ЖУРНАЛ ВІЙСЬКОВОЇ МЕДИЦИНИ» (1. 2024, Т.5) DOI:10.46847/ujmm.2024.1(5)-148 DOI:10.46847/ujmm.2024.1(5)-148 DOI:10.46847/ujmm.2024.1(5)-148 UDC 616.155.2-092 КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine Although HIV- associated venous thrombosis is not as well studied as other HIV-related complications, it can have serious consequences if not recognized and treated in time. The aim of the study is to summarize, analyze and demonstrate the clinical course, diagnostic difficulties and treatment of a rare case of disseminated venous thrombosis in the setting of HIV infection. Materials and methods. Periodical medical publications, patient records, materials of scientific and practical conferences were used. Research methods: historical, bibliographic, systematic approach, analytical, generalization. The patient provided voluntary informed consent for diagnosis and treatment, which is available in the medical record of an inpatient. Materials and methods. Periodical medical publications, patient records, materials of scientific and practical conferences were used. Research methods: historical, bibliographic, systematic approach, analytical, generalization. The patient provided voluntary informed consent for diagnosis and treatment, which is available in the medical record of an inpatient. According to current estimates, for women aged 40 years who are taking modern ART, the standardised life expectancy is 39 years; for 40- year-old men, it is 37 years [3]. Given the above, the number of associated complications, including cardiovascular disease [4-9] and thrombosis [4, 10-17], is increasing significantly. However, despite the success of ART in suppressing viral replication and improving immune function, HIV- infected individuals are at increased risk of thrombotic complications [4, 10-17]. Results. Clinical case. Patient G., born in 1990, on 18.10.23, was admitted to the National Military Medical Clinical Centre "GVKG" (NMMCC "GVKG") with complaints of severe edema, a feeling of swelling and heaviness in both lower extremities, the appearance of vascular pattern on Results. Clinical case. Patient G., born in 1990, on 18.10.23, was admitted to the National Military Medical Clinical Centre "GVKG" (NMMCC "GVKG") with complaints of severe edema, a feeling of swelling and heaviness in both lower extremities, the appearance of vascular pattern on HIV-associated venous thrombosis, including deep vein thrombosis (DVT), pulmonary embolism (PE) and other thrombotic events [18], HIV-associated venous thrombosis, including deep vein thrombosis (DVT), pulmonary embolism (PE) and other thrombotic events [18], ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ «УКРАЇНСЬКИЙ ЖУРНАЛ ВІЙСЬКОВОЇ МЕДИЦИНИ» (1. 2024, Т.5) 48 ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ «УКРАЇНСЬКИЙ ЖУРНАЛ ВІЙСЬКОВОЇ МЕДИЦИНИ» (1 CLINICAL NOTICE the anterior abdominal wall, severe general weakness, dry mouth, fever up to 39.1°C. fragmentarily in the proximal parts, proximal parts of the left branch at the level of compliance with the presence of thrombi. 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine The portal vein is clearly visible at the level up to the level of conflict with the splenic artery. The superior mesenteric vein is clearly contrasted only in the proximal parts, otherwise it is thrombosed and unevenly dilated. The gallbladder, spleen, pancreas, stomach, small and large intestine are also unremarkable. The splenic artery and vein are not dilated, the vein at the level of confluence contains filling defects. Adrenal glands, kidneys, renal vein, renal arteries, bladder, prostate gland are of typical location, without any features. An irregularly shaped saccular aneurysm with clear contours measuring 3.2 × 2.77 × 2.76 cm was detected along the anterior-upper edge of the right renal vein, without any features. The descending aorta and all its branches are evenly contrasted. The inferior vena cava is clearly visible from the level of the renal veins, distally its lumen is sharply narrowed and fragmented. The common iliac, external and internal iliac veins appear unevenly dilated, are not contrasted, and contain small parietal calcifications. In the lumen of the inferior vena cava at the level of the suprarenal segment, a parietal calcification with a conditional diameter of up to 0.7 cm and a length of up to 1.7 cm is determined. From the anamnesis it is known that the deterioration of the condition in the form of swelling of both lower extremities, weakness in the legs, and pain in both legs was noted in August 2023. In September 2023, he noticed a heaviness in his abdomen, on 23.09.2023 he developed a syncopal state, for which he was treated in the Military Medical Clinical Centre of the Northern Region. On 03.10.2023, according to ultrasound and multispiral computer tomography, he was diagnosed with acute thrombosis of the infrarenal division of the inferior vena cava and portal vein. Initially, the patient received anticoagulant therapy: heparin 5000 IU every 4 hours for the first 5 days with a transfer to rivaroxaban 30 mg per day. Indicates a complicated heredity - the presence of lower limb vascular disease in the father, which led to the amputation of one of the limbs. During the mobilisation call-up, HIV testing was not performed (according to the patient). Transferred on 18.10.23 to the NMMCC "GVKG". Objectively on admission: the general condition of moderate severity. Body temperature 38.8 °C. Heart rate 118 per min. Blood pressure 130/80 mmHg, SpO2 98%/ St. 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine 5) ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ 1400 μg/L), GGT (up to 850 U/L) and liver transaminases (ALT - 104 U/L, AST - 111 U/L), which normalised in the course of symptomatic (hepatoprotective, antibacterial) therapy. 1400 μg/L), GGT (up to 850 U/L) and liver transaminases (ALT - 104 U/L, AST - 111 U/L), which normalised in the course of symptomatic (hepatoprotective, antibacterial) therapy. infectious diseases. The procalcitonin test revealed an elevated level of 3.4 ng/mL (25.10.23). RW from 19.10.2023: negative. No viral hepatitis infection was detected. Urine culture of 30.10.2023: bacteriuria was not detected. Blood culture dated 01.11.2023: no growth of microorganisms was detected. Pharyngeal culture of 30.10.2023: Candida albicans 104 CFU/mL. Antibodies to cardiolipin Ig G from 27.10.2023: 3.8 GLP-U/mL (negative at < 10), antibodies to B2-glycoprotein Ig G from 27.10.2023: 1.1 U/mL (negative at < 7), antibodies to B2-glycoprotein Ig M from 27.10.2023: < 2.9 U/mL (negative at < 7). Lupus anticoagulant from 27.10.2023 - 1.906 (normal up to 1.2). The patient was consulted by a rheumatologist - no data were found in favour of antiphospholipid syndrome. The patient continued to receive rivaroxaban (30 mg per day), symptomatic therapy and a course of broad-spectrum antibiotics (25.10.-01.11.2023). After the acute period of thrombosis, a diagnostic search was carried out to identify the cause of such total thrombosis and to make a differential diagnosis between diseases that could provoke thrombosis (diseases of the blood system, connective tissue, infectious diseases, oncological pathology). During the entire observation period, febrile fever of up to 38-39.80C was present. Therefore, our efforts were primarily aimed at finding Table 1 Table The next step in the differential diagnosis was to determine hereditary thrombophilia Polymorphism of thrombophilia genes Result Reference values Factor 2 Prothrombin 20210 G>A G/G G/G – normal genotype Factor 5 Leiden 1691 G>A G/G G/G – normal genotype Factor 5 R2 4070 A>G A/A A/A – normal genotype Factor 5 5279 A>G A/A A/A – normal genotype MTHFR 677 C>T C/C С/С – normal genotype MTHFR 1298 A>T C/C C/C – homozygote The next step in the differential diagnosis was to determine A study of thrombophilia gene polymorphism revealed homozygous carriage of the MTHFR 1298 gene, responsible for the activity of the methylene tetrahydrofolate reductase enzyme. The genetic defect of this gene leads to a decrease in the functional activity of the MTHFR enzyme, which is accompanied by a violation of the folate cycle. 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine localis: skin of both thighs, legs, anterior abdominal wall - cyanotic. In the area of the anterior abdominal wall - increased vascular pattern. There is marked edema of both feet, legs, thighs. Satisfactory pulsation is determined on the arteries of the lower and upper extremities. The symptoms of Homans and Moses are positive on both sides. Elastic bandaging of both lower extremities. At the time of the examination, MSCT showed signs of thrombosis of the right branch of the portal vein, superior mesenteric vein, proximal splenic vein, inferior vena cava below the confluence of the renal veins, saccular aneurysm of the right renal vein. Echocardiography dated 19.10.2023: the heart cavity and valves were unchanged. The left ventricular myocardial contractility was satisfactory (left ventricle ejection fraction - 55%), and no areas of dyskinesia were detected. On the day of admission, according to the ultrasound of the abdomen, there were signs of thrombosis of the v. portae with recanalisation of blood flow, v. cava inferior to the level 6-7 cm above the navel. Hepatomegaly, steatohepatosis, chronic pancreatitis. Taking into account the examination on the day of admission, the diagnosis of acute thrombosis of the portal vein, superior mesenteric vein, right renal vein, infrarenal division of the inferior vena cava, iliofemoral, femoral-popliteal and tibial segments on both sides was clarified. Duplex scan of the veins of the lower extremities dated 18.10.2023: at the time of the examination, ultrasound signs of phlebothrombosis of the deep veins of both lower extremities from the level of the middle third of the lower legs to the level of the confluence of the common iliac veins without signs of recanalisation of blood flow. The haemogram showed normochromic normocytic anemia (80 g/l), slight leukocytosis (10.2 × 109/L), with a shift to neutrophils in the formula, thrombocytosis (480 - 500 × 109/L). The coagulogram showed a periodic decrease in prothrombin index levels, an increase in international normalised ratio, and activated partial thromboplastin time levels. The biochemical blood test showed elevated levels of C-reactive protein (up to 100 mg/L), ferritin (up to MSCT of the chest cavity, abdominal cavity, and pelvic organs dated 18.10.2023. Pulmonary fields, diaphragmatic domes, mediastinal organs, heart, aorta, esophagus, tracheobronchial tree, liver, intra- and extrahepatic ducts, common bile duct without features. Portal vein - 1.9 cm, right branch with signs of thrombosis, contrasted 149 «UKRAINIAN JOURNAL OF MILITARY MEDICINE» (1. 2024, Vol. 150 ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ «УКРАЇНСЬКИЙ ЖУРНАЛ ВІЙСЬКОВОЇ МЕДИЦИНИ» (1. 2024, Т.5) 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine Folate cycle disorders lead to the accumulation of homocysteine in cells and blood plasma. Homocysteine causes an increased risk of thrombosis. lymphocytes was determined on 31.10.23: absolute CD3+/CD4+ (T-helper) 114.0 kl/mL (normal 400-1800 kl/mL); relative CD3+/CD4+ (T-helper) 13.0%, which was indicative of severe immunodeficiency. On 02.11.23, the patient was transferred to the Infectious Diseases Clinic, where he continued treatment. At this time, febrile fever was observed with daily fluctuations of up to 38.0°C, and in the period from 12 to 15 November it reached 39.9°C, which did not affect the patient's general condition. Symptomatically, he was treated with ibuprofen 400mg per day without any significant effect. He received two short courses of fluconazole due to manifestations of oropharyngeal candidiasis. To exclude myeloproliferative diseases (MPDs), which often have thrombotic complications, histological and molecular genetic studies were performed. According to the histological examination of the trepanobiopsy of the hypochondrium dated 07.11.23, no clear signs of myeloproliferative disease were found. No mutations of V617F of the JAK2 gene and types 1, 2 of the SALR gene were detected in peripheral blood cells. He was consulted at the L.V. Gromashevsky Institute of Epidemiology and Infectious Diseases of the National Academy of Medical Sciences of Ukraine and diagnosed: Chronic retroviral infection, clinical stage III, recurrent oral candidiasis. For the first time in his life, he was prescribed lifelong antiretroviral therapy (ART), which was started on 14.11.23 in the dolutegravir/lamivudine/tenofovir During the examination for antibodies to HIV type 1/2 on 19.10.23, a result was obtained that required clarification, which took place on 30.10.23 (positive). He was consulted by an infectious disease specialist, and the number of CD ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ «УКРАЇНСЬКИЙ ЖУРНАЛ ВІЙСЬКОВОЇ МЕДИЦИНИ» (1. 2024, Т.5) CLINICAL NOTICE (DTG/3TC/TDF) regimen 50/300/300 mg, 1 tablet once daily. patient received azithromycin (without significant effect on fever). Along with DTG/3TC/TDF (ART), from 14.11.23 he received isoniazid 300 mg per day as isoniazid preventive therapy (IPT) in combination with a daily dose of B vitamins. All the time, he was treated with elastic bandaging of the lower extremities with the transition to wearing compression stockings, accompanied by rivaroxaban (20 mg per day) and venotonic (diosmin/hesperidin, 1000 mg per day). Increased hepatic transaminases were considered to be a consequence of portal vein thrombosis and were corrected by taking ursodeoxycholic acid at a daily dose of 750 mg. 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine Additionally, the patient underwent a blood test (TORCH) dated 02.11.23: EBV VCA-IgG detected 12.2, EBV VCA-IgM not detected, HSV 1/2 IgG detected 5.5, HSV 1/2 IgM not detected, CMV IgG detected 3.4, CMV IgM not detected, TOXO IgG detected 115.0, TOXO IgM not detected. Blood test by PCR to herpes group from 31.08.23: no herpesvirus genetic material was detected. In terms of treatment, ART was preceded by prophylactic use of trimethoprim/sulfamethoxazole at a dose of 960 mg per day, taking into account low levels of CD4+ T-lymphocytes. Taking into account the impossibility of excluding atypical mycobacteriosis from 7.11.23 to 12.11.23, the In the setting of concomitant ART+IPT, body temperature normalised and periodically rose to subfebrile levels (Figure 1). Figure 1. Combined graph (temperature - treatment measures) 36,5 37 37,5 38 38,5 39 39,5 40 ART + IPT Heparin rivaroxaban diosmin/hesperidin ceftriaxone fl trimethoprim/sulfamethoxazole fluconazole elastic bandaging azithromy Figure 1. Combined graph (temperature - treatment measures) varicose and edematous form, occlusion stage with significant impairment of blood circulation and function. High genetic risk of thrombophilia (C/C - homozygote for MTHFR 1298 mutation). Reactive (drug-induced) hepatitis, cholestatic variant, with moderate liver dysfunction. Non- alcoholic fatty liver disease. Condition after acute kidney injury (AKIN) grade 3 dated 03.10.23. Angiopathy of the retina of both eyes. According to the order of the head of the NMMCC "GVKG", the patient was examined by a therapeutic specialized medical evaluation board and found unfit for military service with exclusion from military registration on the basis of Articles 5a, 42a of Order of the Minister of Defence of Ukraine No. 402 of 2008 (as amended). Diagnosis at discharge: [В20.9] Chronic retroviral infection, clinical stage II, phase of immune decompensation (CD4 T-lymphocytes 114 cells/mL). Oropharyngeal candidiasis. Chronic herpesvirus infection associated with HSV 1/2 (IgG+, IgM-, DNA-), EBV (IgG+, IgM-, DNA-), CMV (IgG+, IgM-, DNA-), latent course, integration phase. To implement the decision of the medical evaluation board, after approval of the certificate of illness, the patient was discharged to the military unit on 30.11.23 with appropriate recommendations for the two main diagnoses. Discussion. This observation confirms the well-known fact that the presence of a chronic infectious disease, namely HIV, increases the risk of venous thrombosis (VT). For example, among PLHIV, the risk of developing VTE is 2-10 times higher [16], and people with low CD4+ cells are more likely to suffer from it [19, 20]. 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine Other risk Primary chronic toxoplasmosis (IgG+, IgM-), latent course. Anaemia of chronic disease, moderate severity. [I82.8] Thrombosis of the inferior vena cava (below the confluence of the renal veins), portal vein, superior mesenteric vein, proximal splenic vein and right renal vein. iliofemoral and popliteal segments on both sides, 151 OURNAL OF MILITARY MEDICINE» (1. 2024, Vol. 5) ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ 151 «UKRAINIAN JOURNAL OF MILITARY MEDICINE» (1. 2024, Vol. 5) ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ plasma levels of D-dimer and sCD14, as well as with HIV RNA levels [36]. factors for VT in PLHIV are malignancies and co- infections [17, 21]. It has been shown that the level of TF- expressing monocytes in PLWH is elevated and significantly higher than in monocytes of healthy individuals [37]. It is a significant fact that ART does not affect the level of TF-expressing monocytes [4], and the rate of thrombotic complications remains constant in PLHIV both on and off ART. According to Virchow's triad, there are three main factors for the development of VT: hypercoagulability, endothelial dysfunction, and blood stasis [22]. In PLHIV, the most important factors in the development of VT are hypercoagulability and endothelial dysfunction [4]. It has been shown that leukocytes, namely monocytes and neutrophils, are involved in the process of thrombus formation [23-25]. This process is called immunothrombosis [25]. As a consequence, activated monocytes express tissue factor (TF), which is a trigger of DVT in pathological conditions [26-28]. Thus, monocytes are a key actor in the activation of TF. In addition, TF-activated extracellular vesicles (EVs) [38] released from activated monocytes can bind to platelets, effectively transferring TF to platelets [39]. Platelets of PLHIV are more likely to contain TF than platelets of healthy people, and the level of TF-positive platelets correlates with TF expression in monocytes [40]. Endothelial cells normally secrete various anticoagulant proteins, such as TF inhibitor, thrombomodulin, endothelial protein C receptor, and antithrombin [29]. The effect of inflammatory mediators on endothelial cells converts the endothelium from an anticoagulant surface to a procoagulant one by decreasing thrombomodulin expression and increasing TF release [30,31]. In summary, immune system dysfunction and chronic inflammation caused by HIV lead to monocyte activation and expression of proinflammatory cytokines such as IL-6. Chronic inflammation leads to endothelial cell dysfunction and platelet activation. Activated monocytes express the procoagulant protein TF. Monocyte TF and monocyte-derived TF-positive EVs can activate the coagulation system. 1Ukrainian Military Medical Academy, Kyiv, Ukraine 2National Military Medical Clinical Centre "MMCC" Kyiv, Ukraine The activated coagulation system and activated platelets contribute to venous thrombosis. PLWH are usually deficient in anticoagulant proteins such as protein S, protein C, heparin cofactor II and antithrombin. The most likely cause of this is opportunistic infections and malignancies [18]. This hypothesis is supported by a clear link between VT and low CD4+ T-cell counts in PLHIV [16]. PLHIV also have autoantibodies to protein S, antiphospholipid antibodies, and lupus anticoagulant [18]. These autoantibodies are also implicated in the impaired anticoagulant function of protein S, protein C and antithrombin [32]. Conclusions: HIV infection remains relevant for Ukraine and requires timely detection in the general population, especially among the contingent to be mobilised. In the clinical case reviewed, the most likely causes of thrombosis were both HIV infection (low CD4 T-cell counts, late presenters - late detection of HIV) and constitutional and genetic prerequisites. 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H., РОЗПОВСЮДЖЕНИЙ ВІЛ-АСОЦІЙОВАНИЙ ВЕНОЗНИЙ ТРОМБОЗ (КЛІНІЧНИЙ ВИПАДОК С.А. Гусєва1, Я.П. Гончаров2, Н.І. Білоус2, В.В. Третьяков2, К.В. Савічан1 Відомості про авторів: Української військово-медичної академії, м. Київ, Україна https://orcid.org/0000-0002-7002-6631 Гончаров Я.П. A, B, C, E, F – полковник медичної служби, кандидат медичних наук, заступник начальника Національного військово-медичного клінічного центру “Головний військовий клінічний госпіталь” – провідний терапевт, м. Київ, Україна https://orcid.org/0009-0009-9564-9825 Білоус Н.І. B, C, D, E – підполковник медичної служби, старший ординатор клініки гематології Національного військово-медичного клінічного центру “Головний військовий клінічний госпіталь”, м.Київ, Україна https://orcid.org/0000-0002-6443-0988 р p // g/ Третьяков В.В. B, C, D, E – полковник медичної служби, кандидат медичних наук, ординатор відділення кишкових інфекцій клініки інфекційних захворювань Національного військово-медичного клінічного центру “Головний військовий клінічний госпіталь”, м.Київ, Україна р p // g/ Третьяков В.В. B, C, D, E – полковник медичної служби, кандидат медичних наук, ординатор відділення кишкових інфекцій клініки інфекційних захворювань Національного військово-медичного клінічного центру “Головний військовий клінічний госпіталь”, м.Київ, Україна ру р Савічан К.В. C, D, E – підполковник медичної служби, PhD, старший викладач кафедри військової терапії факультету підготовки та підвищення кваліфікації Української військово-медичної академії, м. Київ, Україна. E-mail: k.savichan@gmail.com, https://orcid.org/0000-0002-8650-3383. Савічан К.В. C, D, E – підполковник медичної служби, PhD, старший викладач кафедри військової терапії факультету підготовки та підвищення кваліфікації Української військово-медичної академії, м. Київ, Україна. E-mail: k.savichan@gmail.com, https://orcid.org/0000-0002-8650-3383. A – концепція та дизайн дослідження; B – збір даних; C – аналіз та інтерпретація даних; статті; E – редагування статті; F – остаточне затвердження статті. С.А. Гусєва1, Я.П. Гончаров2, Н.І. Білоус2, В.В. Третьяков2, К.В. Савічан1 A, B, C, E, F – полковник медичної служби, кандидат медичних наук, заступник начальника Національного військово-медичного клінічного центру “Головний військовий клінічний госпіталь” – провідний терапевт, м. Київ, Україна https://orcid.org/0009-0009-9564-9825 Білоус Н.І. B, C, D, E – підполковник медичної служби, старший ординатор клініки гематології Information about authors: Guseva S.A. A, C, E, F – DSc, Professor, of the Military Therapy Department of the Staff Retraining and Advanced Training Faculty of the Ukrainian Military Medical Academy Kyiv Ukraine gushem@ukr net Guseva S.A. A, C, E, F – DSc, Professor, of the Military Therapy Department of the Staff Retraining and Advanced Training Faculty of the Ukrainian Military Medical Academy, Kyiv, Ukraine gushem@ukr.net https://orcid.org/0000-0002-7002-6631 p // g/ Goncharov Ya.P. A, B, C, E, F – Colonel MS, PhD, Deputy Head of the National Military Medical C Goncharov Ya.P. A, B, C, E, F – Colonel MS, PhD, Deputy Head of the National Military Medical Clinical Center “Main Military Clinical Hospital” – Leading Internist, Kyiv, Ukraine. https://orcid.org/0009-0009-9564-9825 Bilous N.I. C, D, E, F – LtCol MS, senior resident of the Clinic of Hematology, National Military Medical Clinical Center “Main Military Clinical Hospital”, Kyiv, Ukraine. https://orcid.org/0000-0002-6443-0988 Goncharov Ya.P. A, B, C, E, F – Colonel MS, PhD, Deputy Head of the National Military Medical Clinical Center “Main Military Clinical Hospital” – Leading Internist, Kyiv, Ukraine. https://orcid.org/0009-0009-9564-9825 Bilous N.I. C, D, E, F – LtCol MS, senior resident of the Clinic of Hematology, National Military Medical Clinical Center “Main Military Clinical Hospital”, Kyiv, Ukraine. https://orcid.org/0000-0002-6443-0988 gy y Center “Main Military Clinical Hospital”, Kyiv, Ukraine. https://orcid.org/0000-0002-6443-0988 Tretiakov V.V. B, C, D, E - Col MS, PhD, resident of the Department of Intestinal Infections, Clinic of Infectious Diseases, National Military Medical Clinical Centre "Main Military Clinical Hospital", Kyiv, Ukraine. Savichan K.V. C,D,E – LtCol MS, PhD, Senior Lecturer of the Military Therapy Department of the Staff Retraining and Advanced Training Faculty of the Ukrainian Military Medical Academy, Kyiv, Ukraine. E-mail: k savichan@gmail com https://orcid org/0000-0002-8650-3383 y p , y , p // g/ Tretiakov V.V. B, C, D, E - Col MS, PhD, resident of the Department of Intestinal Infections, Clinic of Infectious Diseases, National Military Medical Clinical Centre "Main Military Clinical Hospital", Kyiv, Ukraine. y p y p // g/ Tretiakov V.V. B, C, D, E - Col MS, PhD, resident of the Department of Intestinal Infections, Clinic of Infectious Diseases, National Military Medical Clinical Centre "Main Military Clinical Hospital", Kyiv, Ukraine. y p y p // g/ Tretiakov V.V. B, C, D, E - Col MS, PhD, resident of the Department of Intestinal Infections, Clinic of Infectious Diseases, National Military Medical Clinical Centre "Main Military Clinical Hospital", Kyiv, Ukraine. Savichan K.V. Адреса для листування: вул. Князів Острозьких, 45/1, буд. 33, м. Київ 01015 С.А. Гусєва1, Я.П. Гончаров2, Н.І. Білоус2, В.В. Третьяков2, К.В. Савічан1 1Українська військова медична академія 2Національний військово-медичний клінічний центр «ГВКГ» Вступ. У 1983 році Роберт Галло (США) та Люк Монтаньє (Франція) відкрили вірус імунодефіциту людини (ВІЛ). На сьогоднішній день близько 39 млн людей живуть з ВІЛ у світі. Завдяки антиретровірусній терапії (АРТ), ВІЛ перетворився на керований хронічний стан для більшості хворих. Однак, зростає кількість супутніх ускладнень, зокрема серцево-судинних захворювань та тромбозів. ВІЛ-асоційовані венозні тромбози, такі як тромбоз глибоких вен та тромбоемболія легеневої артерії, потребують уваги та досліджень через їх серйозні наслідки. Мета роботи: необхідність узагальнити, проаналізувати та продемонструвати особливості клінічного перебігу, труднощі діагностики, а також особливості лікування рідкісного випадку розповсюдженого венозного тромбозу на фоні ВІЛ-інфекції. 154 ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ «УКРАЇНСЬКИЙ ЖУРНАЛ ВІЙСЬКОВОЇ МЕДИЦИНИ» (1. 2024, Т.5) CLINICAL NOTICE Матеріали та методи. Використані періодичні медичні видання, історії хвороби пацієнтів, матеріали науково-практичних конференцій. Методи дослідження: історичний, бібліографічний, системного підходу, аналітичний, узагальнення. Результати. У статті детально проаналізовано дані про важке ускладнення при ВІЛ-інфекції – розповсюджений венозний тромбоз. Для встановлення заключного діагнозу було проведено диференційну діагностику з інфекційними, ревматологічними, мієлопроліферативними захворюваннями. В результаті проведення диференційної діагностики мультидисциплінарною командою лікарів було встановлено гомозигиготне носійство гену MTHFR 1298, який визначає підвищений ризик виникнення тромбозів. Приєднання ВІЛ-інфекції могло бути тригером розвитку тяжкого тромбозу з порушенням функції внутрішніх органів. Висновки. Всі країни світу продовжують стикатися з проблемою ВІЛ-інфекції, яка потребує постійного виявлення серед населення, в тому числі військовослужбовців. У випадку тромбозу, що був розглянутий, причиною могли бути як сама ВІЛ-інфекція (знижені рівні CD4 Т-лімфоцитів, пізнє виявлення ВІЛ), так і генетичні фактори. Мультидисциплінарне ведення пацієнта виявилося ефективним, що дозволило досягти позитивного результату, і його боротьба з захворюванням триватиме. Висновки. Всі країни світу продовжують стикатися з проблемою ВІЛ-інфекції, яка потребує постійного виявлення серед населення, в тому числі військовослужбовців. У випадку тромбозу, що був розглянутий, причиною могли бути як сама ВІЛ-інфекція (знижені рівні CD4 Т-лімфоцитів, пізнє виявлення ВІЛ), так і генетичні фактори. Мультидисциплінарне ведення пацієнта виявилося ефективним, що дозволило досягти позитивного результату, і його боротьба з захворюванням триватиме. Ключові слова: вірус імунодефіциту людини, тромбоз, MTHFR 1298, диференційна діагностика. Ключові слова: вірус імунодефіциту людини, тромбоз, MTHFR 1298, диференційна діагностика. Конфлікт інтересів: відсутній. Conflicts of interest: authors have no conflict of interest to declare. ф р у Conflicts of interest: authors have no conflict of interest to declare. Відомості про авторів: Гусєва С.А. A, C, E, F – доктор медичних наук, професор, професор кафедри військової терапії Української військово-медичної академії, м. Київ, Україна https://orcid.org/0000-0002-7002-6631 Гончаров Я.П. Information about authors: C,D,E – LtCol MS, PhD, Senior Lecturer of the Military Therapy Department of the Staff Savichan K.V. C,D,E – LtCol MS, PhD, Senior Lecturer of the Military Therapy Department of the Staff Retraining and Advanced Training Faculty of the Ukrainian Military Medical Academy, Kyiv, Ukraine. E-mail: k.savichan@gmail.com, https://orcid.org/0000-0002-8650-3383. Savichan K.V. C,D,E – LtCol MS, PhD, Senior Lecturer of the Military Therapy Department of the Staff Retraining and Advanced Training Faculty of the Ukrainian Military Medical Academy, Kyiv, Ukraine. E-mail: k.savichan@gmail.com, https://orcid.org/0000-0002-8650-3383. g , p // g/ A – research concept and design; B – collection and/or assembly of data; C– data analysis and interpretation; D – writing the article; E – critical revision of the article; F – final approval of the article. g p // g/ A – research concept and design; B – collection and/or assembly of data; C– data analysis and interpretation; D – writing the article; E – critical revision of the article; F – final approval of the article. р 155 «UKRAINIAN JOURNAL OF MILITARY MEDICINE» (1. 2024, Vol. 5) ‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡
https://openalex.org/W4240801992	https://www.researchsquare.com/article/rs-10250/latest.pdf	English	null	The Role of Text messaging Intervention in Inner Mongolia among Patients with Type 2 Diabetes Mellitus: A Randomized Controlled Trial	Research Square (Research Square)	2,019	cc-by	8,297	The role of text messaging intervention in Inner Mongolia among patients with type 2 diabetes mellitus: a randomized controlled trial Xuemei Wang Inner Mongolia Medical University Dan Liu Inner Mongolia Medical University Maolin Du (  dumaolin2016@163.com ) Inner Mongolia Medical University https://orcid.org/0000-0003-3740-0043 Ruiqi Hao Inner Mongolia Medical University Huiqiu Zheng Inner Mongolia Medical University Chaoli Yan Inner Mongolia Medical University Research article Keywords: type 2 diabetes mellitus; mHealth; randomized controlled trial Posted Date: May 18th, 2020 DOI: https://doi.org/10.21203/rs.2.19637/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Medical Informatics and Decision Making on May 14th, 2020. See the published version at https://doi.org/10.1186/s12911-020-01129-7 Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Medical Informatics and Decision Making on May 14th, 2020. See the published version at https://doi.org/10.1186/s12911-020-01129-7. Page 1/20 Page 1/20 Abstract Background: Short messages service (SMS) provides a practical medium for delivering content to address patients to adherence to self-management. The aim of study was to design some patient- centered health education messages, evaluate the feasibility of messages, and explore the effect of this model. Methods: The messages were designed by a panel of experts, and SMS Quality Evaluation Questionnaire was used to evaluate their quality. A two-arm randomized controlled trial was conducted to evaluate the effectiveness of this management model. Participants were randomly divided into an intervention group (IG) who received evaluated messages and a control group (CG) who received regular education. The primary outcomes were changes in plasma glucose and control rates, and the secondary outcomes were improvements in diet control, physical activities, weight control, etc. Results: A total of 42 messages covering five main domains: health awareness, diet control, physical activities, living habits and weight control were designed, and the average scores of the messages were 8.0 (SD 0.7), 8.5 (SD 0.6), 7.9 (SD 1.0), 8.0 (SD 0.7), and 8.4 (SD 0.9), respectively. In the SMS intervention, 171 patients with an average age of 55.1 years were involved, including 86 in the CG and 85 in the IG. At 12 months, compared with the control group (CG), the decrease of fasting plasma glucose (FPG) (1.5 vs. 0.4, P = 0.011) and control rate (49.4% vs. 33.3%, P = 0.034), the postprandial glucose (PPG) (5.8 vs. 4.2, P = 0.009) and control rate (57.8% vs. 33.7%, P = 0.002) were better in the intervention group (IG). In terms of self-management, improvements in weight control (49.3% vs. 28.2%, P=0.031), vegetables consumption (87.3% vs. 29.0%, P<0.001), fruits consumption (27.5% vs. 7.4%, P=0.022), and physical activities (84.7% vs. 70.0%, P=0.036) were better in the IG than in the CG. Conclusions: The overall quality of the messages was high. It was effective and feasible to carry out an SMS intervention to improve the behavioral habits of patients with chronic diseases in remote and undeveloped areas. Keywords: type 2 diabetes mellitus; mHealth; randomized controlled trial Trial registration: Clinicaltrials.gov, ChiCTR1900023445. Registered May 28, 2019--Retrospectively registered, http://www.chictr.org.cn/index.aspx Background According to the eighth edition of the Global Diabetes Map released by the International Diabetes Federation, there were 425 million adults worldwide with diabetes in 2017, and this number is expected to increase to 629 million by 2045 [1]. The prevalence of diabetes in China (11.6%) is three percentage points higher than the global prevalence, which is growing especially rapidly in low-and middle-income countries [2,3]. Moreover, individuals with diabetes are at a higher risk of heart disease, stroke, and kidney disease, and diabetes carries a heavy financial burden for these patients’ families [4]. According to a Page 2/20 report on the global medical expenses of patients with diabetes, China ranks second with $110 billion in 2017 [5]. Faced with such a huge expenditure, the level of glycemic control in China also does not inspire optimism, only 32.6% of adult’s plasma glucose levels have been controlled [5]. report on the global medical expenses of patients with diabetes, China ranks second with $110 billion in 2017 [5]. Faced with such a huge expenditure, the level of glycemic control in China also does not inspire optimism, only 32.6% of adult’s plasma glucose levels have been controlled [5]. Type 2 diabetes mellitus (T2DM) and its complications can be controlled through long-term management [6,7], but most diagnosed patients cannot adhere self-management to an extended time, especially after they left hospital. Therefore, real-time health education and reminders are essential. With the development of science and technology, the mobile phone technologies have proposed a possible solution to this problem. Some small-scale studies using information technology for diabetes management have shown the advantages for improving glycemic control [8,9,10,11]. Interventions through application (APP) and short message service (SMS) of mobile phones are common methods. APP is widely used, but the mobile phone must be a smart phone, and 4G network or Wi-Fi is required, and the coverage is relatively limited, especially in underdeveloped area. In contrast, the SMS, it is a feature of all mobile phones that receive information have the advantages of economical, convenient, real-time, and easy operation [12]. In particular, SMS intervention applies to non-smartphones, which is suitable for the economically undeveloped areas. On the other hand, while the rapid advancement of the internet has brought a lot of information and convenience to people, at the same time it has also led to inequality in access to information for different economic, educational and age groups. Background SMS for health education has relatively eliminated this inequality. Inner Mongolia, a remote economically underdeveloped region, with limited medical resources, is located in northern China. Smart phones are not widespread, especially among the elderly. SMS is more suitable for Inner Mongolia. Most herders are far from medical institutions, who have a high demand for health management of the disease. According to the investigation data analysis of a surveillance survey of chronic disease and nutrition in Chinese adults in Inner Mongolia in 2015, the prevalence of diabetes is 8.75%, the treatment rate and control rate are 46.17% and 18.22%, respectively. The application of SMS in self-management not only solves the problem of shortage of medical resources, but also avoids the difficulty of face-to-face instruction caused by poor services accessibility [13]. Therefore, the objective of this study was to design a series of patient-centered health education messages for patients with T2DM, based on American Diabetes Association on Taking Care of Diabetes, following a standard process and then evaluate its effect of the management model by SMS to provide an economic and effective model for elderly diabetes management in poor areas. Patient population Patients with T2DM were all admitted to the Department of Endocrinology at the Affiliated Hospital of Inner Mongolia Medical University from October 2016 to March 2017 were selected for participation in the study. Based on the results of a previous study [14], we expected an effect size of 0.26 in the plasma glucose level between baseline and at the end of intervention. The sample size was estimated with 80% power and a two-sided significance level of α = 0.05. We estimated a 10% dropout rate in the follow-up because of inconvenience or the time-consuming nature of the trial. We calculated a minimum sample size of 73 participants in each group. The inclusion criteria for participants in the SMS quality evaluation were willingness and ability to participate in the SMS quality evaluation. Exclusion criteria were severe illness or inability to complete the SMS assessment. For SMS intervention research participants, the inclusion criteria were as follows: For SMS intervention research participants, the inclusion criteria were as follows: 1) Diagnose with T2DM, and aged ≥ 18 years; 2) Participants or a family member who lived together were literate and had a mobile phone; 2) Participants or a family member who lived together were literate and ha 3) Clear consciousness and normal thinking; and 3) Clear consciousness and normal thinking; and 4) Provided informed consent for the study. The exclusion criteria for participants in the SMS intervention were as follows: 1) Inability to complete the questionnaire because of serious illness; or 2) Participants and their relatives had a lower level of education and could not read text messages. 2) Participants and their relatives had a lower level of education and could not read text messages. The study protocol was approved by the Biomedical Research Ethics Committee of Inner Mongolia Medical University, and all selected participants signed informed consent form. Over the course of the study, staff members protected the personal information of participants. Subjects were free to withdraw, and the researchers ensured that the participants’ rights were not affected. Study design Page 3/20 This was a randomized, single-blind (only the researcher knows the grouping of patients), parallel-group, controlled trial. The study included two aspects, the first was the SMS design, involving the SMS quality evaluation, conducted from October 2016 to April 2017. The second was the SMS intervention, which lasted for 12 months and conducted from May 2017 to April 2018. Page 3/20 SMS design The messages were designed by a panel of experts, including endocrinology, chronic disease management, health education, disease prevention, etc. A total of 42 messages covering five main domains: health awareness, diet control, physical activities, living habits and weight control were designed, respectively. The content of messages was based on the report of the American Diabetes Association on Taking Care of Diabetes [15]. In terms of health awareness, we considered that participants need to have a full understanding of the etiology, process, and complications of diabetes, which is beneficial to the plasma glucose control. For diet control, this was considered to be the most direct factor affecting glycemic control [16,17]. The diet in this study included intake of vegetables, fruits, salt, protein, fat and food, and a low glycemic index, especially for cooking and eating habits. In terms of physical activity and weight control, as we all known that patients with T2DM can effectively reduce plasma glucose and lose weight through exercise. Therefore, we helped patients choose the best mode of exercise, and ensure the best time and frequency of exercise through SMS intervention. Smoking and drinking are almost all risk factors for chronic diseases including diabetes. Studies have shown that plasma glucose can be controlled by quitting smoking and limiting alcohol consumption [18]. Therefore, we intervened on unhealthy living habits. Each text message covered only one topic, and contained 70 words or fewer. The language expression was simple, direct, and easy to understand. Final list of SMS was shown in Additional file 1. Message design was based on the Trans-Theoretical Model (TTM) [19], which is a widely used theory to promote health behavior. For diabetic patients, the pre-contemplation stage is almost non-existent, and combined with the SMS quality evaluation, we directly intervened from the contemplation stage. Therefore, in our study, the intervention was divided into four stages: the contemplation stage, the preparation stage, the action stage, and the reinforcement stage. The contemplation stage focused on education about basic knowledge about diabetes. This stage aimed to make participants aware of the importance of integrated diabetes management and to develop awareness of health management. The preparation and action stages focused on providing tips and suggestions for changing behaviors and on guiding participants to gradually develop the attitudes and actions necessary to change their behaviors. The reinforcement stage aimed to consolidate the changes in behaviors and transform healthy behaviors into habits. SMS design Examples of message in different stages were shown in Additional file 2. Data collection Data were collected at baseline using the Diabetes Questionnaire, which was designed based on the Chinese Adult Chronic Diseases and Nutrition Monitoring Personal Questionnaire (2015). The content mainly included socio-demographic data, health-related behaviors, disease status, and dietary conditions, etc. And biochemical indicators, including fasting plasma glucose (FPG), postprandial glucose (PPG), glycosylated hemoglobin (HbA1c), Total cholesterol, triglyceride, high density lipoprotein-c (HDL-C), and low density lipoprotein-c (LDL-C), were assessed using discharge data. Page 4/20 Outcomes and measurement The main outcomes were the changes of plasma glucose and the control rate of each index. The Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2017 edition) [2], defines the FPG control rate as the proportion of all managed participants with FPG from 4.4 to 7.0 mmol/L and PPG control rate as participants with PPG < 10.0 mmol/L as a percentage of all managed participants. The secondary outcomes were changes in diet control (vegetables and fruits consumption), physical activities (more than 30 minutes per exercise, three to five times per week), living habits (smoking and drinking) and weight control. Each outcome measure was obtained by telephone follow-up. SMS quality evaluation The overall quality of short message was assessed using the SMS Quality Assessment Questionnaire, which included three aspects: the understanding of the text messages, the willingness to act on the text messages, and the current status of the response to the text messages. Specifically, five questions were asked for regarding each short message: Questions 1 and 2 reflected the participant’s understanding of the text message (yes=1, no=0), questions 3 and 4 reflected the participant’s willingness to act on the text message (yes=2, no=0), and question 5 reflected the status of the participant’s response to the text Page 5/20 message (known and have done=1, unknown but have done=2, known but not have done=3, unknown and not have done=4). The setting of the score was based on the principle of scoring the scale and the patients’ demand for message, the higher the demand, the higher the score setting [20]. The total score of each message was calculated as the sum of the scores of the five questions, reflecting the overall quality of the message, with a maximum total of 10 points. We classified >7 as high quality, 4-7 as medium quality and <4 as low quality. The SMS Quality Assessment Questionnaire was shown in Additional file 3. SMS intervention The intervention period was from May 2016 to May 2017. A total of 171 discharged participants with diabetes were randomly divided into two groups using random number. Participants in the intervention group (IG, 85) received evaluated messages, which were sent twice a week, only one message at a time. The content of the text messages during the same stage included five different domains. Combined with the SMS evaluation results, for some text message, more than 60% of participants reported that they did not take action, we added the transmission frequency and repeated every four weeks. Simultaneously, in order to reduce the rate of loss to follow-up, participants in the control group (CG, 86) were sent regular messages, mostly limited to general theoretical knowledge. Examples of regular message that CG received are shown in Additional file 4. Telephone follow-up after each stage of the intervention was conducted. The purpose of the telephone follow-up was to ensure participants compliance and thus to ensure the quality of the study. The follow- up questionnaire was shown in Additional file 5. SMS quality evaluation Table 1 showed the characteristics of the SMS quality evaluation population and the SMS intervention population. There were no significant differences in age, gender, body mass index (BMI), urban and rural, residence, education, and smoking, drinking, or hypertension (all P > 0.05). Table 2 showed the results for the overall quality of the text messages. The average scores for health awareness, diet control, physical activities, living habits, and weight control were 8.0 (SD 0.7), 8.5 (SD 0.6), 7.9 (SD 1.0), 8.0 (SD 0.7), and 8.4 (SD 0.9), respectively. Table 2 showed the results for the overall quality of the text messages. The average scores for health awareness, diet control, physical activities, living habits, and weight control were 8.0 (SD 0.7), 8.5 (SD 0.6), 7.9 (SD 1.0), 8.0 (SD 0.7), and 8.4 (SD 0.9), respectively. Table 3 showed the result of the participants’ understanding of the message content. The participants who completed the SMS quality evaluation, more than 98.0% believed that all kinds of text messages were understandable and approved. Table 4 showed the results of the participants' willingness to act on the message content. Of the participants who completed the SMS quality evaluation, 90.5% indicated they were willing to act on the diet control messages and that they found it helpful and can be persistent, 68.3% indicated they were willing to act on the living habits messages and that they found it helpful and can be persistent. Table 4 showed the results of the participants' willingness to act on the message content. Of the participants who completed the SMS quality evaluation, 90.5% indicated they were willing to act on the diet control messages and that they found it helpful and can be persistent, 68.3% indicated they were willing to act on the living habits messages and that they found it helpful and can be persistent. Table 5 showed the results of the participants’ current status in response to the text messages. The participants who completed the SMS quality evaluation, 79.8% believed that the living habits messages were known but who reported not having taken action. Results A flow diagram of the participant selection process was shown in Figure 1. A total of 72 were selected to complete the SMS quality evaluation questionnaire. And 171 eligible participants were eventually enrolled in the SMS intervention, a total of 146 participants completed the one-year intervention to the end of the follow-up period. Statistical analysis Data were recorded using EpiData 3.1 software. Data analysis was performed using IBM SPSS Statistics, Version 19.0 (IBM Corp, Armonk, NY, USA) software. Continuous variables were expressed as means (standard deviation, SD), and t-test was used for comparisons between the two groups. Categorical variables were expressed as percentages, and c2 test was used for comparisons between the two groups. P ≤ 0.05 was considered statistically significant. Page 6/20 To compare the health behavior of the two groups over time, we converted the behavior change including vegetable and fruit intake, weight control, and physical activities into score at baseline and 12 months. Each instance of behavior change from healthy to unhealthy status was assigned a score of -1, change from unhealthy to healthy status was assigned a score of 1, and no change was assigned a score of 0. The scores for each of the four behaviors were then summed, generating a composite change score that ranged from -4 (when all four behaviors changed from healthy to unhealthy) to 4 (when all four behaviors changed from unhealthy to healthy after the intervention). We then compared the difference in total scores between the two groups. SMS intervention effect evaluation The score of IG ≤ -1 accounted for 20.0% (17/85), while that of CG was 44.2% (38/86). Positive scores indicated improvement in behaviors at 12 months compared to baseline. A total of 67.1% (57/85) of participants in the IG scored ≥ 1, while only 25.6% (22/86) in the CG. The mean of the total score was higher for the IG than for the CG (1.1 vs -0.3, P < 0.001). SMS intervention effect evaluation Table 6 showed there were no statistical differences in baseline characteristics between the two groups (all P < 0.05). The participants were 97 males and 74 females. The average age for the total population Page 7/20 Page 7/20 was 55.1 (SD 10.8) years. The average level of FPG was 9.0 (SD 2.6), and the average level of PPG was 16.6 (SD 4.4). The control rates of FPG and PPG were 28.4% (48/171), 9.6% (16/171), respectively. 97.6% (80/85) in the IG and 90.5% (76/86) in the CG were receiving hypoglycemic therapy. And 36.5% (31/85) in the IG and 33.7% (29/86) in the CG were receiving antihypertensive therapy. was 55.1 (SD 10.8) years. The average level of FPG was 9.0 (SD 2.6), and the average level of PPG was 16.6 (SD 4.4). The control rates of FPG and PPG were 28.4% (48/171), 9.6% (16/171), respectively. 97.6% (80/85) in the IG and 90.5% (76/86) in the CG were receiving hypoglycemic therapy. And 36.5% (31/85) in the IG and 33.7% (29/86) in the CG were receiving antihypertensive therapy. was 55.1 (SD 10.8) years. The average level of FPG was 9.0 (SD 2.6), and the average level of PPG was 16.6 (SD 4.4). The control rates of FPG and PPG were 28.4% (48/171), 9.6% (16/171), respectively. 97.6% (80/85) in the IG and 90.5% (76/86) in the CG were receiving hypoglycemic therapy. And 36.5% (31/85) in the IG and 33.7% (29/86) in the CG were receiving antihypertensive therapy. was 55.1 (SD 10.8) years. The average level of FPG was 9.0 (SD 2.6), and the average level of PPG was 16.6 (SD 4.4). The control rates of FPG and PPG were 28.4% (48/171), 9.6% (16/171), respectively. 97.6% (80/85) in the IG and 90.5% (76/86) in the CG were receiving hypoglycemic therapy. And 36.5% (31/85) in the IG and 33.7% (29/86) in the CG were receiving antihypertensive therapy. Table 7 showed changes in plasma glucose, and diabetes risk factors over the entire intervention period. At 12 months, compared with the CG, the decrease in the FPG (1.5 vs. 0.4, P = 0.011), and the PPG (5.8 vs. 4.2, P = 0.009) were better in the IG. Table 8 showed the results of plasma glucose control rate and health behaviors improvements at 12 month. The FPG control rate (49.4% vs. 33.3%, P = 0.034), and the PPG control rate (57.8% vs. SMS intervention effect evaluation 33.7%, P = 0.002) were better in the IG, compared with the CG. In terms of self-management, improvements in weight control (49.3% vs. 28.2%, P = 0.031), vegetables consumption (87.3% vs. 29.0%, P < 0.001), fruits consumption (27.5% vs. 7.4%, P = 0.022), and physical activities (84.7% vs. 70.0%, P = 0.036) were better in the IG than in the CG. There was no difference between the two groups in terms of quitting smoking or drinking (both P > 0.05). Table 8 showed the results of plasma glucose control rate and health behaviors improvements at 12 month. The FPG control rate (49.4% vs. 33.3%, P = 0.034), and the PPG control rate (57.8% vs. 33.7%, P = 0.002) were better in the IG, compared with the CG. In terms of self-management, improvements in weight control (49.3% vs. 28.2%, P = 0.031), vegetables consumption (87.3% vs. 29.0%, P < 0.001), fruits consumption (27.5% vs. 7.4%, P = 0.022), and physical activities (84.7% vs. 70.0%, P = 0.036) were better in the IG than in the CG. There was no difference between the two groups in terms of quitting smoking or drinking (both P > 0.05). Figure 2 showed the comparison of FPG and PPG changes between the baseline and the end of the four intervention stages. The FPG level in the IG was superior to that in the CG at the end of the preparation stage, the end of the action stage, and the end of the reinforcement stage (P < 0.05). The PPG level in the IG was superior to that in the CG at the end of the action stage and the end of the reinforcement stage (P < 0.05). Figure 3 showed the composition of behavior change scores for two groups. Negative scores indicated deterioration in behaviors at 12 months compared to baseline. The score of IG ≤ -1 accounted for 20.0% (17/85), while that of CG was 44.2% (38/86). Positive scores indicated improvement in behaviors at 12 months compared to baseline. A total of 67.1% (57/85) of participants in the IG scored ≥ 1, while only 25.6% (22/86) in the CG. The mean of the total score was higher for the IG than for the CG (1.1 vs -0.3, P < 0.001). Figure 3 showed the composition of behavior change scores for two groups. Negative scores indicated deterioration in behaviors at 12 months compared to baseline. SMS quality evaluation Patients with T2DM need long-term self-management, especially in the community. High-quality messages not only to meet the needs of participants, but also to drive active behavior changes among diabetes and to ensure the effective of an SMS intervention on diabetes. In order to ensure the quality of messages, we made a judgment through the SMS quality evaluation questionnaire. The results showed that, all of the messages had a mean score of approximately 8 points, indicating the overall quality of the message was high. Diet control messages had the highest willingness to act, consistent with the patient’s higher awareness of diet control, and believed that diets were the most direct and effective way to glycemic control [21]. A high number of participants reported that the content of weight control and diet Page 8/20 control messages were "unknown and not have done". Inner Mongolia lacks fresh vegetables and fruits due to its remote location, and it is a minority community containing 49 ethnic groups, people lived there like wine and meat. Especially for elderly, who knew very little about the glycemic index of food, and had a fuzzy choice of vegetables and fruits. In addition, patients’ self-management was limited by medical resources and education [22]. The proportion of participants who reported not having taken action on diet control, living habits, and weight control messages were relatively high, which were consistent with their lower willingness to act on the one hand. Although participants have a certain understanding of glycemic control, they may also lack systematic theoretical knowledge or fail to establish corresponding healthy behaviors. Health beliefs and behaviors establishment must be based on correct knowledge of diabetes. Without this knowledge, the importance of glycemic control cannot be translated into behavioral improvement, but only into conception. Especially for middle-aged and elderly patients, it takes a process to completely change the bad habits they have cultivated over the years. Therefore, we need to carry out comprehensive systemic interventions to encourage patients to increase their knowledge regarding treatment attitudes and then expand this to changes in behavior. SMS intervention effect evaluation The SMS intervention successfully improved participants’ continuous exercise and weight control, which was similar to previous results [23]. Sustained message alerts can increase participants subjective initiative [24]. Some participants reported that, SMS served as a reminder to guide them to choose appropriate activities and methods, it will gradually develop the habit of continuous exercise and help control weight. The SMS messages contained a large amount of dietary information, covering all aspects of a balanced diet, which guided participants to choose foods that were beneficial to glycemic control. SMS intervention did not affect smoking cessation or alcohol restriction, which was consistent with previous research [25,26,27]. An in-depth analysis showed that 18.2% (8/44) of smokers in this study said they would quit but not act within one year. In addition, 36.4% (16/44) said that they did not want to quit smoking. Among the drinkers, 79.6% (43/54) were employed, and they said drinking is a social necessity. Therefore, society and the media need to take measures to control tobacco and alcohol. Consistent with a study in India [28], the SMS intervention group had a greater improvement in awareness of diabetes risk factors. Participants had high adherence when health care professionals were able to use mobile phones to maintain close contact with participants to provide individualized education and management [29]. It has been observed that Behavior change was a gradual, phased, and complex process, which was characterized by jumps or reversals in between periods of progress [30], suggesting that we have to implement long patients behavioral intervention to promote the transformation of patient behavior. The successful reductions in FPG and PPG levels observed in our study can be attributed to the synergistic effects of improved health behaviors. Participants might be unaware of the conditions or factors that aggravate plasma glucose levels. Previous studies have shown [31] that better self- management behavior among patients with diabetes is associated with better glycemic control. Participants in the IG had increased knowledge of the etiology, development process, complications, and influencing factors of diabetes—a first step in the process of glycemic control. Limited knowledge about Page 9/20 Page 9/20 diabetes resulted in poorer protective practices against the disease [32]. Diabetes and hypertension are lifestyle diseases. Hypertension is the most common complication of diabetes [33], and may worsen the condition of patients with diabetes [34]. SMS intervention effect evaluation Therefore, because of the common risk factors between two diseases, significant content on the management of BP have been added to the design of SMS messages, and for a long time, effective diabetes management can also promote stability of BP. We found that the levels of FPG and PPG were different between the two groups after the second and third stages by comparing of the IG and CG at each intervention stage. This finding demonstrated the effectiveness of the application of the TTM, which provided targeted intervention education based on the physical activities intentions and behavioral characteristics of participants at different stages. The contemplation stage aimed to impart knowledge to the participants, the preparation stage prompted participants to elevate their knowledge to therapeutic attitudes and to develop plans to change their behaviors, and the action stage aimed to further elevate therapeutic attitudes to behavior changes. The goal was to gradually improve the participants’ multiple objective physiological indicators and reduce the risk of recurrence. Conclusions The overall quality of the SMS content we designed was high enough to meet the needs of participants. Our findings support the feasibility, acceptability, and preliminary effectiveness of a low-cost, low-burden text messaging intervention to promote the effective control of plasma glucose and to prevent the degradation of health behaviors over time. This model has the potential to significantly reduce the required medical resources and to be widely popularized in economically backward areas. Limitations The sample size of this study needs to be further expanded, and considering that we only selected hospitalized patients, we will select some community patient to expand the sample size in the next step. In addition, the main outcome indicators were FBG and PBG, but owing to HbA1C is the gold standard for measurement for the assessment of glycemic control, which can effectively reflect the situation over the past 1-2 months blood sugar control in diabetics, we will consider HbA1C as the main outcome indicator in the next study. Availability of data and materials The datasets used and/or analyzed during this study are available from the corresponding author upon reasonable request. Consent for publication Not applicable. Authors’ contributions XW conceived and design the protocol. XW and DL conducted the statistical analysis and prepared the manuscript. MD, RH, CY contributed to data collection. HZ and DL participated in clearing and analyzing the data. All authors contributed to the writing, reviewing and final approval of the manuscript. Funding This study was supported by Inner Mongolia Science and Technology Project (Intelligent Health Monitoring and Modern Medical Information System Development Based on Internet of Things Technology). The funder played a role in the design of the study and collection, analysis of data and in writing the manuscript. Abbreviations T2DM: type 2 diabetes mellitus SMS: short message service IG: intervention group CG: control group Page 10/20 SD: standard deviation BP: blood pressure FBG: fasting blood glucose PBG: postprandial blood glucose SBP: systolic blood pressure DBP: diastolic blood pressure Ethics approval and consent to participate The study protocol was approved by the Biomedical Research Ethics Committee of Inner Mongolia Medical University, and all selected participants signed informed consent forms. Competing interests The authors’ declare that they have no competing interests. Acknowledgements Page 11/20 In addition, we thank Jennifer Barrett, Ph.D, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. Contributor information Xuemei Wang, Email: wangxm_zsu@163.com. Dan Liu, Email: liudan_summer@163.com Maolin Du, Email: dumaolin2016@163.com Ruiqi Hao, Email: haorqimmu@163.com Xuemei Wang, Email: wangxm_zsu@163.com. Dan Liu, Email: liudan_summer@163.com Maolin Du, Email: dumaolin2016@163.com Ruiqi Hao, Email: haorqimmu@163.com Huiqiu Zheng, Email: zhxqqjye1988@163.com Chaoli Yan, Email: aliceyzl@126.com Chaoli Yan, Email: aliceyzl@126.com Chaoli Yan, Email: aliceyzl@126.com References Page 12/20 Page 12/20 8. Fang R, Deng X. Electronic messaging intervention for management of cardiovascular risk factors in type 2 diabetes mellitus: a randomised controlled trial. Journal of Clinical Nursing. 2018; 27(3-4): 612-620. doi: 10.1111/jocn.13962. 9. Wu I X Y, Kee J C Y, Threapleton D E, et al. Effectiveness of smartphone technologies on glycaemic control in patients with type 2 diabetes: systematic review with meta-analysis of 17 trials. Obesity Reviews. 2018; 19(6): 825-838. doi: 10.1111/obr.12669. 10. Morita P P, Yeung M S, Ferrone M, et al. A Patient-Centered Mobile Health System That Supports Asthma Self-Management (breathe): Design, Development, and Utilization. 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Tables Table 1 Comparison the characteristics of the SMS quality evaluation population and the SMS intervention population SMS intervention populations （N = 171） SMS quality evaluation population (N = 72) c2 P value Age 0.001 0.982 <50 52 (30.4) 22 (30.6) ³50 119 (69.6) 50 (69.4) Gender 0.400 0.527 male 97 (56.7) 44 (61.1) female 74 (43.3) 28 (38.9) BMI 0.761 a <18.5 4 (2.4) 3 (3.7) 18.5~24.0 44 (26.3) 19 (23.2) ³24.0 119 (71.3) 60 (73.2) Distribution 0.957 0.328 urban 133(77.8) 60 (83.3) rural 38 (22.2) 12 (16.7) Education 0.412 0.521 below high school 68 (40.0) 32 (44.4) high school and above 102 (60.0) 40 (55.6) Marital status 0.773 a married 161 (94.2) 67 (93.1) other 10(5.8) 5 (6.9) Employment status 2.605 0.106 employed 90 (52.6) 46 (63.9) non-employed 81 (47.4) 26 (36.1) Smoking 0.011 0.915 yes 44 (25.7) 19 (26.4) no 127 (74.3) 53 (73.6) Drinking 1.317 0.251 yes 58(33.9) 30 (41.7) no 113 (66.1) 42 (58.3) Hypertension 0.262 0.609 yes 65 (39.6) 26 (36.1) no 99 (60.4) 46 (63.9) SMS: short message service; BMI: body mass index. a Fisher probabilities method Table 2 Comprehensive quality of the message content (%). Page 15/20 Main domain Average score (SD) Maximum score Minimum score Health awareness 8.0 (0.7) 9.6 6.6 Diet control 8.5 (0.6) 9.8 6.8 Physical activities 7.9(1.0) 10.0 6.8 Living habits 8.0(0.7) 9.2 6.8 Weight control 8.4(0.9) 10.0 5.5 Table 3 Participants’ understanding of the message content (%). Main domain understandable and approved cannot understand but approved understandable but disapproved cannot understand and approved Health awareness 99.8 0.0 0.2 0.0 Diet control 99.9 0.0 0.1 0.0 Physical activities 99.7 0.0 0.3 0.0 Living habits 98.5 0.0 1.5 0.0 Weight control 99.5 0.0 0.5 0.0 Table 3 Participants’ understanding of the message content (%). Table 4 Participants’ willingness to act on the message content (%). Main domain Helpful and persistent Unhelpful but persistent Helpful but cannot insist Unhelpful and cannot insist Health awareness 90.1 1.8 7.3 0.8 Diet control 90.5 1.8 5.8 1.8 Physical activities 80.8 1.6 7.3 10.3 Living habits 68.3 1.4 26.2 4.4 Weight control 81.3 1.8 12.7 4.2 Table 4 Participants’ willingness to act on the message content (%). Table 5 Current status of the participant in response to the text message content (%). Tables Main domain Known and have done Unknown but have done Known but not have done Unknown and not have done Health awareness 44.6 2.4 36.7 16.3 Diet control 29.6 7.0 20.8 42.5 Physical activities 40.4 6.8 17.2 35.7 Living habits 8.7 1.6 79.8 9.9 Weight control 28.5 6.7 20.1 44.7 Table 5 Current status of the participant in response to the text message content (% Main domain Known and have d Unknown but have d Known bu urrent status of the participant in response to the text message content (%). Page 16/20 Table 6 Baseline characteristics of the participants in the intervention group and control group. Tables aseline characteristics of the participants in the intervention group and control group Total IG (n=85) CG (n=86) t/c2 P value Demographic, Mean(SD) Gender (Male/Female) 97/74 42/43 55/31 3.682 0.055 Age (years) 55.1 (10.8) 55.4 (9.7) 54.7 (11.8) -0.416 0.678 BMI (Kg/m2) 25.8 (3.8) 25.9 (3.5) 25.8 (4.1) -0.255 0.799 Indicators, Mean (SD) FPG (mmol/L) 9.0 (2.6) 9.2 (2.8) 8.8 (2.4) -0.954 0.342 PPG (mmol/L) 16.6 (4.4) 16.8 (4.6) 16.4 (4.3) -0.599 0.550 SBP (mmHg) 132.3 (17.0) 131.0 (16.1) 134.3 (17.8) 1.137 0.257 DBP (mmHg) 80.8 (11.8) 79.8 (9.8) 81.8 (13.5) 1.034 0.303 FPG control (%) 48 (28.4) 25 (29.8) 23 (27.1) 0.152 0.697 PPG control (%) 16 (9.6) 10 (12.0) 6 (7.2) 1.107 0.293 SBP control (%) 90 (60.8) 51 (68.0) 39 (53.4) 3.298 0.069 DBP control (%) 92 (63.4) 50 (68.5) 42 (58.3) 1.613 0.204 Total cholesterol (mg/l) 4.7 (1.2) 4.8 (1.2) 4.7 (1.2) -0.747 0.456 Triglycerides (mg/l) 1.9 (1.3) 1.9 (1.3) 1.9 (1.2) 0.275 0.783 HDL-C (mg/l) 1.1 (0.3) 1.1 (0.2) 1.1 (0.3) -1.062 0.290 LDL-C (mg/l) 3.0 (0.9) 3.0 (0.9) 2.9 (1.0) -0.739 0.461 HbAlC (%) 9.2 (2.0) 9.2 (2.0) 9.1 (2.0) -0.339 0.735 Risk factors Hypertension (%) 65 (39.6) 32 (39.0) 33 (40.2) 0.025 0.873 Dyslipidemia (%) 96 (56.1) 46 (54.1) 50 (58.1) 0.126 0.722 Smoking (%) 44 (25.7) 23 (27.1) 21 (24.4) 0.156 0.693 Drinking (%) 58 (33.9) 26 (30.6) 32 (37.2) 0.361 0.836 Overweight (%) 97 (58.1) 50 (61.0) 47 (55.3) 0.553 0.457 Pain (%) 94 (55.3) 41 (48.2) 53 (62.4) 3.427 0.064 Anxiety (%) 82 (48.2) 43 (50.6) 39 (45.9) 0.377 0.539 Hypoglycemic therapy (%) 156 (94.0) 80 (97.6) 76 (90.5) 2.534 0.111 Medication way (%) 3.058 0.217 insulin 80 (51.9) 46 (57.5) 34 (45.9) Oral medication + insulin 48 (31.2) 20 (25.0) 28 ( 37.8) Oral medication 26 (16.9) 14 (17.5) 12 (16.2) Antihypertensive therapy (%) 60 (35.1) 31 (36.5) 29 (33.7) 0.672† CG: control group; IG: intervention group; SD: standard deviation; BMI: body mass index; FPG: fasting plasma glucose; PPG: postprandial glucose; SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL-C: high density lipoprotein-c; LDL-C: low density lipoprotein-c. †Fisher probabilities method Table 7 Change in primary outcomes during the intervention. Tables Total CG IG tb P value At 12 month Change a At 12 month Change a At 12 month Change a Primary outcomes, Mean(SD) FPG (mmol/L) 8.0 (2.1) -0.9 (2.9) 8.4 (2.2) -0.4 (2.8) 7.7 (1.9) -1.5 (3.0) 2.556 0.011* PPG (mmol/L) 11.1 (2.8) -5.4 (4.8) 11.9 (3.2) -4.2 (4.7) 10.4 (2.2) -5.8 (5.1) 2.633 0.009* P < 0.05. a change = Endpoint value – Baseline value; b The t value was the comparative result of the changes between the CG and the IG. FPG: fasting plasma glucose; PPG: postprandial glucose; CG: control group; IG: intervention group; SD: standard deviation. Table 7 Change in primary outcomes during the intervention. between the CG and the IG. FPG: fasting plasma glucose; PPG: postprandial glucose; CG: control group; IG: intervention group; SD: standard deviation. G. se; PPG: postprandial glucose; CG: control group; IG: intervention group; SD: Page 17/20 Page 17/20 Control rate of plasma glucose and improvements of health behaviors at 12 month. Total CG IG t/c2 P value Primary outcomes FPG control (%) 70 (41.4) 28 (33.3) 42 (49.4) 4.501 0.034 PPG control (%) 76 (45.8) 28 (33.7) 48 (57.8) 9.708 0.002* Secondary outcomes Body Weight (%) 6.972 0.031* Increased from Baseline 27 (19.0) 17 (23.9) 10 (14.1) Decreased from Baseline 55 (38.7) 20 (28.2) 35 (49.3) Unchanged from Baseline 60 (42.3) 34 (47.9) 26 (36.6) Vegetables consumption (%) 49.081 <0.001** Increased from Baseline 82 (58.6) 20 (29.0) 62 (87.3) Decreased from Baseline 19 (13.6) 16 (23.2) 3 (4.2) Unchanged from Baseline 39 (27.9) 33 (47.8) 6 (8.5) Fruits consumption (%) 7.634 0.022* Increased from Baseline 15 (16.0) 4 (7.4) 11 (27.5) Decreased from Baseline 44 (46.8) 26 (48.1) 18 (45.0) Unchanged from Baseline 35 (37.2) 24 (44.4) 11 (27.5) Physical activities (%) 4.407 0.036* Continuous exercise 110 (77.5) 49 (70.0) 61 (84.7) Discontinuous exercise 32 (22.5) 21 (30.0) 11 (15.3) Quit smoking (%) 12 (25.5) 3 (13.6) 9 (36.0) 3.078 0.079 Quit drinking (%) 17 (39.5) 9 (34.6) 8 (47.1) 0.666 0.415 P < 0.05; *P < 0.001 FPG: fasting plasma glucose; PPG: postprandial glucose; CG: control group; IG: intervention group. of plasma glucose and improvements of health behaviors at 12 month. Additional Files Additional file 1 Final list of SMS Additional file 2 Stages of change, type of content, and number of SMS text messages per key domain Additional file 3 SMS evaluation questionnaire Additional file 2 Stages of change, type of content, and number of SMS text messages per key domain Additional file 3 SMS evaluation questionnaire Additional file 4 Examples of regular messages Additional file 5 Follow-up record sheet Figure 3 Composite change in scores by intervention group. Figure 2 Comparison of FPG and PPG levels at the end of each intervention stage. Comparison of FPG and PPG levels at the end of each intervention stage. Comparison of FPG and PPG levels at the end of each intervention stage. Figures Page 18/20 Figure 1 Study flow diagram. Page 19/20 Page 19/20 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. CONSORT2010Checklist.doc Additionalfile2.xls Additionalfile2.xls Additionalfile3.xls Additionalfile5.xls Additionalfile4.xls Additionalfile1.xls Page 20/20 Page 20/20
https://openalex.org/W3205545286	https://figshare.com/articles/journal_contribution/The_role_of_invasion_and_urbanization_gradients_in_shaping_avian_community_composition/20635449/1/files/36841359.pdf	English	null	The role of invasion and urbanization gradients in shaping avian community composition	Journal of urban ecology	2,021	cc-by	7,264	AUTHOR(S) A M Rogers, A S Griffin, F Lermite, B Van Rensburg, Carla Archibald, S Kark The role of invasion and urbanization gradients in shaping avian community composition AUTHOR(S) A M Rogers, A S Griffin, F Lermite, B Van Rensburg, Carla Archibald, S Kark PUBLICATION DATE 01-01-2021 HANDLE 10536/DRO/DU:30158615 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B The role of invasion and urbanization gradients in shaping avian community composition The role of invasion and urbanization gradients in shaping avian community composition AUTHOR(S) The role of invasion and urbanization gradients in shaping avian community composition Andrew M. Rogers ,1,* Andrea S. Griffin,2 Franc¸oise Lermite,2 Berndt van Rensburg,1,3 Carla Archibald1,4 and Salit Kark1 1The Biodiversity Research Group, The School of Biological Sciences, Centre for Biodiversity and Conservation Science, The University of Queensland, Brisbane, QLD 4072, Australia, 2School of Psychology, University of Newcastle, University Drive, Callaghan, NSW, Australia, 3Department of Zoology, DST-NRF Centre for Invasion Biology, University of Johannesburg, Johannesburg, South Africa and 4Centre for Integrative Ecology, School of Life & Environmental Sciences, Deakin University, Melbourne Burwood Campus, VIC, Australia *Corresponding author. E-mail: a.munro.rogers@gmail.com Submitted: 20 November 2020; Received (in revised form): 2 September 2021. Accepted: 9 September 2021 Submitted: 20 November 2020; Received (in revised form): 2 September 2021. Accepted: 9 September 2021 Abstract The extent to which native species utilize urban environments depends on species responses to multiple threatening pro- cesses. Here, we aimed to quantify changes in bird communities in response to changing habitat structure, invasive species and aggressive native species. We conducted surveys in two independently invaded regions with similar patterns of urban development. The study regions were New South Wales (NSW) and Queensland (QLD), Australia. We observed 127 species in NSW and 144 species in QLD. Most species (NSW 83 and QLD 84) are urban adapters making use of some or all urban sub- environments. Urban avoiders, species only found in remnant vegetation, were the second largest group (urban avoiders: NSW 23 and QLD 31). We found the lowest richness in the most urban sites (urban exploiters: NSW 10 and QLD 15). Using generalized linear mixed models, we found a non-signiﬁcant relationship between species richness and the abundance of aggressive species like the common myna and noisy miners, Manorina melanocephala, but a signiﬁcant positive correlation with the percentage of shrub cover at a site. As there is a gradual loss of species with increasing urbanization, retaining higher complexity in vegetation structure in urban areas will support large numbers of species and could help mitigate the potential impacts of aggressive urban-adapted species and habitat loss. Key words: invasive species, habitat change, urban conservation, community ecology r 2021 V C The Author(s) 2021. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 10536/DRO/DU:30158615 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Journal of Urban Ecology, 2021, 1–9 doi: 10.1093/jue/juab030 Research Article V The Author(s) 2021. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION environments are heterogeneous and individual species ability to use sub-environments within a city may differ depending on species-specific responses, size of the city, the patterns of urban development and the intensity of disturbance within urban habitat (Bolger et al. 1997; Sewell and Catterall 1998; Blair 2004; Fournier et al. 2020; Heggie-Gracie et al. 2020). Species responses to urbanization can be divided into three cat- egories: urban exploiters, urban adapters and urban avoiders (Blair 1996; Kark et al. 2007). Urban ‘Exploiters’ are species that can capitalize on the altered resources within urban environ- ments and reach high densities in modified environments. Urban ‘Adapters’ are species that can make limited use of urban areas, or do well in lightly transformed environments. Urban ‘Avoiders’ are native species that are most sensitive to habitat change and prefer to occur in unmodified areas. However, urban Factors such as habitat change, competition with invasive species and interactions with aggressive native urban adapted species are not uniform across urban environments (Garden et al. 2006, 2010), resulting in local and landscape-scale gradients 1 2 \| Journal of Urban Ecology, 2021, Vol. 7, No. 1 2012), and it is unknown whether the reported impacts are con- sistent across its range. of disturbance (Garaffa et al. 2009; Luck et al. 2013). The extent to which species use different parts of urban environments has important implications for conserving species in and around ur- ban centres (Bolger et al. 1997). Understanding how urban com- munities vary within and among different cities can help identify important habitat features that promote urban biodi- versity (Shwartz et al. 2008; Sushinsky et al. 2013). Despite the impact of common myna and noisy miner on Australian bird communities having been demonstrated, most studies of impact have been derived from atlas data (at a very large spatial scale) or from single city field studies. Studies across multiple cities in Australia are lacking. As habitat quality is likely to vary within and between cities (Ferenc et al. 2014), conserving urban species in Australia requires understanding how species respond to variation in urbanization. Here we ex- plore how bird richness changed along local urban gradients (sub-environments: urbanized, park and semi-natural edge hab- itats) and how vegetation structure, common myna presence and native noisy miner presence influenced species richness across urban sites. Study area In this study, we focused on south-east Queensland and cen- tral New South Wales, which have relatively similar patterns of urban development and independent invasion histories for the common myna (Fig. 1). Both regions are also within the na- tive range of the noisy miner. We selected study sites in each region along an invasion gradient. Sites that had been invaded before 1970 (‘source’ sites) and those invaded after 1990 (‘front sites’). We selected nine cities across the two regions repre- senting five source locations and four front locations (Fig. 1). Within each city, we surveyed along an urbanization gradient, with one survey site in each of three different sub- environments; at the edge of remnant vegetation (‘edge’), in an open park (‘park’) and a heavily urbanized area (‘urban’). We chose areas within each city that contained the three sub- environments within 1 km of each other. Edge sites were areas adjacent to remnant native vegetation. Park sites were charac- terized by large amounts of mowed turf and few large trees. Urban sites were areas with high human activity, higher pro- portions of sealed surface (concrete or asphalt) and a large number of buildings and other artificial structures. Overall, we surveyed 27 sites, including 9 locations within the two regions (QLD, NSW) 3 sub-environments (edge, park, urban) within each location (see Fig. 1). Two species associated with declines in native species rich- ness and abundance are the native noisy miner (Manorina mela- nocephala) and the introduced common myna (Acridotheres tristis) (MacDonald and Kirkpatrick 2003; Tindall et al. 2007; Kath et al. 2009; Mac Nally et al. 2014). Evidence of significant impacts of the common myna in Australia is generally restricted to com- petition for nesting sites (Pell and Tidemann 1997a; Rogers et al. 2020), although they show aggressive behaviour around food resources and are associated with declines in native species (Grarock et al. 2014). Noisy miners are attributed to changing species composition and driving declines of small-bodied birds at both local and landscape scales (Montague-Drake et al. 2011; Mac Nally et al. 2014). The noisy miners achieve this through the aggressive exclusion of other birds from breeding territories (Maron et al. 2013; Haythorpe et al. 2014). Competition between myna, miners and other species show important differences across sites with varying levels of urbanization (Sol et al. 2012; Haythorpe et al. INTRODUCTION We followed up by asking how are bird spe- cies distributed across the urban exploiters, adapters and avoiders categories in two regions of Australia. In Australia, urban expansion over the last 200 years has oc- curred relatively recently (compared to places like Europe), and nearly 90% of the Australian population now live in cities (Australian Bureau of Statistics 2012). The changing Australian landscapes have driven significant changes in bird communi- ties due to both species introductions and changes in native species distributions (Joyce et al. 2018). Around 60 species have been introduced to Australia, while 24 of these have established breeding populations (McKinney and Kark 2017); however, the impact of most of these established species on native bird spe- cies has not been studied. In addition to non-native species introductions, habitat modification associated with humans has facilitated the range expansion and spread of many native species (Major and Parsons 2010). Several of these native urban adapted birds are highly aggressive (Montague-Drake et al. 2011), further impacting urban birds in addition to the changes brought about by habitat change and introduced species (Haythorpe et al. 2014). Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest on 18 November 2021 Urban exploiters, adapters, avoiders along gradients \| 3 Urban exploiters, adapters, avoiders along gradients \| 3 Figure 1: Study sites were located within nine cities across the states of Queensland (QLD) and New South Wales (NSW). Study sites were located along an invasion gra- dient (front, source) and within each city (edge, park, urban) representing the urban gradients within each city. Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest on 18 November 2021 Figure 1: Study sites were located within nine cities across the states of Queensland (QLD) and New South Wales (NSW). Study sites were located along an invasion gra- dient (front, source) and within each city (edge, park, urban) representing the urban gradients within each city. We quantified local habitat structure and the cover around each survey point using eight circular vegetation plots with a di- ameter of 30 m. We selected points for the vegetation plots by generating a random distance (within 100 m) and the direction of the centre of site. If that point was not accessible (a building or other structure was in the way), the nearest accessible point to the selected point was used. Because tree density is an im- portant habitat factor for both common myna and noisy miners (Grarock et al. 2014; Haythorpe et al. 2014), we counted the num- ber of trees within each plot and where possible identified to a genus for individual trees over 5 cm in diameter. Additionally, within each plot, we estimated the percent ground cover of shrub (woody vegetation between 5 cm and 3 m in height), turf, and sealed ground (i.e. asphalt and concrete). We calculated species richness using data pooled across the 2 years of surveys. Species abundance was calculated using all birds observed utilizing a site during surveys but excluding birds flying over. We calculated average survey richness with the ‘specnumber’ function in the ‘biodiversityR’ package (Oksanen et al. 2018) in the program R (R Core Team 2015). We calculated total site richness using the SJack2 species richness estimator as it makes no assumptions about underlying species distribu- tions and has been shown to be robust even when calculating richness with small sample sizes (Hellmann and Fowler 1999; Magurran 2004). To quantify changes in vegetation across sites, we used sev- eral approaches. The native vegetation at our sites would have historically been open eucalypt woodland characterized by 50– 80% crown cover (Australia’s state of the forests report 2018). Study area 2014) with both species being highly successful in sub-urban environments such as those created in city parks with scattered trees and large areas of open habitat (Pell and Tidemann 1997b; Grey et al. 1998). In each study site, the study team and volunteers familiar with the local birds carried out monthly bird surveys between August 2014 and August 2016. Each survey consisted of a 10- min point count with an unlimited radius, during which we recorded the total number of birds that were seen and heard. For each observation, we recorded bird activity as flying over- head, flying through, walking, perched. We conducted bird sur- veys within 3 h of sunrise and did not survey on days with heavy rain or high winds. To minimize the effect of different observers, we pooled data over the 2 years for the analysis. We excluded birds observed only flying over a site, and not actively using a site from the analysis. For example, a raptor flying over- head but foraging would be included, but waterfowl flying high overhead would be excluded. To calculate the abundance, we used the total number of birds recorded within a 100 m radius of the point count, averaged over the 24 surveys for each site. We calculated common myna and noisy miner abundance by avearchivraging the total number of birds observed per survey, within 100 m of the survey point, over the 2 years. In Australia, the common myna is still expanding its geo- graphic range, creating an invasion gradient as mynas spread from long invaded ‘source’ areas to the expanding invasion ‘front’ (Fogarty et al. 2011; Perkins 2012; Berthouly-Salazar et al. 2013). Source and front populations often have different impacts on local species (Urban et al. 2008; Tayleur 2010; Sullivan 2014), but the impacts of the common myna across its range have not been studied. The common myna is a member of the starling family (Sturnidae) and was introduced starting the 19th century into several locations across eastern Australia, and it has been gradually spreading across its potential range that includes the entire east coast of the continent (Martin 1996). The invasion gradient of the common myna also occurs along a landscape-scale gradient of urbanization, with larger cities generally having been invaded earlier and rural towns more recently. Previous studies on the myna have occurred in long invaded areas (Pell and Tidemann 1997a; Grarock et al. Urban exploiters, adapters, avoiders along gradients \| 3 To quantify variation in the vegetation cover surrounding the study sites, we used remotely sensed data of the normalized difference vegetation index (NDVI; Supplementary materials, Table S1) sensu Bino et al. (2008). We used NDVI data from NASA’s LandSat satellite, which produces images with a 30 m 30 m pixel resolution. We downloaded NDVI images for all sites for the years 2009 –2014. We evaluated each image for cloud cover, and only images that had less than 10% cloud cover were used. We found 14 cloud-free images to include in the analysis. For each survey site, we calculated NDVI by averaging the NDVI value for pixels in a 100 m2 area centred on each survey site. The relationship between habitat characteristics, the abun- dance of aggressive species, and species richness at each site was explored using Generalized Linear Mixed Models (Zuur et al. 2009) using the package ‘lme4’ (De Boeck et al. 2011) and ‘lmerTest’ (Kuznetsova et al. 2015). We used the SJack2 richness estimate for each site as the dependent variable. Explanatory variables include common myna abundance, noisy miner abun- dance, average tree density, average percent shrub ground cover and average NDVI. To account for the nested nature of the study design, we included the city as a random factor. We tested for collinearity between explanatory variables and included varia- bles in the final model if they had a variance inflation factor of 4 \| Journal of Urban Ecology, 2021, Vol. 7, No. 1 4 \| Journal of Urban Ecology, 2021, Vol. 7, No. 1 less than four (Crawley 2007). The percent ground cover of shrub, turf and sealed ground showed high collinearity. Both percent ground cover shrubs and sealed surface showed the largest differences between sub-environments (Supplementary materials, Fig. S2), so we selected used shrub cover in the GLMMs to help distinguish between sites. In Australia, areas with higher shrub cover have been shown to support greater bird richness in Australia (see Archibald et al. 2017). The correla- tion between explanatory variables in the final model, along with the full model results are included in supplementary mate- rials (Supplementary materials, Table S2). (Fig. 2). Total bird abundance was highly variable across sites and showed no consistent pattern between regions (Fig. 2). The abundance of the common myna and noisy miner showed high variation along the urban gradient between regions and across front/source locations (Fig. 3). Urban exploiters, adapters, avoiders along gradients \| 3 However, noisy miners were simi- larly or more abundant compared to the common myna. Across the invasion gradient, the common myna was more abundant in source sites on average but these differences are not signifi- cant (Fig. 3). RESULTS Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest on 18 November 2021 Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by gues Urban exploiters, adapters and avoiders Based on species rank abundance across all urban sub- environments in each region (Supplementary materials, Table S3), we found a gradual loss of species with the increasing ur- banization of sub-environments at local scales (Fig. 4). We cate- gorized urban adapters as species that occurred in some, but not all of the urban and park sites. Adapters were the largest group, with 83 species in QLD and 84 species in NSW. Urban adapters, therefore, represent 60–70% of the urban bird assemb- lages in our study regions. Urban avoiders, species found only the edge sites, were the next largest group with 35 species in NSW and 42 species in QLD that occurred in the least developed sites. We found that urban exploiters were the group with the smallest number of species with 10 birds in NSW and 16 in QLD. These species accounted for 8.5% and 11.6% of the total species pool in NSW and QLD but accounted for 42% and 52% of all indi- viduals birds observed in the respective regions. The two study regions shared six of the ten most abundant urban exploiters including the Australian magpie (Gymnorhina tibicen), laughing kookaburra (Dacelo novaeguineae) and magpie-lark (Grallina Bird surveys In total, we conducted 648 surveys (24 for each of the 27 sites) and recorded 168 species across all study locations. Regionally, we found 132 species in our 15 Queensland sites and 117 species in the 12 New South Wales (NSW) sites. Across all sites, we found 129 native and five invasive species (Supplementary materials, Table S3). The introduced bird species in the QLD sites were the common myna, common starling (Sturnus vulga- ris) and the rock dove (Columba livia). In NSW introduced species included the common myna, common starling, common black- bird (Turdus merula), European goldfinch (Carduelis carduelis) and rock dove. Species richness along the urbanization gradient showed that edge sites contained the most species, followed by the park then urban sites (Fig. 2). The pattern of decreasing rich- ness with increasing levels of urbanization was consistent after pooling the data for each region, and front vs. sources locations Figure 2: Avian species richness (SJack2) and abundance calculated for each sub-environment (edge, park, urban) representing the invasion gradient within the front and source locations of the two regions (QLD and NSW) combined (a, c), and the two regions with front and source locations combined (b, d). Horizontal lines in the boxes represent median values, and box boundaries indicate 25th and 75th percentiles. Whiskers span two standard deviations of the mean. Non-overlapping notches indicate signiﬁcant differences between medians. Figure 2: Avian species richness (SJack2) and abundance calculated for each sub-environment (edge, park, urban) representing the invasion gradient within the front and source locations of the two regions (QLD and NSW) combined (a, c), and the two regions with front and source locations combined (b, d). Horizontal lines in the boxes represent median values, and box boundaries indicate 25th and 75th percentiles. Whiskers span two standard deviations of the mean. Non-overlapping notches indicate signiﬁcant differences between medians. Urban exploiters, adapters, avoiders along gradients \| 5 Figure 3: The mean observed abundance of the common myna and the noisy miner in Queensland and New South Wales across the (a) urban gradient and (b) front-source locations. Horizontal lines in the boxes represent median values, and box boundaries indicate 25th and 75th percentiles. Whiskers span two stan- dard deviations of the mean. Non-overlapping notches indicate signiﬁcant dif- ferences between medians. structure (Bolger et al. 1997; Chace and Walsh 2006; Shwartz et al. 2008; Shanahan and Possingham 2009; Archibald et al. 2017). Bird surveys Importantly, the high numbers of urban adapters highlight the need for conservation in modified environments and the benefit of retaining green space for native wildlife (Edwards et al. 2011; Ives et al. 2016). We found weak support for the impact of common myna and noisy miners on avian community richness and more sup- port for the importance of habitat structure. Earlier work (Grarock et al. 2014a) investigating common myna impacts along an urban gradient in Canberra found that the common myna in combination with habitat change was a driver of changes in bird communities, but that work did not report the impact of noisy miners. The low abundance of mynas we ob- served across some sites, especially in Queensland, relative to other parts of its range (Grarock et al. 2013), highlights impor- tant variation in the common myna’s success across the conti- nent. Nevertheless, the higher average abundance of noisy miners compared to common mynas across our urban gradients means that the baseline levels of aggression by native noisy miners need to be taken into account when assessing impacts of non-native species (Mac Nally et al. 2012; Haythorpe et al. 2014). Both species showed a non-significant but negative corre- lation with shrub cover, such that increasing the structural di- versity of habitat fragments may deter these species from increasing the value of such habitat to other birds. Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest on 18 November 2021 In both study regions, total species richness was lower in larger urban areas (sites in the greater Brisbane and greater Newcastle areas) compared to our more rural study sites. This pattern may be due to shorter distances from our rural sites to intact forests, allowing more species to filter into urban sub- environments (Fontana et al. 2011; MacGregor-Fors et al. 2011). However, the same pattern of species loss with increasing ur- banization intensity in both large and small cities suggests that maintaining remnant habitat as cities grow will have significant conservation benefits for many birds (Shanahan and Possingham 2009; Rayner et al. 2015; Archibald et al. 2017). An important finding is that of higher bird diversity in edge sites across local gradients of urbanization (within 1 km), such that even small patches of habitat with complex vegetation struc- ture are likely to support more diverse bird assemblages in ur- ban environments (Shwartz et al. 2008). Bird communities across disturbance gradients In this study, we were interested in how urban bird communi- ties respond to habitat modification, invasive species and ag- gressive native species across multiple Australian cities. We found consistent patterns of species loss and increasing biotic homogenization with increasing urbanization across our inva- sion gradients, and across the two study regions, a result found across many urban environments (Blair 1996; Kark et al. 2007; Fontana et al. 2011, Heggie-Gracie et al. 2020). However, the ma- jority of bird species across our study sites were urban adapters, making some use of some but not all urban sub-environments. Sub-environments that had more complex habitat structure (shrub ground cover); a finding that lends further support to the importance of urban green space with complex vegetation Bird surveys Figure 3: The mean observed abundance of the common myna and the noisy miner in Queensland and New South Wales across the (a) urban gradient and (b) front-source locations. Horizontal lines in the boxes represent median values, and box boundaries indicate 25th and 75th percentiles. Whiskers span two stan- dard deviations of the mean. Non-overlapping notches indicate signiﬁcant dif- ferences between medians. cyanoleuca), the galah (Eolophus roseicapilla) and the noisy miner and rainbow lorikeet (Trichoglossus moluccanus). Species richness and sources of disturbance We found that across all 27 survey sites, species richness was positively correlated with per cent shrub ground cover (estimate ¼ 0.52, df ¼ 15.68, P < 0.00). A non-significant negative correla- tion was found between common myna abundance and noisy miner abundance with species richness and remnant vegeta- tion (Supplementary materials, Fig. S1), and we found no signifi- cant relationships in the generalized linear mixed models. Individual species tolerance to urbanization contributed to a pattern of gradual species loss across urban environments (Blair 1996; Joyce et al. 2018). Conservation efforts to support the ur- ban adapters, therefore, stand to benefit between 60% and 70% of birds that are found in urban environments. Conservation efforts targeting these urban adapters should be a priority as Joyce et al. (2018) found the long-term population trends of many common birds in SE Queensland are declining. The rela- tively recent establishment of Australian cities, within the last 200 years, and may mean that there remains an extinction debt in more rural–urban areas (Szabo et al. 2011; Hanski 2013; Soga and Koike 2013). Mitigating the loss of species from urban envi- ronments will require better incentives and policies to protect and improve the remaining fragments for birds and other wild- life (Bolger et al. 1997; Rodewald and Shustack 2008; Dale 2018). Figure 4: Presence and absence (black squares are present) for species (each column) in each sub-environment, for Queensland (QLD) New South Wales (NSW). Sites are grouped and coloured by sub-site with an edge, park and urban being green, light green and grey, respectfully. Urban exploiters (species that occurred at all sites) are 8.5% and 11.6% of the regional species pool (10 NSW; 15 QLD). Many more species are urban adapters and occurred in some but not all the sites (84 NSW; 83 QLD) representing 71.7% and 62.8% of NSW and QLD species, respectfully. Urban avoiders, species found only in edge sites were the second largest group (23 NSW; 31 QLD). The urban exploiters (species recorded at all sites) made up 42% and 52% of the total individuals recorded in NSW and QLD, respectfully. Table 1: Generalized linear model residuals for testing the relation- ship between species richness and local habitat structure and the presence of aggressive urban-adapted species (the common myna and noisy miner). Models used a negative binomial error distribution and a log link relative impact of each species on bird communities, both mynas and miners show preferences for more open habitats so efforts to maintain or restore habitat should mitigate the impact of these two species. Patterns of common myna abundance were highly variable across source-front gradients. The pattern of increasing com- mon myna abundance with increasing urbanization is consis- tent with other studies from across its range (Sol et al. 2012; Grarock et al. 2013). Common myna can reach high densities in cities, and in some parts of its invasive range, it is one of the most common urban birds (Grarock et al. 2012). The variation of common myna abundance at landscape scales remains a key gap in our understanding of common myna invasion dynamics in Australia. The impact of the introduced common myna on lo- cal bird assemblages has been largely inferred from patterns of increasing myna density and decreasing bird abundance (Tindall et al. 2007; Grarock et al. 2012). This poor understanding of the drivers of myna abundance in different regions limits what conclusions can be made about common myna impacts across its introduced range (Newson et al. 2011; Baker 2017). Estimate Std. Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest on 18 November 2021 Downloaded from https://academic.oup.com/jue/article/7/1/juab030/6382020 by guest Patterns of urban habitat use by alien and despotic species Beyond changes in habitat, interactions between species can also drive changes in community composition (Tindall et al. 2007; Kath et al. 2009; McClure et al. 2011; Maron et al. 2013), 6 \| Journal of Urban Ecology, 2021, Vol. 7, No. 1 FUNDING A.M.R. was supported by the Australian Government International Post-graduate Research Scholarship. The re- search was supported by an Australian Government Discovery Grant, project number DP140100524. Fogarty, S., Cote, J., and Sih, A. (2011) ‘Social Personality Polymorphism and the Spread of Invasive Species: A Model’, The American Naturalist, 177: 273–87. Conﬂict of interest statement. None declared. Conﬂict of interest statement. None declared. Fontana, S. et al. (2011) ‘How to Manage the Urban Green to Improve Bird Diversity and Community Structure’, Landscape and Urban Planning, 101: 278–85. REFERENCES Australian Bureau of Statistics (2012) Year book Australia, 2012. Canberra: ABS. https://www.abs.gov.au/ausstats/abs@.nsf/mf/ 1301.0, accessed 3 August 2019. Bino, G. et al. (2008) ‘Accurate Prediction of Bird Species Richness Patterns in an Urban Environment Using Landsat-Derived NDVI and Spectral Unmixing’, International Journal of Remote Sensing, 29: 3675–700. Data availability Garden, J. G., McAlpine, C. A., and Possingham, H. P. (2010) ‘Multi-Scaled Habitat Considerations for Conserving Urban Biodiversity: Native Reptiles and Small Mammals in Brisbane, Australia’, Landscape Ecology, 25: 1013–28. All data used in the analysis are included in the supplemen- tary data Grarock, K. et al. (2013) ‘Does Human-Induced Habitat Modiﬁcation Inﬂuence the Impact of Introduced Species? A Case Study on Cavity-Nesting by the Introduced Common Myna (Acridotheres Tristis) and Two Australian Native Parrots’, Environmental Management, 52: 958–70. Consent for publication All authors gave ﬁnal approval for publication. Fauna: A Focus on Spatially Explicit Processes’, Austral Ecology, 31: 126–48. Ethics approval (include appropriate approvals or waivers) Fournier, B., Frey, D., and Moretti, M. (2020) ‘The Origin of Urban Communities: From the Regional Species Pool to Community Assemblages in City’, Journal of Biogeography, 47: 615–29. University of Queensland Animal Ethics approval number 454/13. Garaffa, P. I., Filloy, J., and Bellocq, M. I. (2009) ‘Landscape and Urban Planning Bird Community Responses along Urban – Rural Gradients: Does the Size of the Urbanized Area Matter? ’, Landscape and Urban Planning, 90: 33–41. Garden, J. et al. (2006) ‘Review of the Ecology of Australian Urban Fauna: A Focus on Spatially Explicit Processes’, Austral Ecology, 31: 126–48 Supplementary data Supplementary data are available at JUECOL online. error df p (Intercept) 18.872 19.42 17.015 0.345 NDVI 41.488 32.767 17.803 0.222 Common myna abundance 5.685 3.216 14.71 0.098 Noisy miner abundance 0.304 4.033 20.532 0.941 Percent ground cover: shrubs 0.525 0.141 15.681 0.002 Total number of trees 0.018 0.038 15.185 0.637 creating additional challenges for conservation management in urban areas (Grarock et al. 2014). Of the two aggressive spe- cies we focused on here, the common myna and noisy miner, the later was more abundant at all spatial scales of the study except in urban sites in NSW. Noisy miners aggression can in- fluence bird community composition at large spatial scales (Grey et al. 1998; Montague-Drake et al. 2011). In contrast, the common myna in experimental studies quantifying aggres- sive interactions around food sources has shown that mynas are less aggressive than many urban adapted native species (Sol et al. 2012). Most of the impact of common myna is re- stricted to competition for natural tree hollows (Pell and Tidemann 1997a; Grarock et al. 2013). Regardless of the Importantly, both the noisy miner and common myna prefer areas of habitat with an open understory, represented in our sites by areas of grass or sealed surfaces (concrete or asphalt). We did not find a significant correlation between these species and shrubs in our study (likely due to the small habitat patch sizes we were working in); however, the pattern of lower myna and miner abundance in areas with more shrubs is supported by previous work (Pell and Tidemann 1997b; Grey et al. 1998). 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https://openalex.org/W4386830342	https://ntb.gpntb.ru/jour/article/download/1187/900	Russian	null	The fundamentals of the book studies (The review of the book “Neurobibliology: The first effort of the cognitive study” by V. P. Leonov)	Naučnye i tehničeskie biblioteki	2,023	cc-by	2,989	БИБЛИОГРАФИИ. ОБЗОРЫ. РЕЦЕНЗИИ УДК 002.2 https://doi.org/10.33186/1027-3689-2023-9-82-91 Scientific and Technical Libraries, 2023, № 9 Ю. Н. Столяров1, 2, 3 Ю. Н. Столяров , , 1Российская государственная библиотека, Москва, Российская Федерация 2Научный и издательский центр «Наука» РАН, Москва, Российская Федерация 3ГПНТБ России, Москва, Российская Федерация yn100@narod.ru, https://orcid.org/0000-0002-9597-4275 1Российская государственная библиотека, Москва, Российская Федерация 2Научный и издательский центр «Наука» РАН, Москва, Российская Федерация 3ГПНТБ России, Москва, Российская Федерация yn100@narod.ru, https://orcid.org/0000-0002-9597-4275 Аннотация. Книга состоит из глав: «Книга как врождённая программа челове- ка», «Книговедение в поисках смыслов: книга как внешний когнитом?», «Книга и музыка как внешний когнитом и вид познания», «О нейробиологии чтения и “книге мозга”», «Нейрофилософия и нейрокниговедение: проблемы на стыке наук», «О междисциплинарности книговедения: от Библиологоса к Нейрокни- говедению», «Нейрокниговедение как междисциплинарное научное направле- ние изучения книги и когнитивных процессов». Рецензент подвергает критическому анализу ключевые положения книги. Утверждение, что «книга – врождённая программа человека», расходится с теорией эволюционного происхождения речи, а также с практикой. На зага- дочный для автора вопрос о том, как именно происходит письменная запись устной речи, дан ответ в документологии: звук представляет собой знак, явля- ется «представителем» мысли. Пользоваться этим знаком для продолжительно- го сохранения изначального смысла невозможно, поэтому пришлось найти ему более подходящий эквивалент. Им стала буква, то есть преобразованный знак звука, или код. Далее развивается концепция книги как внешнего когнитома, то есть эле- мента совокупности биомедицинских, технологических и экзистенциальных – познавательных способностей мозга, что приводит автора к идее представить книговедение в соединении нейронаук и гуманитарного знания. Методологи- ческой основой книговедения и библиографоведения должна стать теория когнитивных систем. Российской академии наук нужен междисциплинарный аналитический центр изучения феномена книги. Для нового научного направ- ления предложен термин «книговедение нейронаук». Кроме того, предлагается Scientific and Technical Libraries, 2023, № 9 82 создать междисциплинарную науку когнитологию, соединяющую в аспекте познания психологию, компьютерную науку, лингвистику, антропологию, нейронауку, философию, педагогику, книговедение и библиографию. Когнитом представляет собой внутренний когнитом, а книга – когнитом внешний. Такая трактовка когнитома совпадает с концепцией К. Поппера о трёх мирах, один из которых – мир знаний, отвлечённых от носителя знания. Ссылка на эту кон- цепцию существенно усилила бы позицию В. П. Леонова. Работа № 720000Ф.99.1.БН60АВ03000 выполнена в рамках государ- ственного задания ГПНТБ России на 2023 г. № 075-01235-23-00 от 29.12.2022 г. на тему № 1021062311368-2-5.8.3 «Развитие электронного библиотековедения как научной и учебной дисциплины в условиях трансфор- мации библиотечных фондов, справочно-библиографического и документного обслуживания в цифровой среде (FNEG-2022-0004)». Ключевые слова: нейрокниговедение, когнитом, К. Поппер, В. П. Леонов, биб- лиографоведение, документология Для цитирования: Столяров Ю. Н. Глубинные основы книговедения (рецензия на книгу В. П. Научные и технические библиотеки, 2023, № 9 BIBLIOGRAPHIES. REVIEWS UDC 002.2 https://doi.org/10.33186/1027-3689-2023-9-82-91 Scientific and Technical Libraries, 2023, № 9 Ю. Н. Столяров1, 2, 3 Леонова «Нейрокниговедение: опыт когнитивного исследова- ния») // Научные и технические библиотеки. 2023. № 9. С. 82–91. https://doi.org/10.33186/1027-3689-2023-9-82-91 83 Научные и технические библиотеки, 2023, № 9 Научные и технические библиотеки, 2023, № 9 The fundamentals of the book studies (The review of the book “Neurobibliology: The first effort of the cognitive study” by V. P. Leonov) Yury N. Stolyarov1, 2, 3 1Russian State Library, Moscow, Russian Federation 2Scientific and Publishing Center “Nauka” of Russian Academy of Sciences, Moscow, Russian Federation 3Russian National Public Library for Science and Technology, Moscow, Russian Federation yn100@narod.ru, https://orcid.org/0000-0002-9597-4275 Yury N. Stolyarov1, 2, 3 1Russian State Library, Moscow, Russian Federation 2Scientific and Publishing Center “Nauka” of Russian Academy of Sciences, Moscow, Russian Federation 3Russian National Public Library for Science and Technology, Moscow, Russian Federation yn100@narod.ru, https://orcid.org/0000-0002-9597-4275 Abstract. The book [in Russian] comprises three chapters, i. e. “The book as a hu- man innate program”, “The bibliology in search of the meanings: The book is an outer cognitome?”, “The bibliology and music as an outer cognitome and form of knowledge”, “On the reading neurobiology and “brain book”, “Neurphilosophy and neurobibliology: The problems at the interface of disciplines”, “On the interdisci- plinarity of bibliology: From Bibliologos to Neurobibliology”, and “Neurbibliology as the interdisciplinary studies of the book and cognitive processes”. The reviewer dissects and analyzes the key provisions of the book. The as- sumption that “the book is the human innate program” contradicts to the speech evolutionary descent theory and to the practice. The documentology also answers to the question surprisingly mysterious to the book author (“what are the origins of the writing?”) – the sound is a sign and “represents” the thought. It is impossi- ble to preserve the initial meaning for the long time so the appropriate equivalent had to be found in the form of the letters as the transformed sound signs, or the code. Further, V. Leonov develops the concept of the book as an outer cognitome, i. e. the element of integrated biomedical, technological, and existential – brain cognitive power which makes him to view the bibliology on the conjunction of neurosciences and the humanities. The theory of cognitive systems has to become the methodological foundation of the bibliology and bibliography, and the Rus- sian Academy of Sciences needs an interdisciplinary analytic center for studies of the book phenomenon. The author suggests the term “bibliology of neurosci- Scientific and Technical Libraries, 2023, № 9 84 ences” for the new discipline. Besides, he suggests that a new interdisciplinary science of cognitology has to integrate psychology, computer science, linguistics, anthropology, neuroscience, philosophy, pedagogy, bibliology, and bibliography in the aspect of knowledge. The cognitome is the innate cognitome while the book is an outer cognitome. This interpretation coincides with that of K. Научные и технические библиотеки, 2023, № 9 The fundamentals of the book studies (The review of the book “Neurobibliology: The first effort of the cognitive study” by V. P. Leonov) Popper’s three worlds, one of which is the world of “products of thought”, the objects in their own right. The reference to this concept would significantly strengthen Leonov’s hand. The paper No. 720000F.99.1.BN60AV03000 is prepared within the frame- work of the Government Order to RNPLS&T for 2023 No. 075-01235-23-00 of December 29, 2022, theme No. 1021062311368-2-5.8.3 “Development of e- librarianship as a scholarly and academic discipline in the context of transfor- mation of library collections, reference bibliographic and document services in the digital environment (FNEG-2022-0004)”. Keywords: neurobibliology, cogntome, Karl Popper, Valery P. Leonov, bibliography, documentology Keywords: neurobibliology, cogntome, Karl Popper, Valery P. Leonov, bibliography, documentology Cite: Stolyarov Y. N. The fundamentals of the book studies (The review of the book “Neurobibliology: The first effort of the cognitive study” by V. P. Leonov) // Scien- tific and technical libraries. 2023. No. 9. P. 82–91. https://doi.org/10.33186/ 1027-3689-2023-9-82-91 Несмотря на эпизодические всплески глубокой теоретической мысли, книговедение возникло и развивается как отчётливо эмпириче- ская наука. Валерий Павлович Леонов предпринял очередную успеш- ную попытку поставить его на высокий абстрактный уровень, включив в область современной нейропсихологии. В недавно изданной книге «Нейрокниговедение: опыт когнитивного исследования» (2023) [1] развиваются идеи, давно занимающие автора и высказанные им в предыдущих публикациях. Общий ход мысли автора прослеживается в самой структуре книги, состоящей из семи глав: «Книга как врождённая программа человека», «Книговедение в поисках смыслов: книга как внешний когнитом?», «Книга и музыка как внешний когнитом и вид познания», «О нейробиологии чтения и “книге мозга”», «Нейрофилосо- фия и нейрокниговедение: проблемы на стыке наук», «О междисци- плинарности книговедения: от Библиологоса к Нейрокниговедению», 85 «Нейрокниговедение как междисциплинарное научное направление изучения книги и когнитивных процессов». Новый труд В. П. Леонова имеет методологическое значение, поскольку посвящён исследованию сущности книги. Проверим же на прочность основные постулаты автора. «Нейрокниговедение как междисциплинарное научное направление изучения книги и когнитивных процессов». Новый труд В. П. Леонова имеет методологическое значение, поскольку посвящён исследованию сущности книги. Проверим же на прочность основные постулаты автора. Книга, по утверждению В. П. Леонова, «устроена по аналогии с мозгом, то есть это гиперсеть» [1. С. 7]. Принимаем это умозаключение как вполне корректное. Но следующее утверждение – «современный человек появился на свет уже с речью» [Там же. С. 9] несколько напря- гает: все известные дети-маугли изначально речью не владели. Происхождение книги В. П. Леонов связывает с происхождением речи. Однако признаем, что речь вторична, она является средством выражения мысли. Сначала должна возникнуть неосязаемая мысль, лишь потом появляется необходимость довести её до окружающих – путём жестов, мимики, позы (первая сигнальная система) или слова (вторая сигнальная система). Речь возникла в процессе эволюции че- ловека как биологического вида, утверждает В. П. Леонов. Этот тезис тоже согласуется с данными науки. Но тогда возникает вопрос: на ка- ком основании книгу (точнее, сначала речь) можно считать космиче- ским субъектом, то есть чем-то привнесённым в сознание человека извне? И если «книга – врождённая программа человека», то это утверждение расходится с теорией эволюционного происхождения речи, а также с практикой: как уже отмечено, дети-маугли в результате отсутствия общения с себе подобными, воспитания, любви и других форм человеческого взаимодействия вместо речи обладают звукопод- ражанием. Научные и технические библиотеки, 2023, № 9 Keywords: neurobibliology, cogntome, Karl Popper, Valery P. Leonov, bibliography, documentology Чем младше возраст, в котором ребёнок оказался в изоля- ции от человеческого сообщества, тем труднее развить в нём челове- ческий интеллект, эмоции и привить нормы поведения. Никакой кос- мической программы во всех известных такого рода случаях не обнаруживается. Зато с эволюционной концепцией возникновения речи синдром Маугли коррелируется отлично. Следующий вопрос, которым задаётся В. П. Леонов, относится к появлению письменности как записи устной речи. Автору такое появ- ление представляется загадочным, поскольку «текст являет слово, а не конкретный мир, который он отражает. Говоря иначе, он обращён не к видимому, а к слышимому. Написанное закрепляет звук, а не форму» [1. С. 10]. В документологии эта загадка разгадана: звук Scientific and Technical Libraries, 2023, № 9 86 представляет собой знак, то есть «материальный предмет (явление, действие, событие), который выступает в процессе коммуникации как представитель другого предмета, свойства или отношения и использу- ется для передачи, переработки и хранения информации» [2]. Иными словами, звук (речи) выступает представителем мысли. Пользоваться этим знаком для продолжительного сохранения изначального смысла невозможно, поэтому пришлось найти ему более подходящий эквива- лент. Им стала буква, то есть преобразованный знак звука. Преобразо- ванный знак иначе именуют кодом. Изобретение буквенного кода в определённый момент эволюционного развития цивилизации – есте- ственное и закономерное явление, позволяющее обойтись без поту- сторонней помощи и разъясняющее то, что кажется загадочным. С идеей о том, что возникновение книги как врождённой про- граммы человека предусмотрено природой, можно согласиться в том смысле, что в геноме человека заложена способность пользоваться кодом (буквенным или иным; сейчас их количество измеряется сотня- ми, а в будущем ещё увеличится). «Процесс постижения смысла книги по внешним проявлениям В. П. Леонов подкрепляет ссылкой на Н. А. Рубакина (1862–1946) и В. П. Зинченко (1931–2014). Построения В. П. Зинченко действительно во многом смыкаются с теорией Н. А. Рубакина. Свойство книги как врождённой программы человека и книги как отчуждённой от челове- ка сущности должно исследоваться, по убеждению В. П. Леонова, пси- хологией. Но она, по его словам, к этому пока не готова, и автор уве- рен, что в таком случае следует призвать на помощь искусство – преж- де всего поэзию и музыку. Делает он это, как всегда, весьма убеди- тельно. 87 Научные и технические библиотеки, 2023, № 9 Научные и технические библиотеки, 2023, № 9 Далее подробно развивается концепция книги как внешнего ко- гнитома (по академику К. В. Анохину (род. Keywords: neurobibliology, cogntome, Karl Popper, Valery P. Leonov, bibliography, documentology 1957), элемента совокупно- сти – биомедицинских, технологических и экзистенциальных – позна- вательных способностей мозга), что приводит к идее изучения книго- ведения в соединении средств нейронаук и гуманитарного знания. Это обусловлено тем, что «мозг не только обрабатывает информацию, он же её и создаёт. Книга как внешний когнитом – один из результатов такого создания» [1. С. 19]. В таком случае, по В. П. Леонову, методоло- гической основой книговедения и библиографоведения (библиотеко- ведение почему-то опущено) должна стать теория когнитивных систем. Что же, сказано убедительно. К массиву книг, частным случаем которо- го является библиотечный фонд, вполне приложимы понятия гиперсе- тевой теории мозга. Дальше автор возвращается к тому, что книга в каждом из нас уже есть, и автору остаётся «её лишь перевести в про- цессе чтения» [Там же. С. 21]. Как это сделать, неизвестно, однако вновь возникает аллюзия c библиопсихологией Н. А. Рубакина – с той разницей, что Рубакин более убедителен: снятие противоречий между «внешним когнитомом и субъективным опытом читателя» он возлагал на библиотекаря. Действительно, автор-то лишён возможности выйти на читателя и повлиять на него. Одних только нейронаук для изучения феномена книги, по убеж- дению В. П. Леонова, недостаточно, их надо подкрепить знанием фило- софии, социологии, лингвистики, цифровых методов гуманитарных наук. Какие конкретно знания имеются в виду, автор оставляет за скоб- ками, он ограничивается предложением создать в структуре Россий- ской академии наук соответствующий междисциплинарный аналитиче- ский центр. Правда, в главе «Нейрофилософия и нейрокниговедение: проблемы на стыке наук» подсказка даётся: проблема заключается в том, чтобы понять и объяснить, как сознание связано с мозгом. При этом область сознания определяется автором как «своего рода ме- тасфера по отношению к другим возможным сферам научного рас- смотрения (философии, социологии, педагогики, книговедения, биб- лиографии)» [Там же. С. 42]. С точки зрения автора, в исследование сознания большой вклад могут внести книговеды, поскольку при чте- нии в сознании происходит перегруппировка нейронных сетей. Пред- ложен и термин для этого нового научного направления: «книговеде- Scientific and Technical Libraries, 2023, № 9 88 ние нейронаук». В главе «О нейробиологии чтения и “книге мозга”» со ссылкой преимущественно на современные зарубежные исследования обращено внимание на возможность понять содержание происходя- щих в мозгу процессов по физическим показаниям. В конечном счёте возникает потребность в создании междисциплинарной науки когнито- логии, соединяющей в аспекте познания психологию, компьютерную науку, лингвистику, антропологию, нейронауку, философию, педагогику, книговедение и библиографию [1. С. 45]. В этот ряд «просятся» также гносеология, эпистемология, не говоря о документологии, которую В. П. Леонов почему-то во внимание не принимает. Научные и технические библиотеки, 2023, № 9 Keywords: neurobibliology, cogntome, Karl Popper, Valery P. Leonov, bibliography, documentology Демонстрируя осведомлённость о существовании концепции Библиологоса («разума мира книг»), автор ограничивается самым кратким изложением её су- щества, даже не высказывая отношения к ней и не связывая со своей генеральной идеей, после чего вновь возвращается к междисципли- нарности, обходящейся без идеи Библиологоса и Библиосферы. Отвлёкшись от оценки сомнительного тезиса, что разум – надстройка над мозгом как физической материей [Там же. С. 57], со- средоточусь на особенно дорогой для В. П. Леонова мысли: если когни- том представляет собой материю сознания, или внутренний когнитом, то книга – это когнитом внешний. Такая трактовка когнитома совпада- ет с концепцией трёх миров К. Поппера (1902–1994), один из кото- рых – мир знаний, отвлечённых от носителя знания. Ссылка на эту концепцию существенно усилила бы позицию В. П. Леонова, тем более что, судя по другим его публикациям, труды этого выдающегося фило- софа и социолога Валерию Павловичу хорошо известны и высоко им оцениваются. В документологии они рассматриваются как базисная методологическая основа, предшествующая взглядам классика этой науки П. Отле (1868–1944) [3]. В документологии дан ответ на основной вопрос, сформулирован- ный В. П. Леоновым в самом конце книги [1. С. 63]: «Где кончается буквенный код и начинается собственно информация?» Вопрос этот некорректен в самой постановке: Книга как частный случай Документа в общем случае представляет собой единство идеальной информации и её материального (не только кодового) воплощения. Документология дозрела до понимания того, что информация составляет сущность до- кумента, но сущность эта проявляется, среди прочего, в виде знаков. 89 Напомню, что преобразованный знак (так сказать, «знак знака») есть код. Поставленный автором сакраментальный вопрос документология формулирует несколько иначе и тем снимает его: «Где кончается бук- венный код, там кончается и передаваемая им информация». Или ина- че: «Собственно информация начинается там же, где начинается бук- венный код, призванный визуализировать, материализовать неосязае- мую, невесомую, невидимую, вообще иначе никак не воспринимаемую информацию». Пожеланием углубиться в фундаментальные основы документоло- гии – явно близкой к книговедению дисциплины, тем более что В. П. Леонов признаёт междисциплинарность книговедения, – и хоте- лось бы закончить эту статью. P. S. Валерию Павловичу Леонову как библиографу экстра-класса удалось разыскать действительный источник, в котором опубликована блестящая мысль Л. Н. Толстого о том, что суть книги вечна и состоит она в увековечении мысли посредством видимых знаков. В каких бы то ни было других ограничениях в понимании книги Л. Н. Толстой необ- ходимости не видит. Более того, В. П. Леонов полностью приводит факсимиле статьи «Книги» и тем делает бесценный подарок всем книговедам и книголю- бам. Keywords: neurobibliology, cogntome, Karl Popper, Valery P. Leonov, bibliography, documentology За это Валерию Павловичу отдельное спасибо! 1. Леонов В. П. Нейрокниговедение: опыт когнитивного исследования / Библиотека Российской академии наук. Санкт-Петербург : БАН, 2023. 79 с. 2. 3.1.1 знак // ГОСТ 7.0-99. Информационно-библиотечная деятельность, библиография. Термины и определения. URL: http://docs.cntd.ru (дата обращения: 06.07.2023). 3. Столяров Ю. Н. Документология: причины появления, этапы развития // Научные и технические библиотеки. 2021. № 1. С. 15–26. https://doi.org/10.33186/1027-3689- 2021-1-15-26. References 1. Leonov V. P. Nei`roknigovedenie: opy`t kognitivnogo issledovaniia / Biblioteka Rossii`skoi` akademii nauk. Sankt-Peterburg : BAN, 2023. 79 s. 2. 3.1.1 znak // GOST 7.0-99. Informatcionno-bibliotechnaia deiatel`nost`, bibliografiia. Terminy` i opredeleniia. URL: http://docs.cntd.ru (data obrashcheniia: 06.07.2023). 2. 3.1.1 znak // GOST 7.0-99. Informatcionno-bibliotechnaia deiatel`nost`, bibliografiia. Terminy` i opredeleniia. URL: http://docs.cntd.ru (data obrashcheniia: 06.07.2023). 3. Stoliarov Iu. N. Dokumentologiia: prichiny` poiavleniia, e`tapy` razvitiia // Nauchny`e i tekhnicheskie biblioteki. 2021. № 1. S. 15–26. https://doi.org/10.33186/1027-3689-2021- 1-15-26. Список источников 1. Леонов В. П. Нейрокниговедение: опыт когнитивного исследования / Библиотека Российской академии наук. Санкт-Петербург : БАН, 2023. 79 с. 2. 3.1.1 знак // ГОСТ 7.0-99. Информационно-библиотечная деятельность, библиография. Термины и определения. URL: http://docs.cntd.ru (дата обращения: 06.07.2023). 3. Столяров Ю. Н. Документология: причины появления, этапы развития // Научные и технические библиотеки. 2021. № 1. С. 15–26. https://doi.org/10.33186/1027-3689- 2021-1-15-26. Scientific and Technical Libraries, 2023, № 9 90 Научные и технические библиотеки, 2023, № 9 Информация об авторе / Information about the author Столяров Юрий Николаевич – доктор пед. наук, профессор, главный научный сотрудник Российской государственной библиотеки, Научного и издатель- ского центра «Наука» РАН, ГПНТБ России, заслуженный работник высшей школы Российской Федерации, Москва, Российская Федерация yn100@narod.ru Столяров Юрий Николаевич – доктор пед. наук, профессор, главный научный сотрудник Российской государственной библиотеки, Научного и издатель- ского центра «Наука» РАН, ГПНТБ России, заслуженный работник высшей школы Российской Федерации, Москва, Российская Федерация yn100@narod.ru Yury N. Stolyarov – Dr. Sc. (Pedagogy), Professor, Chief Re- searcher, Russian State Library; Chief Researcher, Scientific and Publishing Center “Nauka” of Russian Academy of Sciences; Chief Researcher, Russian National Public Library for Science and Technology, Moscow, Russian Federation yn100@narod.ru 91 Научные и технические библиотеки, 2023, № 9 Научные и технические библиотеки, 2023, № 9
https://openalex.org/W2603315599	https://www.revistas.usp.br/clinics/article/download/130844/127283	English	null	Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma	Clinics	2,017	cc-by	3,534	Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma Omer Bilgehan Poyrazoglu,I Ahmet Cumhur Dulger,II,* Bilge Sumbul GultepeIII I Lokman Hekim Hospital, General Surgery, Van, Turkey. II Yuzuncu Yil University, School of Medicine, Department of Gastroenterology, Van, Turkey. III Bezmialem Vakif University, School of Medicine, Microbiology, Istanbul, Turkey. OBJECTIVE: Esophageal squamous cell carcinoma is one of the most common esophageal diseases in the developing world, but the relationship between esophageal squamous cell carcinoma and Helicobacter pylori infection remains a neglected topic. The primary objective of this study was to determine the association between Helicobacter pylori infection and esophageal squamous cell carcinoma. A second purpose was to determine the incidence and factors associated with Helicobacter pylori infection following esophagectomy. METHOD: The microorganism was identiﬁed by testing the gastric biopsy materials from 95 esophageal squamous cell carcinoma patients (66 females; 39 were esophagectomized) for urease activity in a medium containing urea and a power of hydrogen detection reagent and comparing the results with those from a healthy population. Differences in patient characteristics were assessed with chi-square tests and t-tests for categorical and continuous factors, respectively. RESULTS: The patients with esophageal squamous cell carcinoma had a signiﬁcantly lower prevalence of Helicobacter pylori compared with the healthy population (po0.001). The naive and esophagectomized patients, in contrast, showed no signiﬁcant differences in Helicobacter pylori infection (p40.005). Patients with esophageal squamous cell carcinoma showed a signiﬁcant association between leukocytosis and hypoglobu- linemia and the presence of Helicobacter pylori infection (p=0.023 and p=0.045, respectively). CONCLUSION: These results suggest that Helicobacter pylori is not an etiological factor in patients with esophageal squamous cell carcinoma. We found a statistically signiﬁcant negative correlation between esoph- ageal squamous cell cancer and Helicobacter pylori infection. These ﬁndings may guide new strategies for esophageal squamous cell carcinoma therapy. KEYWORDS: Helicobacter pylori; Esophageal Squamous Cell Carcinoma; Turkey. KEYWORDS: Helicobacter pylori; Esophageal Squamous Cell Carcinoma; Turkey. Poyrazoglu OB, Dulger AC, Gultepe BS.Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma. Clinics. 2017; 72(3):150-153 Poyrazoglu OB, Dulger AC, Gultepe BS.Helicobacter Pylory infection in patients with esophageal squamous cell carcinoma. Clinics. 2017; 72(3):150-153 Received for publication on July 12, 2016; First review completed on October 10, 2016; Accepted for publication on December 19, 2016 Corresponding author. E-mail: acdulger@gmail.com Received for publication on July 12, 2016; First review completed on October 10, 2016; Accepted for publication on December 19, 2016 Corresponding author. E-mail: acdulger@gmail.com *Corresponding author. E-mail: acdulger@gmail.com CLINICAL SCIENCE CLINICAL SCIENCE ’ INTRODUCTION cancer (in 0.1 to 3%), and gastric mucosa-associated lym- phoid-tissue (MALT) lymphoma (in o0.01%) (4). Recent studies have demonstrated a high frequency of HP infection in gastric cancer patients, suggesting that presence of HP may function as a driver of the events contributing to oncogen- esis in gastric adenocarcinomas (5,6). Furthermore, an inverse association has been established between Cag A-positive HP infection and the risk of esophageal adenocarcinoma (7). HP infection plays crucial roles in gastric carcinogenesis; however, the impact of HP on ESCC is not well understood. Esophageal squamous cell carcinoma (ESCC), one of the most aggressive digestive system tumors, is associated with numer- ous factors, including advanced age, achalasia, Plummer-Vinson syndrome, low socioeconomic status, high-starch diets lacking in fruits and vegetables, alcohol abuse, tobacco use, previous head and neck squamous cell carcinoma, and radiation ther- apy (1). It is also an important cause of mortality in the Asian esophageal cancer belt and in the eastern part of Turkey (2). p g p y Helicobacter pylori (HP), a gram-negative bacterium found on the gastric mucosa, was first isolated 30 years ago (3). Infection with HP is one cause of duodenal or gastric ulcers (reported to develop in 1 to 10% of infected patients), gastric The available data, derived from studies using serologic tests, are conflicting with respect to any association between HP and ESCC (8-10). The aim of the present study was to examine the potential correlation between ESCC and HP infection and to compare the presence of HP in naive ESCC patients and their esophagectomized counterparts. Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No potential conﬂict of interest was reported. DOI: 10.6061/clinics/2017(03)04 ’ DISCUSSION Our study revealed a strong association between HP infec- tion and a reduced risk of ESCC. In the Turkish adult popula- tion, the prevalence of HP infection is higher than reported in Western countries. A previous investigation of 4622 Turkish subjects indicated an HP infection prevalence of 82.5% (12). The prevalence of HP infection in the present study was ’ RESULTS endemic. In total, 95 ESCC patients (65 women, aged 32- 92 years) were evaluated in our clinic from July 2012 to July 2015. All the patients were diagnosed with ESCC based on established endoscopic and histopathological criteria. Of these, 39 had undergone a subtotal esophagectomy. In this esophagectomy group, esophageal reconstruction was per- formed through a subcutaneous route in 10 patients, through a retrosternal route in 25, and through a posterior mediast- inal route in 4. The reconstructed esophagus was a wide gastric tube, as described by Holscher (11). The mean age was 52.96±11.81 years in the control group (100 women), 59.53±13.93 years in the naive ESCC group (36 females), and 55.95±11.53 years in esophagectomized group (28 females). Nearly two-thirds of the ESCC patients were female. Descriptive statistics and comparison results according to the presence of HP are shown in Table 1. g p HP infection was observed in 39 (68.4%) of the 57 naive ESCC patients, 27 (69.2%) of the 39 esophagectomized patients, and 128 (85%) of the 151 dyspeptic control patients (Figure 2). We found a significantly lower rate of HP in patients with ESCC compared with the dyspeptic subjects (po0.0001), whereas no statistically significant difference was detected between the naive ESCC patients and their esopha- gectomized counterparts (p40.005). We also found no gender differences between the groups (p40.0005). Postoperatively, the patients typically underwent upper gastrointestinal endoscopy 6 weeks after surgery to deter- mine the presence or absence of HP infection. p Gastric biopsy samples were tested for urease activity using a commercial Hp-fast test kit (GI Supplys, Camp Hill, PA, USA) consisting of a urea-containing medium and a power of hydrogen (pH) detection reagent. g p Significantly higher levels of leukocytes and serum glob- ulin were also found in the ESCC patients diagnosed with HP compared with those without HP infection (p=0.023 for leukocytes and p=0.045 for serum globulin). p y g p g The control group comprised 151 dyspeptic subjects (100 women and 51 men, aged 30–85 years). Controls were also required to be medical-treatment free for at least 6 months from the time of study entry. Antrum biopsies with normal endoscopic evaluations were examined for HP using the same method. The prognostic values of the presence of HP and other clinicopathologic factors were also evaluated. ’ PATIENTS AND METHODS This retrospective trial was conducted at a university medical center in a large metropolitan area near the Iranian border of Turkey, where both ESCC and HP infection are DOI: 10.6061/clinics/2017(03)04 150 CLINICS 2017;72(3):150-153 CLINICS 2017;72(3):150-153 HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. CLINICS 2017;72(3):150-153 similar (85%). Squamous cell carcinomas are usually detected in the proximal two-thirds of the esophagus. They predomi- nantly affect elderly people and usually present as dysphagia, odynophagia, and unintentional weight loss (13). Worldwide, esophageal cancer ranks fifth in mortality among all malig- nancies, and ESCC remains the most common type. cancers are the most prevalent malignancies in both females and males in eastern part of Turkey (16). The probable culprit factors for ESCC in this region are low educational and socioeconomic status; the consumption of smoked, salted, hot, fatty foods; overconsumption of hot tea; cigarette smok- ing; poor intake of fresh fruits and vegetables, and poor hygienic conditions (17). Previous studies have shown that the eastern part of Turkey has one of the highest rates of both ESCC and HP infection. An HP infection rate of 36% was reported for gastric biopsy specimens in patients with gastric carcinoma from the Van region (18). cancers are the most prevalent malignancies in both females and males in eastern part of Turkey (16). The probable culprit factors for ESCC in this region are low educational and socioeconomic status; the consumption of smoked, salted, hot, fatty foods; overconsumption of hot tea; cigarette smok- ing; poor intake of fresh fruits and vegetables, and poor hygienic conditions (17). Previous studies have shown that the eastern part of Turkey has one of the highest rates of both ESCC and HP infection. An HP infection rate of 36% was reported for gastric biopsy specimens in patients with gastric carcinoma from the Van region (18). In Asia, upper gastrointestinal cancers constitute a major group of malignancies with high rates of morbidity and mortality. The esophageal cancer belt, originating in the Far East and extending through middle Asia and the Near East, encompasses many countries, including northern China, northern Iran and the eastern part of Turkey (14). The pre- dominant histopathological type of esophageal cancer is the squamous cell type in the endemic Asian regions, with incidence rates that may vary 200-fold among different populations within the same defined region due to cultural practices. More than 80% of ESCC patients in the rural areas of Asia also present at advanced stages that are not amenable to curative therapies; hence the need for novel preventive strategies is urgent (15). CLINICS 2017;72(3):150-153 g g ( ) Various environmental factors, including cigarette smok- ing and excessive alcohol intake, can be associated with an increased risk of developing ESCC (19). HP is considered one of the most important human carcinogens for the upper gastrointestinal tract and the stomach (20). At present, a few reports have indicated a possible relationship between HP and ESCC, but most of these were performed using non- endoscopic (serologic) techniques (7). A recent Chinese study reported an HP seropositivity of 35.3% in ESCC patients, which was lower than that of the control groups (40% and 59%) (21). A recent meta-analysis also found an association between CagA-negative HP strains and a marginally signif- icant increased risk of ESCC (7). In contrast, a prospective and serological study from China showed no association between HP and ESCC (22). Another recent meta-analysis from China showed an association between HP infection and a decreased risk of ESCC in Eastern populations and a decreased risk of esophageal adenocarcinoma (EAC) in the overall population (23). The Van region of eastern Turkey is located in the western end of the esophageal cancer belt. Esophageal and gastric Figure 1 - Traditional large oven (Tandir). p p The current study revealed no association between HP infection and ESCC among people living in the eastern part of Turkey. The low prevalence of HP in these ESCC patients was similar to that reported in the Chinese meta-analyses. We also found a predominance of ESCC in female patients, in agreement with a previous Turkish study (17). This phe- nomenon has been linked to the use of the traditional large ovens (tandirs) that use smoke to cook meals (Figure 1). The mechanisms by which these ovens induce esophageal carci- nogenesis remain undefined, but previous work suggests that smoke may play role similar to that of cigarette smoking (17). We conclude that an effective follow-up strategy for HP-negative female Asian adults will be necessary if ESCC screening is to yield public health benefits. The higher levels of leucocytes and serum globulin among ESCC patients with HP may also reflect an emerging phenomenon that requires additional investigation to determine the underlying causa- tive factors. Figure 1 - Traditional large oven (Tandir). Figure 1 - Traditional large oven (Tandir). Figure 2 - Rate of HP according to groups. The H. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. CLINICS 2017;72(3):150-153 Statistical analysis h The two groups were compared using the Mann-Whitney U-test. The differences were considered statistically signifi- cant at po0.005. Table 1 - Descriptive statistics and comparison results according to Helicobacter pylori status. HP1 n Mean Std. Dev Min. Max. p Age (years) + 65 57.02 11.514 32 92 0.239 - 30 60.43 15.939 23 87 Total 95 58.09 13.084 23 92 Hemoglobin (gr/dL) + 63 12.50 2.025 8 17 0.371 - 30 12.11 1.876 9 17 Total 93 12.38 1.976 8 17 Hematocrit + 63 37.20 5.616 24 49 0.427 - 30 36.21 5.502 28 49 Total 93 36.88 5.569 24 49 Leukocytes (/mm3) + 63 7.598 3.8817 1.8 25.0 0.023 - 30 5.803 2.4689 2.0 11.0 Total 93 7.019 3.5760 1.8 25.0 Platelets (/mm3) + 63 270.000 123.839 67.000 702.000 0.150 - 30 616.687 324.542 79.000 178.000 Total 93 443.343 184.309 67.000 178.000 ALT2 (U/L) + 63 18.94 17.766 6 142 0.284 - 30 15.27 8.026 6 39 Total 93 17.75 15.362 6 142 AST3 (U/L) + 62 33.02 48.837 2 341 0.089 - 30 17.57 6.976 9 38 Total 92 27.98 40.833 2 341 Albumin (g/dL) + 58 3.71 0.734 2 5 0.882 - 25 3.69 0.667 2 5 Total 83 3.71 0.711 2 5 Globulin (g/dL) + 47 3.07 0.533 2 4 0.045 - 19 2.73 0.772 1 4 Total 66 2.97 0.625 1 4 Calcium (mg/dL) + 57 8.92 1.362 1 13 0.370 - 27 9.17 0.639 8 11 Total 84 9.00 1.181 1 13 Std. Dev: Standard deviation 1 Helicobacter pylori, 2 Alanine transaminase, 3 Aspartate transaminase Table 1 - Descriptive statistics and comparison results according to Helicobacter pylori status. tistics and comparison results according to Helicobacter pylori status. 151 HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. infection status changed from preoperatively positive to postoperatively negative and that this changing pattern was linked to the eradication of HP via the perioperative administration of antibiotics (25). Conversely, a report from China showed a low incidence of HP infection in the gastric conduit in patients who underwent esophagectomy, pylor- oplasty, and reconstruction. The authors of that study con- cluded that this phenomenon was mostly due to the chronic reflux of bile after pyloroplasty (26). carcinoma in a Taiwanese population. Am J Gastroenterol. 2005;100 (3):588-93, http://dx.doi.org/10.1111/j.1572-0241.2005.40623.x. carcinoma in a Taiwanese population. Am J Gastroenterol. 2005;100 (3):588-93, http://dx.doi.org/10.1111/j.1572-0241.2005.40623.x. p g j 10. Henrik Simán J, Forsgren A, Berglund G, Floren CH. Helicobacter pylori infection is associated with a decreased risk of developing oesophageal neoplasms. Helicobacter. 2001;6(4):310-6, http://dx.doi.org/10.1046/j.1523- 5378.2001.00041.x. 11. Holscher AH, Voit H, Buttermann G, Siewert JR. Function of the intra- thoracic stomach as esophageal replacement. World J Surg. 1998;12(6): 835-44, http://dx.doi.org/10.1007/BF01655491. p g 12. Ozaydin N, Turkyilmaz SA, Cali S. Prevalence and risk factors of Heli- cobacter pylori in Turkey: a nationally-representative, cross-sectional, screening with the 13C-Urea breath test. BMC Public Health. 2013;13:1215, http://dx.doi.org/10.1186/1471-2458-13-1215. Our findings were in concordance with previous reports on HP status and gastric tube cancer patients. In our Turkish population, HP infection was not associated with ESCC and had a similar pattern to that reported for Asian populations. HP infection may not contribute to the development of ESCC in patients who reside in rural areas of Asia, especially not in females. Furthermore, esophageal damage could be dimin- ished by the prior presence of HP in areas with a high risk of ESCC. HP treatment has been associated with impro- ved thrombocyte levels. An increase in platelet counts was observed in only 6.7% of treated patients. In the current study, we found no relationship between HP status and thrombocyte levels (27,28). p g 13. Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med. 2003;349(23): 2241-52, http://dx.doi.org/10.1056/NEJMra035010. p g 14. Lambert R, Hainaut P. The multidisciplinary management of gastro- intestinal cancer. Epidemiology of oesophagogastric cancer. Best Pract Res Clin Gastroenterol. 2007;21(6):921-45, http://dx.doi.org/10.1016/ j.bpg.2007.10.001. j pg 15. Ke L. Mortality and incidence trends from esophagus cancer in selected geographic areas of China circa 1970-90. Int J Cancer. 2002;102(3):271-4, http://dx.doi.org/10.1002/ijc.10706. p g j 16. Koca T, Arslan D, Basaran H, Cerkesli AK, Tastekin D, Sezen D, et al. CLINICS 2017;72(3):150-153 CLINICS 2017;72(3):150-153 ’ AUTHOR CONTRIBUTIONS y J ( ) 19. Lambert R, Hainaut P. Esophageal cancer: cases and causes (part I). Endoscopy. 2007;39(6):550-5, http://dx.doi.org/10.1055/s-2007-966530. Poyrazoglu OB was involved in developing the concept and design of the study and writing the manuscript. Dulger AC was involved in imple- menting the study and collecting data. Dulger AC and Gultepe BS were involved in collecting and processing the data. All the authors read and approved the ﬁnal manuscript. py p g 20. Goodwin CS, Mendall MM, Northfield TC. Helicobacter pylori infection. Lancet. 1997;349(9047):265-9, http://dx.doi.org/10.1016/S0140-6736(96) 07023-7. 21. Wu IC, Wu DC, Yu FJ, Wang JY, Kuo CH, Yang SF, et al. Association between Helicobacter pylori seropositivity and digestive tract cancers. World J Gastroenterol. 2009;15(43):5465-71, http://dx.doi.org/10.3748/ wjg.15.5465. jg 22. Kamangar F, Qiao YL, Blaser MJ, Sun XD, Katki H, Fan JH, et al. Heli- cobacter pylori and oesophageal and gastric cancers in a prospective study in China. Br J Cancer. 2007;96(1):172-6, http://dx.doi.org/10.1038/ sj.bjc.6603517. HP in esophageal squamous cell carcinoma Poyrazoglu OB et al. Dietary and demographical risk factors for oesophageal squamous cell carcinoma in the Eastern Anatolian region of Turkey where upper gas- trointestinal cancers are endemic. Asian Pac J Cancer Prev. 2015;16(5): 1913-7, http://dx.doi.org/10.7314/APJCP.2015.16.5.1913. p g J 17. Turkdogan MK, Akman N, Tuncer I, Uygan I, Kösem M, Ozel S, et al. Epidemiological aspects of endemic upper gastrointestinal cancers in eastern Turkey. Hepatogastroenterology. 2005;52(62):496-500. y Finally, further studies are needed to address the impact of HP infection on ESCC and its natural history. y Finally, further studies are needed to address the impact of HP infection on ESCC and its natural history. y p g gy 18. Türkdog˘an K, Alici S, I˙lhan M, Dilek H, Akman E, Ayakta H, et al. Helicobacter pylori infection in gastric carcinoma in the Van region of Turkey. Turk J Gastroenterol. 1999;10(1):36-9. CLINICS 2017;72(3):150-153 pylori-fast test (Hp-fast test) is based on the detection of HP urease activity and has a high sensitivity (85%) and specificity (495%) for detecting HP infection. As this test is considered cost-effective and suitable for endoscopy units (24), we used the Hp-fast test to establish HP infection. The current data are limited regarding any changes in the prevalence of HP in patients who have undergone esopha- gectomy. We therefore performed gastroduodenoscopy with pathological examination of the biopsy specimens obtained from the gastric conduit. We observed that the rate of HP infection was lower in esophagectomized ESCC patients than in the control subjects (19% and 78%, respectively; po0.001). The rate of HP infection was similar, however, between the naive ESCC patients and the esophagectomized patients (p40.005). A Japanese study reported that the HP Figure 2 - Rate of HP according to groups. 152 ’ REFERENCES 1. Layke JC, Lopez PP. Esophageal cancer: a review and update. Am Fam Physician. 2006;73(12):2187-94. j j 23. Xie FJ, Zhang YP, Zheng QQ, Jin HC, Wang FL, Chen M, et al. Helico- bacter pylori infection and esophageal cancer risk: an updated meta- analysis. World J Gastroenterol. 2013;19(36):6098-107, http://dx.doi.org/ 10.3748/wjg.v19.i36.6098. y 2. Onuk MD, Oztopuz A, Memik F. Risk factors for esophageal cancer in eastern Anatolia. Hepatogastroenterology. 2002;49(47):1290-2. p g gy 3. Unidentified curved bacilli on gastric epithelium in active chronic gas- tritis. Lancet. 1983;1(8336):1273-5. jg 24. Onders RP. Detection methods of Helicobacter pylori: accuracy and costs. Am Surg. 1997;63(8):665-8. 4. Vakil N, Malfertheiner P, Chey WD. Helicobacter pylori infection. N Engl J Med. 2010;363(6):595, http://dx.doi.org/10.1056/NEJMc1006158. g 25. Mori N, Fujita H, Sueyoshi S, Aoyama Y, Yanagawa T, Shirouzu K. Helicobacter pylori infection influences the acidity in the gastric tube as an esophageal substitute after esophagectomy. Dis Esophagus. 2007;20(4): 333-40, http://dx.doi.org/10.1111/j.1442-2050.2007.00718.x. J ( ) p g J 5. Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med. 2002;347(15):1175-86, http://dx.doi.org/10.1056/NEJMra020542. p g 6. Peek RM Jr, Blazer MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. 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Helicobacter pylori infection and gastric atrophy: risk of adenocarcinoma and squamous-cell carcinoma of the esophagus and adenocarcinoma of the gastric cardia. J Natl Cancer Inst. 2004;96(5):388-96, http://dx.doi. org/10.1093/jnci/djh057. 28. Sultan S, Irfan SM, Kaker J, Hasan M. Efficacy of helicobacter pylori eradication as an upfront treatment of secondary immune thrombocyto- penia: an experience from Pakistan. Med J Malaysia. 2016;71(2):53-6. g j j 9. Wu DC, Wu IC, Lee JM, Hsu HK, Kao EL, Chou SH, et al. Helicobacter pylori infection: a protective factor for esophageal squamous cell 153
https://openalex.org/W4282831217	https://www.nature.com/articles/s41598-022-14484-4.pdf	English	null	Author Correction: Exceptional longevity in northern peripheral populations of Wels catfish (Siluris glanis)	Scientific reports	2,022	cc-by	523	www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: Exceptional longevity in northern peripheral populations of Wels catfish (Siluris glanis) OPEN Kristofer Bergström, Oscar Nordahl, Peter Söderling, Per Koch‑Schmidt, Tobias Borger, Petter Tibblin & Per Larsson Correction to: Scientific Reports https://doi.org/10.1038/s41598-022-12165-w, published online 16 May 2022 The original version of this Article contained a repeated error where Silurus glanis was incorrectly written as Siluris glanis in the title, “Exceptional longevity in northern peripheral populations of Wels catfish (Siluris glanis)” now reads: “Exceptional longevity in northern peripheral populations of Wels catfish (Silurus glanis)” In the Introduction, 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 Author(s) 2022 now reads: “Here, we investigate growth trajectories and longevity in northern peripheral populations of Wels catfish (Silurus glanis, hereafter catfish).” In the Reference list, 17. IUCN (International Union for Conservation of Nature) 2008. Siluris glanis. The IUCN Red List of Threatened Species. Version 2021–3 (2010). https://www.iucnredlist.org. (Accessed 25 February 2021). 33. Alp, A., Kara, C. & Büyükcapar, H. M. Reproductive biology in a Native European Catfish, Siluris glanis L., 1758, population in Menzelet Resevoir. Turk. J. Vet. Ani. Sci. 28, 613 (2004). 17. IUCN (International Union for Conservation of Nature) 2008. Silurus glanis. The IUCN Red List of Threatened Species. Version 2021–3 (2010). https://www.iucnredlist.org. (Accessed 25 February 2021). 33. Alp, A., Kara, C. & Büyükcapar, H. M. Reproductive biology in a Native European Catfish, Silurus glanis L., 1758, population in Menzelet Resevoir. Turk. J. Vet. Ani. Sci. 28, 613 (2004). The original Article has been corrected. \| https://doi.org/10.1038/s41598-022-14484-4 Scientific Reports \| (2022) 12:9812 Scientific Reports \| (2022) 12:9812 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 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 Author(s) 2022 https://doi.org/10.1038/s41598-022-14484-4 Scientific Reports \| (2022) 12:9812 \|
https://openalex.org/W4289979090	https://www.biorxiv.org/content/biorxiv/early/2022/08/05/2022.08.04.502722.full.pdf	English	null	Geometric variation of the human tibia-fibula: A public dataset of tibia-fibula surface meshes and statistical shape model	bioRxiv (Cold Spring Harbor Laboratory)	2,022	cc-by	16,799	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Abstract 17 45 Conclusion. Important variations that could increase the risk of tibial stress injury w 46 observed in the tibia and tibia-fibula SSM. These included general tibial thickness, mids 47 thickness, tibial length and medulla cavity diameter (indicative of cortical thickness). Fur 48 research is warranted to better understand the effect of these tibial and fibula sh 49 characteristics on tibial stress, loading and injury risk. This SSM and associated code has b 50 provided in an open-source dataset. The associated code includes three example applications 51 generation of a random sample; (ii) reconstruction of trabecular surfaces; and (iii) reconstruc 52 from palpable landmarks. The developed tibial surface models and statistical shape model wil 53 made available for use at: https://simtk.org/projects/ssm_tibia. 54 55 Background. Variation in tibia geometry is a risk factor for tibial stress fractures. Geometric 18 variability in bones is often quantified using statistical shape modelling. Statistical shape models 19 (SSM) offer a method to assess three-dimensional variation of structures and identify the source 20 of variation. Although SSM have been used widely to assess long bones, there is limited open- 21 source datasets of this kind. Overall, the creation of SSM can be an expensive process, that 22 requires advanced skills. A publicly available tibia shape model would be beneficial as it enables 23 researchers to improve skills. An opensource SSM could benefit health, sport and medicine with 24 the potential to assess geometries suitable for medical equipment, and aid in clinical diagnosis. 25 This study aimed to: (i) quantify tibial geometry using a SSM; and (ii) provide the SSM and 26 associated code as an open-source dataset. 27 Methods. Lower limb computed tomography (CT) scans from the right tibia-fibula of 30 28 cadavers (male n = 20, female n =10) were obtained from the New Mexico Decedent Image 29 Database. Tibias were segmented and reconstructed from the CT scans into both cortical and 30 trabecular sections. Fibulas were segmented as a singular surface. The segmented bones (cortical 31 and trabecular tibia; and fibula) were used to develop three SSM of the: (i) tibia; (ii) combined 32 tibia and fibula; and (iii) tibial trabecular. Principal component analysis (PCA) was applied to 33 obtain the three SSM, with the principal components (PCs) that explained 95% of the geometric 34 variation retained. 35 Results. Geometric variation of the human tibia-fibula: A public 1 dataset of tibia-fibula surface meshes and statistical shape 2 model 3 Meghan Keast1, Jason Bonacci1, Aaron Fox1 4 5 1 Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University 6 75 Pigdons Road, Waurn Ponds, 3216, VIC, Australia 7 8 9 10 Corresponding Author: 11 Meghan Keast1 12 Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University 13 75 Pigdons Road, Waurn Ponds, 3216, VIC, Australia 14 Email address: mf.keast@gmail.com 15 16 Geometric variation of the human tibia-fibula: A public 1 dataset of tibia-fibula surface meshes and statistical shape 2 model 3 Meghan Keast1, Jason Bonacci1, Aaron Fox1 4 5 1 Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University 6 75 Pigdons Road, Waurn Ponds, 3216, VIC, Australia 7 8 9 10 Corresponding Author: 11 Meghan Keast1 12 Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University 13 75 Pigdons Road, Waurn Ponds, 3216, VIC, Australia 14 Email address: mf.keast@gmail.com 15 16 Geometric variation of the human tibia-fibula: A pub 1 dataset of tibia-fibula surface meshes and statistical sha 2 model 3 Meghan Keast1, Jason Bonacci1, Aaron Fox1 4 5 1 Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University 6 75 Pigdons Road, Waurn Ponds, 3216, VIC, Australia 7 8 9 10 Corresponding Author: 11 Meghan Keast1 12 Centre for Sport Research, School of Exercise and Nutrition Sciences, Deakin University 13 75 Pigdons Road, Waurn Ponds, 3216, VIC, Australia 14 Email address: mf.keast@gmail.com 15 16 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Abstract 17 Abstract 17 Background. Variation in tibia geometry is a risk factor for tibial stress fractures. Geome 18 variability in bones is often quantified using statistical shape modelling. Statistical shape mod 19 (SSM) offer a method to assess three-dimensional variation of structures and identify the sou 20 of variation. Although SSM have been used widely to assess long bones, there is limited op 21 source datasets of this kind. Overall, the creation of SSM can be an expensive process, 22 requires advanced skills. A publicly available tibia shape model would be beneficial as it enab 23 researchers to improve skills. An opensource SSM could benefit health, sport and medicine w 24 the potential to assess geometries suitable for medical equipment, and aid in clinical diagno 25 This study aimed to: (i) quantify tibial geometry using a SSM; and (ii) provide the SSM 26 associated code as an open-source dataset. 27 Methods. Lower limb computed tomography (CT) scans from the right tibia-fibula of 28 cadavers (male n = 20, female n =10) were obtained from the New Mexico Decedent Im 29 Database. Tibias were segmented and reconstructed from the CT scans into both cortical 30 trabecular sections. Fibulas were segmented as a singular surface. The segmented bones (cort 31 and trabecular tibia; and fibula) were used to develop three SSM of the: (i) tibia; (ii) combi 32 tibia and fibula; and (iii) tibial trabecular. Principal component analysis (PCA) was applied 33 obtain the three SSM, with the principal components (PCs) that explained 95% of the geome 34 variation retained. 35 Results. Overall size variation was the main source of variation of all three models accoun 36 for 90.31%, 84.24% and 85.06%, respectively. Other sources of geometric variation in the t 37 surface models included overall (PC3) and midshaft thickness (PC2); prominence and size of 38 condyle plateau, tibial tuberosity, and anterior crest (PC4); and axial torsion of the tibial s 39 (PC5). Further variations in the tibia-fibula model included midshaft thickness of the fib 40 (PC2); fibula head position relative to the tibia (PC2); tibia and fibula anterior-poste 41 curvature (PC4); fibula posterior curvature (PC5); tibia plateau rotation (PC5); and interosse 42 width (PC6). The main sources of variation in the tibia-trabecular model other than general 43 included variation in the medulla cavity diameter (PC2); overall thickness (PC3); and the rela 44 volume of proximal and distal ends compared to middle (PC4). Introduction 56 Variations in tibial geometry such as smaller tibias (Crossley et al., 1999; 83 Beck et al., 2014) and a thinner mid diaphysis (Popp et al., 2019) have been cited as a risk factor 84 for tibial stress injury. Quantifying tibial anatomy using SSM and providing this as an open 85 resource can assist in progressing our understanding of risk factors for tibial stress injury, as well 86 as identify relevant interventions to reduce the risk of these injuries in running populations. 87 Therefore, the aims of this paper are: (i) to quantify tibial geometry using a SSM; and (ii) 88 provide the SSM and associated code as an open-source dataset. This paper and associated code 89 will also demonstrate potential applications for the SSM to guide potential use. 90 91 Methods 92 Lower limb computed tomography (CT) scans from the right tibia and fibula of 30 cadavers 93 (male n = 20, female n =10) were obtained from the New Mexico Decedent Image Database 94 (Edgar et al., 2020). The images included were from individuals with a mean (± standard 95 Statistical shape models (SSM) offer a method to assess three-dimensional variation of 57 structures, including the sources of variation in bones. Although SSM have been used widely to 58 assess long bones (Zhang et al., 2014a; Nolte et al., 2020; Bruce et al., 2021) there is limited 59 open-source datasets of this kind. SSM that use medical imaging can be costly and the funding or 60 facilities needed to produce high quality imagery can be inaccessible. Segmentation of medical 61 images and bone surface reconstruction requires specialized software, skill, and extensive time. 62 Further, transforming the segmented data into three-dimensional models requires an intermediate 63 level of computer coding knowledge. Attainment of coding proficiency is a steep learning curve 64 that requires extensive mentorship. Overall, the creation of SSM can be an expensive and time 65 costly process, that requires advanced skills. A publicly available tibial shape model would be 66 beneficial as it enables researchers who may be affected by the aforementioned hurdles to 67 improve skills and adapt the code to meet their needs. Openly sharing code, data and 68 instructional papers is beneficial to all fields of research (Pisani et al., 2016; Pronk, 2019). Abstract 17 Overall size variation was the main source of variation of all three models accounting 36 for 90.31%, 84.24% and 85.06%, respectively. Other sources of geometric variation in the tibia 37 surface models included overall (PC3) and midshaft thickness (PC2); prominence and size of the 38 condyle plateau, tibial tuberosity, and anterior crest (PC4); and axial torsion of the tibial shaft 39 (PC5). Further variations in the tibia-fibula model included midshaft thickness of the fibula 40 (PC2); fibula head position relative to the tibia (PC2); tibia and fibula anterior-posterior 41 curvature (PC4); fibula posterior curvature (PC5); tibia plateau rotation (PC5); and interosseous 42 width (PC6). The main sources of variation in the tibia-trabecular model other than general size 43 included variation in the medulla cavity diameter (PC2); overall thickness (PC3); and the relative 44 volume of proximal and distal ends compared to middle (PC4). 45 Conclusion. Important variations that could increase the risk of tibial stress injury were 46 observed in the tibia and tibia-fibula SSM. These included general tibial thickness, midshaft 47 thickness, tibial length and medulla cavity diameter (indicative of cortical thickness). Further 48 research is warranted to better understand the effect of these tibial and fibula shape 49 characteristics on tibial stress, loading and injury risk. This SSM and associated code has been 50 provided in an open-source dataset. The associated code includes three example applications: (i) 51 generation of a random sample; (ii) reconstruction of trabecular surfaces; and (iii) reconstruction 52 from palpable landmarks. The developed tibial surface models and statistical shape model will be 53 made available for use at: https://simtk.org/projects/ssm_tibia. 54 55 55 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Introduction 56 Introduction 56 Statistical shape models (SSM) offer a method to assess three-dimensional variation of 57 structures, including the sources of variation in bones. Although SSM have been used widely to 58 assess long bones (Zhang et al., 2014a; Nolte et al., 2020; Bruce et al., 2021) there is limited 59 open-source datasets of this kind. SSM that use medical imaging can be costly and the funding or 60 facilities needed to produce high quality imagery can be inaccessible. Segmentation of medical 61 images and bone surface reconstruction requires specialized software, skill, and extensive time. 62 Further, transforming the segmented data into three-dimensional models requires an intermediate 63 level of computer coding knowledge. Attainment of coding proficiency is a steep learning curve 64 that requires extensive mentorship. Overall, the creation of SSM can be an expensive and time 65 costly process, that requires advanced skills. A publicly available tibial shape model would be 66 beneficial as it enables researchers who may be affected by the aforementioned hurdles to 67 improve skills and adapt the code to meet their needs. Openly sharing code, data and 68 instructional papers is beneficial to all fields of research (Pisani et al., 2016; Pronk, 2019). SSM 69 can also further research in health, sport, and medicine with the potential to create a sample of 70 computer simulated tibias, assess geometries suitable for medical equipment, and aid in clinical 71 diagnosis. 72 73 The ability to characterize and understand bone shape and geometric variation is also an 74 important aspect in skeletal research. Bone stress injuries caused by exercise are one area that 75 may benefit from an increased understanding of bone shape and individual variation. Individuals 76 who engage in high volumes of running, through recreational running or sport participation, are 77 at an elevated risk for lower limb bone stress injuries (Rizzone et al., 2017). Tibial stress 78 reactions and tibial stress fractures are the fifth and ninth most common running-related injuries, 79 respectively (Taunton, 2002). Tibial stress injuries result, in part, from the mechanical fatigue of 80 bone after cyclic loading (Burr et al., 1991). Tibial stress injuries are multifactorial in nature and 81 can be caused by several intrinsic and extrinsic factors, one of these being skeletal geometry 82 (Popp et al., 2019). Introduction 56 SSM 69 can also further research in health, sport, and medicine with the potential to create a sample of 70 computer simulated tibias, assess geometries suitable for medical equipment, and aid in clinical 71 diagnosis. 72 The ability to characterize and understand bone shape and geometric variation is also an 74 important aspect in skeletal research. Bone stress injuries caused by exercise are one area that 75 may benefit from an increased understanding of bone shape and individual variation. Individuals 76 who engage in high volumes of running, through recreational running or sport participation, are 77 at an elevated risk for lower limb bone stress injuries (Rizzone et al., 2017). Tibial stress 78 reactions and tibial stress fractures are the fifth and ninth most common running-related injuries, 79 respectively (Taunton, 2002). Tibial stress injuries result, in part, from the mechanical fatigue of 80 bone after cyclic loading (Burr et al., 1991). Tibial stress injuries are multifactorial in nature and 81 can be caused by several intrinsic and extrinsic factors, one of these being skeletal geometry 82 (Popp et al., 2019). Variations in tibial geometry such as smaller tibias (Crossley et al., 1999; 83 Beck et al., 2014) and a thinner mid diaphysis (Popp et al., 2019) have been cited as a risk factor 84 for tibial stress injury. Quantifying tibial anatomy using SSM and providing this as an open 85 resource can assist in progressing our understanding of risk factors for tibial stress injury, as well 86 as identify relevant interventions to reduce the risk of these injuries in running populations. 87 Therefore, the aims of this paper are: (i) to quantify tibial geometry using a SSM; and (ii) 88 provide the SSM and associated code as an open-source dataset. This paper and associated code 89 will also demonstrate potential applications for the SSM to guide potential use. 90 91 Methods 92 Lower limb computed tomography (CT) scans from the right tibia and fibula of 30 cadavers 93 (male n = 20, female n =10) were obtained from the New Mexico Decedent Image Database 94 (Edgar et al., 2020). The images included were from individuals with a mean (± standard 95 Methods 92 Lower limb computed tomography (CT) scans from the right tibia and fibula of 30 cadavers 93 (male n = 20, female n =10) were obtained from the New Mexico Decedent Image Database 94 (Edgar et al., 2020). The images included were from individuals with a mean (± standard 95 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint deviation) age of 28.7± 6.7, living weight of 70.22 ± 11.36kg, and living height of 176.06 ± 96 11.61cm. Individuals whose records indicated participation in impact-based physical activities 97 throughout life were selected for inclusion (i.e. team sports, dancing, recreational running and 98 walking). This criterion was included in an effort to ensure participants were sampled from a 99 generally active population. Participants were excluded if there was any noticeable damage to the 100 right tibia-fibula or the inclusion of medical devices (e.g. plates, rods, or screws). The tibia and 101 fibulas were segmented from the CT images using Mimics innovation suite (Materialise, Leuven, 102 Belgium). Tibias were segmented into two surfaces representing the outer boundaries of the 103 trabecular and cortical bone, while fibulas were segmented as one surface representing the outer 104 shape of the entire bone. Cortical bone was classified as all visible hard bone, while trabecular 105 was all remaining soft bone and the medulla cavity. The medulla cavity was included in the 106 trabecular bone segmentation as this enabled us to describe changes in size and shape of the 107 entire trabecular surface and medulla cavity without needing to create a cross section of the tibia. 108 Segmentation was done using a combination of image intensity thresholding with manual 109 corrections. Methods 92 It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint interpret the variation explained by each PC, the mean surface was compared to surfaces 136 generated by manipulating the retained PC up to plus/minus three standard deviations. Animated 137 heatmaps of each PC component, where colour variation indicated the relative amount of 138 position change of the surface nodes, were also used to assist with interpretation of PCs (see 139 supplementary files 1, 2 and 3). 140 141 Results 142 143 Tibia SSM 144 The first five PCs accounted for 95.80% of the geometric variation across the tibial surfaces (see 145 Table 1). Mean and peak error in surface node position (in mm), and the Jaccard index for the 146 reconstructed tibia surfaces using a reduced component set were 1.76 [1.66, 1.86 95% CIs], 4.70 147 [4.41, 4.98 95% CIs] and 0.773 [0.771, 0.776 95% CIs], respectively (see Figure 1). 148 149 Tibia - Fibula SSM 150 The first seven PCs accounted for 95.49% of the geometric variation across the tibial surfaces 151 (see Table 2). Mean and peak error in surface node position (in mm), and the Jaccard index for 152 the reconstructed tibia-fibula surfaces using a reduced component set were 1.89 [1.81, 1.97 95% 153 CIs], 5.67 [5.30, 5.99 95% CIs] and 0.751 [0.743, 0.759 95% CIs], respectively (see Figure 2). 154 155 Tibia - Trabecular SSM 156 The first four PCs accounted for 95.17% of the geometric variation across the tibia trabecular 157 (see Table 3). Mean and peak error in surface node position (in mm), and the Jaccard index for 158 the reconstructed trabecular surfaces using a reduced component set were 2.05 [1.96, 2.14 95% 159 CIs], 5.63 [5.31, 5.95 95% CIs] and 0.759 [0.753, 0.764 95% CIs], respectively (see Figure 3). 160 interpret the variation explained by each PC, the mean surface was compared to surfaces 136 generated by manipulating the retained PC up to plus/minus three standard deviations. Methods 92 Landmarks on the lateral and medial malleoli, lateral and medial tibial condyle, 110 fibula head, tibial tuberosity, anterior aspect of the tibia at 25%, 50% and 75% of the distance 111 between the medial condyle and malleolus, and lateral fibula diaphysis 25% proximally from the 112 malleoli were registered from the medical images on the segmented surfaces (Bruce et al., 2021). 113 The segmented bones (cortical and trabecular tibia; and fibula) were used to develop three SSM 115 of the: (i) tibia; (ii) combined tibia and fibula; and (iii) tibial trabecular. The procedures used to 116 create the SSM were applied identically across all three models. Prior to development of the 117 SSM, the bones were aligned to the tibial reference system as per International Society of 118 Biomechanics recommendations (Wu et al., 2002). This was done to simplify later steps in the 119 surface registration processes. Surfaces were remeshed to have a matching number of points 120 (tibia n = 3500; fibula n = 1500; trabecular n = 1500). Nodal correspondence and registration 121 were performed in MATLAB (R2019b, Mathworks, MA, United States). All surfaces were 122 registered against the same target mesh, which was selected due to being the closest to the 123 sample mean (Bruce et al., 2021). Nodal correspondence was performed using the coherent drift 124 point algorithm (Myronenko & Song, 2010). This algorithm non-rigidly registers (i.e. rotates, 125 translates and scales) each surface mesh to the target mesh so that corresponding points between 126 surfaces can be identified. The surface meshes were then rigidly aligned using a Procrustes 127 analysis with no scaling applied. 128 Principal component analysis (PCA) was applied to the surface sets to obtain the three SSM. The principal components (PCs) that accounted for > 95% of geometric variation in each SSM were retained (Smoger et al., 2017). Mean and peak error in surface node position, and the Jaccard index (i.e. a measure of volumetric similarity where values range from 0 to 1 indicating no to perfect similarity, respectively) (Real & Vargas, 1996)(mean ± 95% confidence intervals [CIs]) of reconstructed surfaces using the reduced component set were calculated for each SSM. To . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Methods 92 Lower scores described external/lateral rotation at the distal end of the bone and internal/medial rotation at the proximal end. Higher PC5 scores describ internal/medial rotation at distal end of the tibia and external/lateral rotation at proximal end of the tibia. PC – principal component 163 164 165 166 Figure 1. Mean and peak error in surface node position, and the Jaccard index for the 167 reconstructed tibia surfaces using a reduced component set. Error variables are presented in mean 168 (dashed line) and 95% confidence intervals (dotted lines). 169 170 ia ribed ia. ribed ribed ribed inence s also rease ribed at the d at the he an Table 1. Percentage of variance and interpretations of each principal component for the tibia 161 SSM. 162 Principal component % of variance Geometric variation observed Interpretation PC1 90.32 General size variation (length and width). Lower PC1 scores described a longer and wider tibia. Higher PC1 scores describ a shorter and thinner tibia. PC2 2.30 Midshaft thickness. Lower PC2 scores described a thicker midshaft. Higher PC2 scores describ a thinner midshaft. PC3 1.30 General overall thickness. Lower PC3 scores described an overall thicker tibia. Higher PC2 scores describ an overall thinner tibia. PC4 0.95 Prominence and size of condyle plateau and tibial tuberosity. Anterior crest prominence. Lower PC4 scores described a decreased prominence and size of the condyles, tibial plateau, and tibial tuberosity. Lower scores also described an overall decrease in the anterior crest prominence. Higher PC4 scores describ an increase in the promine and size of the condyles, tibial plateau, and tibial tuberosity. Higher scores a described an overall increa in the anterior crest prominence. PC5 0.93 Axial rotation. Lower PC5 scores described axial rotation of the tibia shaft. Lower scores described external/lateral rotation at the distal end of the bone and internal/medial rotation at the proximal end. Higher PC5 scores describ internal/medial rotation at distal end of the tibia and external/lateral rotation at proximal end of the tibia. ia ribed ia. ribed ribed ribed inence s also rease ribed at the d at the Higher PC4 scores describ an increase in the promine and size of the condyles, tibial plateau, and tibial tuberosity. Higher scores a described an overall increa in the anterior crest prominence. ribed inence s also rease Lower PC5 scores described axial rotation of the tibia shaft. Methods 92 Animated 137 heatmaps of each PC component, where colour variation indicated the relative amount of 138 position change of the surface nodes, were also used to assist with interpretation of PCs (see 139 supplementary files 1, 2 and 3). 140 interpret the variation explained by each PC, the mean surface was compared to surfaces 6 generated by manipulating the retained PC up to plus/minus three standard deviations. Animated 7 heatmaps of each PC component, where colour variation indicated the relative amount of 8 position change of the surface nodes, were also used to assist with interpretation of PCs (see 9 supplementary files 1, 2 and 3). 0 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Table 1. Percentage of variance and interpretations of each principal component for the tibia 161 SSM. 162 Principal component % of variance Geometric variation observed Interpretation PC1 90.32 General size variation (length and width). Lower PC1 scores described a longer and wider tibia. Higher PC1 scores describ a shorter and thinner tibia. PC2 2.30 Midshaft thickness. Lower PC2 scores described a thicker midshaft. Higher PC2 scores describ a thinner midshaft. PC3 1.30 General overall thickness. Lower PC3 scores described an overall thicker tibia. Higher PC2 scores describ an overall thinner tibia. PC4 0.95 Prominence and size of condyle plateau and tibial tuberosity. Anterior crest prominence. Lower PC4 scores described a decreased prominence and size of the condyles, tibial plateau, and tibial tuberosity. Lower scores also described an overall decrease in the anterior crest prominence. Higher PC4 scores describ an increase in the promine and size of the condyles, tibial plateau, and tibial tuberosity. Higher scores a described an overall increa in the anterior crest prominence. PC5 0.93 Axial rotation. Lower PC5 scores described axial rotation of the tibia shaft. Methods 92 Lower scores described external/lateral rotation at the distal end of the bone and internal/medial rotation at the proximal end. Higher PC5 scores describ internal/medial rotation at distal end of the tibia and external/lateral rotation at proximal end of the tibia. ribed at the d at the proximal end. PC – principal component 163 164 165 166 Figure 1. Mean and peak error in surface node position, and the Jaccard index for the 167 he 164 165 166 Figure 1. Mean and peak error in surface node position, and the Jaccard index for the 167 reconstructed tibia surfaces using a reduced component set. Error variables are presented in mean 168 (dashed line) and 95% confidence intervals (dotted lines). 169 170 he an Figure 1. Mean and peak error in surface node position, and the Jaccard index for the 167 reconstructed tibia surfaces using a reduced component set. Error variables are presented in mean 168 (dashed line) and 95% confidence intervals (dotted lines). 169 170 he an . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Table 2. Percentage of variance and interpretations of each principal component for the tibia- 171 fibula SSM. 172 Principal component % of variance Geometric variation observed Interpretation PC1 84.24 General size variation (length and width) of tibia and fibula. Lower PC1 scores described a longer and wider tibia and fibula Higher PC1 scores described a shorter and thinner tibia and fibula. PC2 3.50 Midshaft thickness of fibula. Fibula head position. Lower PC2 scores described a thinner fibula midshaft, with a more anterior head relative to the tibia. Higher PC2 scores described a thicker fibula midshaft, with a more posterior head relative to the tibia. PC3 2.60 General overall thickness of tibia and fibula. Lower PC3 scores described an overall thicker tibia and fibula, Higher PC3 scores described an overall thinner tibia and fibula. PC4 1.91 Tibia and Fibula anterior-posterior curvature. Lower PC4 scores described posterior curvature of the fibula at the proximal end, the fibula head becomes more anterior. Lower scores also describe a straighter tibia. Higher PC3 scores describe a straighter fibula, with a more posterior head. The tibia displays anterior curvature. Higher PC6 scores describe a thicker tibia with increased condyle prominence. Higher PC3 scores described an overall thinner tibia and fibula. Higher PC6 scores describe a thicker tibia with increased condyle prominence. Higher PC5 scores describe a more posteriorly curved fibula, and a more externally rotated tibial plateau. PC – principal component Higher PC7 scores describes a bigger/thicker gap between the tibia and fibula at the distal end. Higher PC3 scores describe a straighter fibula, with a more posterior head. The tibia displays anterior curvature. Methods 92 182 Principal component % of variance Geometric variation observed Interpretation PC1 85.07 General size variation (length and width). Lower PC1 scores describe a longer and wider trabecular bone. Higher PC1 scores describ shorter and thinner trabecu bone. PC2 7.50 Medulla cavity diameter. Lower PC2 scores describe an increased diameter of the medulla cavity. Higher PC2 scores describ decreased diameter of the medulla cavity. PC3 1.50 General overall thickness Lower PC3 scores describe an overall thicker trabecular bone. Higher PC3 scores describ an overall thinner trabecul bone. PC4 1.10 Relative volume of proximal and distal ends compared to the middle. Lower PC4 scores describe an increased trabecular bone volume at the ends of the bone compared to the middle of the bone. This indicates a decreased medulla cavity diameter and a potential thicker cortical bone at the midshaft of the tibia. Higher PC4 scores describ more consistent volume across the bone, this indic an increased medulla cavit diameter and potentially thinner cortical bone at the midshaft of the tibia. PC – principal component 183 184 185 186 Figure 3. Mean and peak error in surface node position, and the Jaccard index for the 187 reconstructed tibia-trabecular surfaces using a reduced component set. Error variables are 188 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 189 he ribe a ecular ribe a he ribe cular ribe a icates vity the he re Table 3. Percentage of variance and interpretations of each principal component for the 181 trabecular SSM. 182 Principal component % of variance Geometric variation observed Interpretation PC1 85.07 General size variation (length and width). Lower PC1 scores describe a longer and wider trabecular bone. Higher PC1 scores describ shorter and thinner trabecu bone. PC2 7.50 Medulla cavity diameter. Lower PC2 scores describe an increased diameter of the medulla cavity. Higher PC2 scores describ decreased diameter of the medulla cavity. PC3 1.50 General overall thickness Lower PC3 scores describe an overall thicker trabecular bone. Higher PC3 scores describ an overall thinner trabecul bone. PC4 1.10 Relative volume of proximal and distal ends compared to the middle. Lower PC4 scores describe an increased trabecular bone volume at the ends of the bone compared to the middle of the bone. This indicates a decreased medulla cavity diameter and a potential thicker cortical bone at the midshaft of the tibia. Methods 92 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Table 3. Percentage of variance and interpretations of each principal component for the 181 trabecular SSM. 182 Principal component % of variance Geometric variation observed Interpretation PC1 85.07 General size variation (length and width). Lower PC1 scores describe a longer and wider trabecular bone. Higher PC1 scores describ shorter and thinner trabecu bone. PC2 7.50 Medulla cavity diameter. Lower PC2 scores describe an increased diameter of the medulla cavity. Higher PC2 scores describ decreased diameter of the medulla cavity. PC3 1.50 General overall thickness Lower PC3 scores describe an overall thicker trabecular bone. Higher PC3 scores describ an overall thinner trabecul bone. PC4 1.10 Relative volume of proximal and distal ends compared to the middle. Lower PC4 scores describe an increased trabecular bone volume at the ends of the bone compared to the middle of the bone. This indicates a decreased medulla cavity diameter and a potential thicker cortical bone at the midshaft of the tibia. Higher PC4 scores describ more consistent volume across the bone, this indic an increased medulla cavit diameter and potentially thinner cortical bone at the midshaft of the tibia. PC – principal component 183 184 185 186 Figure 3. Mean and peak error in surface node position, and the Jaccard index for the 187 reconstructed tibia-trabecular surfaces using a reduced component set. Error variables are 188 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 189 he ribe a ecular ribe a he ribe cular ribe a icates vity the he re . CC-BY 4.0 International license available under a ( y p ) g p y p p p p y Table 3. Percentage of variance and interpretations of each principal component for the 181 trabecular SSM. Methods 92 PC5 1.36 Fibula posterior curvature. Tibia plateau rotation. Lower PC5 scores describe a straighter fibula, flattening in an anterior direction. Lower scores also describe internal rotation of the tibial plateau. Higher PC5 scores describe a more posteriorly curved fibula, and a more externally rotated tibial plateau. PC6 1.03 Tibia upper to midshaft thickness. Tibia condyle prominence. Lower PC6 scores describe a thinner tibia, with a decreased condyle prominence. Higher PC6 scores describe a thicker tibia with increased condyle prominence. PC7 0.85 Interosseous width. Lower PC7 scores describe a smaller/thinner gap between the tibia and fibula at the distal end. Higher PC7 scores describes a bigger/thicker gap between the tibia and fibula at the distal end. PC – principal component 173 Higher PC2 scores described a thicker fibula midshaft, with a more posterior head relative to the tibia. Higher PC3 scores describe a straighter fibula, with a more posterior head. The tibia displays anterior curvature. Lower PC7 scores describe a smaller/thinner gap between the tibia and fibula at the distal end. 173 PC – principal component . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint 174 175 Figure 2. Mean and peak error in surface node position, and the Jaccard index for the 176 reconstructed tibia-fibula surfaces using a reduced component set. Error variables are presented 177 in mean (dashed line) and 95% confidence intervals (dotted lines). 178 179 he ed 174 175 176 177 178 179 180 Figure 2. Mean and peak error in surface node position, and the Jaccard index for the 176 reconstructed tibia-fibula surfaces using a reduced component set. Error variables are presented 177 in mean (dashed line) and 95% confidence intervals (dotted lines). 178 179 he ed 180 . Discussion 190 The impact that a smaller tibia has on tibial stress injuries has been 219 cited in both athletic (Crossley et al., 1999; Beck et al., 2014) and military populations (Giladi et 220 al., 1991; Beck et al., 2000). However, size of bone is often proportionate to individuals’ height 221 and mass (Duyar & Pelin, 2003). The population used for this study had a height and mass range 222 of 160 to 200cm and 49.9 to 93kg, respectively. It is likely that the smaller tibias we observed 223 were related to height and mass and this may not automatically be indicative of elevated tibial 224 stress injury risk. Despite having smaller tibias, shorter individuals would likely produce reduced 225 forces and moments during running given their lighter mass and bone segment length. 226 Nonetheless, our shape model demonstrates that tibial size, a commonly cited risk factor for 227 tibial stress injuries (Giladi et al., 1991; Crossley et al., 1999; Beck et al., 2000, 2014), is a 228 primary source of variation in tibial geometry. What is likely problematic is when an individual 229 Several of the observed variations in tibial geometry could relate to increased risk of tibial stress 216 injury. Smaller and thinner tibial geometry was the predominant source of variation found in our 217 SSM, which can impact the bones’ ability to withstand bending forces created during exercise 218 (Nordin & Frankel, 2012). The impact that a smaller tibia has on tibial stress injuries has been 219 cited in both athletic (Crossley et al., 1999; Beck et al., 2014) and military populations (Giladi et 220 al., 1991; Beck et al., 2000). However, size of bone is often proportionate to individuals’ height 221 and mass (Duyar & Pelin, 2003). The population used for this study had a height and mass range 222 of 160 to 200cm and 49.9 to 93kg, respectively. It is likely that the smaller tibias we observed 223 were related to height and mass and this may not automatically be indicative of elevated tibial 224 stress injury risk. Despite having smaller tibias, shorter individuals would likely produce reduced 225 forces and moments during running given their lighter mass and bone segment length. Methods 92 Higher PC4 scores describ more consistent volume across the bone, this indic an increased medulla cavit diameter and potentially thinner cortical bone at the midshaft of the tibia. PC i i l 183 he ribe a ecular ribe a he ribe cular ribe a icates vity the Higher PC1 scores describ shorter and thinner trabecu bone. ribe a ecular Higher PC2 scores describ decreased diameter of the medulla cavity. ribe a he Higher PC4 scores describ more consistent volume across the bone, this indic an increased medulla cavit diameter and potentially thinner cortical bone at the midshaft of the tibia. ribe a icates vity the midshaft of the tibia. PC – principal component 183 184 185 186 Figure 3. Mean and peak error in surface node position, and the Jaccard index for the 187 reconstructed tibia-trabecular surfaces using a reduced component set. Error variables are 188 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 189 he re PC – principal component 183 184 185 186 Figure 3. Mean and peak error in surface node position, and the Jaccard index for the 187 reconstructed tibia-trabecular surfaces using a reduced component set. Error variables are 188 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 189 he re Figure 3. Mean and peak error in surface node position, and the Jaccard index for the 187 reconstructed tibia-trabecular surfaces using a reduced component set. Error variables are 188 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 189 he re Figure 3. Mean and peak error in surface node position, and the Jaccard index for the 187 reconstructed tibia-trabecular surfaces using a reduced component set. Error variables are 188 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 189 he re . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Discussion 190 The purpose of this study was to explore tibial geometry using a SSM and provide the SSM and 191 associated code as an open-source dataset. The data presented and associated code can be found 192 online at https://simtk.org/projects/ssm_tibia. Our results indicated that overall size variation 193 (length and width) was the main source of variation of all three models (i.e. tibia, tibia and 194 fibula, and tibial trabecular) accounting for 90.31%, 84.24% and 85.06%, respectively. This 195 aligns with previous SSM of the tibia where overall size was the main source of variation in tibia 196 (Quintens et al., 2019) and tibia-fibula models (Bruce et al., 2021). Other sources of geometric 197 variation in our tibia surface models included overall and midshaft thickness; prominence and 198 size of the condyle plateau, tibial tuberosity and anterior crest; and axial torsion of the tibial 199 shaft. Sources of variation noted in the tibia model were also evident in the tibia-fibula model, 200 however inclusion of the fibula introduced variations in midshaft thickness of the fibula; fibula 201 head position relative to the tibia; tibia and fibula anterior-posterior curvature; fibula posterior 202 curvature; tibia plateau rotation; and interosseous width. However, these variations only 203 accounted for a small proportion of overall tibia-fibula geometric variation. Lastly, sources of 204 variation in the tibia-trabecular model included variation in the medulla cavity diameter; overall 205 thickness; and the relative volume of proximal and distal ends compared to middle. Previous 206 research has identified both similar and different variations in tibial geometry compared to the 207 outcomes from our research(Tümer et al., 2019; Quintens et al., 2019). Similar to the variations 208 identified in our tibia model, past studies identified variations in the ‘bow’ of the tibia in the 209 anterior posterior direction (i.e. anterior posterior shaft curvature) (Quintens et al., 2019), as well 210 as changes in the orientation of the tibial plateau (Quintens et al., 2019). Variation in the 211 prominence of the condyles as well as changes to the tibial tuberosity and overall thickness of the 212 tibia were also evident in past isolated studies of the tibia (Tümer et al., 2019; Quintens et al., 213 2019). 214 The purpose of this study was to explore tibial geometry using a SSM and provide the SSM and 191 associated code as an open-source dataset. Discussion 190 The data presented and associated code can be found 192 online at https://simtk.org/projects/ssm_tibia. Our results indicated that overall size variation 193 (length and width) was the main source of variation of all three models (i.e. tibia, tibia and 194 fibula, and tibial trabecular) accounting for 90.31%, 84.24% and 85.06%, respectively. This 195 aligns with previous SSM of the tibia where overall size was the main source of variation in tibia 196 (Quintens et al., 2019) and tibia-fibula models (Bruce et al., 2021). Other sources of geometric 197 variation in our tibia surface models included overall and midshaft thickness; prominence and 198 size of the condyle plateau, tibial tuberosity and anterior crest; and axial torsion of the tibial 199 shaft. Sources of variation noted in the tibia model were also evident in the tibia-fibula model, 200 however inclusion of the fibula introduced variations in midshaft thickness of the fibula; fibula 201 head position relative to the tibia; tibia and fibula anterior-posterior curvature; fibula posterior 202 curvature; tibia plateau rotation; and interosseous width. However, these variations only 203 accounted for a small proportion of overall tibia-fibula geometric variation. Lastly, sources of 204 variation in the tibia-trabecular model included variation in the medulla cavity diameter; overall 205 thickness; and the relative volume of proximal and distal ends compared to middle. Previous 206 research has identified both similar and different variations in tibial geometry compared to the 207 outcomes from our research(Tümer et al., 2019; Quintens et al., 2019). Similar to the variations 208 identified in our tibia model, past studies identified variations in the ‘bow’ of the tibia in the 209 anterior posterior direction (i.e. anterior posterior shaft curvature) (Quintens et al., 2019), as well 210 as changes in the orientation of the tibial plateau (Quintens et al., 2019). Variation in the 211 prominence of the condyles as well as changes to the tibial tuberosity and overall thickness of the 212 tibia were also evident in past isolated studies of the tibia (Tümer et al., 2019; Quintens et al., 213 2019). 214 215 Several of the observed variations in tibial geometry could relate to increased risk of tibial stress 216 injury. Smaller and thinner tibial geometry was the predominant source of variation found in our 217 SSM, which can impact the bones’ ability to withstand bending forces created during exercise 218 (Nordin & Frankel, 2012). Discussion 190 226 Nonetheless, our shape model demonstrates that tibial size, a commonly cited risk factor for 227 tibial stress injuries (Giladi et al., 1991; Crossley et al., 1999; Beck et al., 2000, 2014), is a 228 primary source of variation in tibial geometry. What is likely problematic is when an individual 229 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint with greater height/mass demonstrates these smaller tibial properties. Reducing tibial forces and 230 moments during running in individuals displaying this anatomical characteristic is likely 231 beneficial for minimising tibial stress injury risk. 232 with greater height/mass demonstrates these smaller tibial properties. Reducing tibial forces and 230 moments during running in individuals displaying this anatomical characteristic is likely 231 beneficial for minimising tibial stress injury risk. 232 233 PC2 of the trabecular model described the diameter of the medulla cavity. Changes in the 234 diameter could indicate changes in overall bone width in addition to thickness of the cortical 235 bone (i.e. enlarged medulla cavity equating to reduced cortical bone thickness). Cortical bone 236 thickness is cited as a risk factor for tibial stress injuries in military (Cosman et al., 2013) and 237 athletic populations (Beck et al., 2014). Similar to smaller and thinner tibial geometries, 238 decreased cortical thickness could impact a bones ability to withstand loads. Reduced thickness 239 of the cortical bone could reduce the overall stiffness of the tibia and make it more susceptible to 240 compression and bending forces during exercise (Nordin & Frankel, 2012). PC3 of both the tibia 241 and tibia-fibula model described changes in overall thickness of the tibia, while PC2 of the tibia 242 model described changes in midshaft thickness. General tibia thickness may be an important 243 factor for tibial stress injuries (Giladi et al., 1991). Discussion 190 248 Decreased overall tibial thickness and thickness of the midshaft likely decrease the tibias’ ability 249 to withstand bending forces created during exercise, potentially increasing the risk of tibial stress 250 injury. Promoting running technique that minimizes tibial forces and moments in individuals 251 with these geometries could likely be important for reducing tibial stress injury risk. 252 253 Additional areas of geometric variation we observed in the tibia, such as axial torsion of the tibia, 254 interosseous width, and anterior-posterior curvature have not been considered in relation to tibial 255 stress injury risk. Further, we have limited understanding of how changes in fibula geometry 256 influence tibial stress. Previous research has identified that the fibula potentially acts in a bracing 257 fashion, by restricting medial and posterior motion of the tibial plateau relative to the malleoli 258 (Haider, Baggaley & Edwards, 2020). It is unknown if changes in fibula geometry might impact 259 its ability to provide this support. Further research is warranted to better understand the effect of 260 these additional tibial shape characteristics and fibula geometry on tibial stress, loading and 261 injury risk. 262 263 Practical Applications of the SSM 264 The dataset and associated code provided at htt // i tk / j t / tibi can be used in 265 PC2 of the trabecular model described the diameter of the medulla cavity. Changes in the 234 diameter could indicate changes in overall bone width in addition to thickness of the cortical 235 bone (i.e. enlarged medulla cavity equating to reduced cortical bone thickness). Cortical bone 236 thickness is cited as a risk factor for tibial stress injuries in military (Cosman et al., 2013) and 237 athletic populations (Beck et al., 2014). Similar to smaller and thinner tibial geometries, 238 decreased cortical thickness could impact a bones ability to withstand loads. Reduced thickness 239 of the cortical bone could reduce the overall stiffness of the tibia and make it more susceptible to 240 compression and bending forces during exercise (Nordin & Frankel, 2012). PC3 of both the tibia 241 and tibia-fibula model described changes in overall thickness of the tibia, while PC2 of the tibia 242 model described changes in midshaft thickness. General tibia thickness may be an important 243 factor for tibial stress injuries (Giladi et al., 1991). Discussion 190 However, tibial midshaft thickness is likely a 244 more important variation than overall tibia thickness for tibial stress injury risk. Tibial stress 245 injuries most commonly occur in the mid to distal third of the tibial shaft (Coady & Micheli, 246 1997; Beck, 1998). A past study found that when adjusting for body size, thinner mid–diaphysis 247 of the tibia is an important factor in the incidence of tibial stress injuries (Popp et al., 2019). 248 Decreased overall tibial thickness and thickness of the midshaft likely decrease the tibias’ ability 249 to withstand bending forces created during exercise, potentially increasing the risk of tibial stress 250 injury. Promoting running technique that minimizes tibial forces and moments in individuals 251 with these geometries could likely be important for reducing tibial stress injury risk. 252 253 g j y 233 PC2 of the trabecular model described the diameter of the medulla cavity. Changes in the 234 diameter could indicate changes in overall bone width in addition to thickness of the cortical 235 bone (i.e. enlarged medulla cavity equating to reduced cortical bone thickness). Cortical bone 236 thickness is cited as a risk factor for tibial stress injuries in military (Cosman et al., 2013) and 237 athletic populations (Beck et al., 2014). Similar to smaller and thinner tibial geometries, 238 decreased cortical thickness could impact a bones ability to withstand loads. Reduced thickness 239 of the cortical bone could reduce the overall stiffness of the tibia and make it more susceptible to 240 compression and bending forces during exercise (Nordin & Frankel, 2012). PC3 of both the tibia 241 and tibia-fibula model described changes in overall thickness of the tibia, while PC2 of the tibia 242 model described changes in midshaft thickness. General tibia thickness may be an important 243 factor for tibial stress injuries (Giladi et al., 1991). However, tibial midshaft thickness is likely a 244 more important variation than overall tibia thickness for tibial stress injury risk. Tibial stress 245 injuries most commonly occur in the mid to distal third of the tibial shaft (Coady & Micheli, 246 1997; Beck, 1998). A past study found that when adjusting for body size, thinner mid–diaphysis 247 of the tibia is an important factor in the incidence of tibial stress injuries (Popp et al., 2019). Discussion 190 It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint 270 Case One: Generating Surface Samples 271 Obtaining CT and/or MRI scans to develop a large sample of bone surfaces can be costly from 272 both a time and financial perspective. Further, the imaging facilities to obtain these data may not 273 be available to all. In this case study, we provide an application that uses the SSM of the tibia- 274 fibula to create a sample set of tibia-fibula surfaces representing standardized variation across all 275 and/or a selection of the model components. The ‘simulatedPopulations.m’ function provided 276 with our dataset allows users to generate a select number of surfaces from a pre-loaded shape 277 model. While the example provided uses the tibia-fibula shape model, this process could be 278 applied to any of the shape models included in our dataset. The function is structured to allow 279 users to: (i) load the desired shape model; (ii) set the desired number of samples (i.e. n) to be 280 generated; (iii) select the components of the model to be perturbed in creating the surface 281 variations; (iv) set the magnitude of variation to perturb the model components by (in standard 282 deviations); and (v) set the output options. The function takes these user inputs and randomly 283 samples values for the chosen components within the standard deviation bounds provided to 284 reconstruct n samples. Users can select to output a visual representation of the surfaces with 285 heatmaps applied to highlight the areas of variation (see Figure 4), as well as outputting the 286 three-dimensional surfaces in STL format. 287 288 The SSM developed in our study represent the major sources of variation in the bone surfaces, 289 and this model can be used to produce a representative sample of surfaces or a ‘simulated 290 population.’ The ability to perturb or manipulate specific components of the shape models also 291 allows for samples to be generated with isolated geometric variations of interest (e.g. specifically 292 isolating variation in mid-shaft tibial thickness). 293 294 270 Case One: Generating Surface Samples 271 Obtaining CT and/or MRI scans to develop a large sample of bone surfaces can be costly from 272 both a time and financial perspective. Further, the imaging facilities to obtain these data may not 273 be available to all. Discussion 190 However, tibial midshaft thickness is likely a 244 more important variation than overall tibia thickness for tibial stress injury risk. Tibial stress 245 injuries most commonly occur in the mid to distal third of the tibial shaft (Coady & Micheli, 246 1997; Beck, 1998). A past study found that when adjusting for body size, thinner mid–diaphysis 247 of the tibia is an important factor in the incidence of tibial stress injuries (Popp et al., 2019). 248 D d ll ibi l hi k d hi k f h id h f lik l d h ibi ’ bili 249 Decreased overall tibial thickness and thickness of the midshaft likely decrease the tibias’ ability 49 to withstand bending forces created during exercise, potentially increasing the risk of tibial stress 50 injury. Promoting running technique that minimizes tibial forces and moments in individuals 51 with these geometries could likely be important for reducing tibial stress injury risk. 52 Additional areas of geometric variation we observed in the tibia, such as axial torsion of the tibia, 254 interosseous width, and anterior-posterior curvature have not been considered in relation to tibial 255 stress injury risk. Further, we have limited understanding of how changes in fibula geometry 256 influence tibial stress. Previous research has identified that the fibula potentially acts in a bracing 257 fashion, by restricting medial and posterior motion of the tibial plateau relative to the malleoli 258 (Haider, Baggaley & Edwards, 2020). It is unknown if changes in fibula geometry might impact 259 its ability to provide this support. Further research is warranted to better understand the effect of 260 these additional tibial shape characteristics and fibula geometry on tibial stress, loading and 261 injury risk. 262 The dataset and associated code provided at https://simtk.org/projects/ssm_tibia can be used in 265 several ways to assist with skeletal focused research of the tibia-fibula complex. This paper does 266 not have the scope to highlight each and every potential application, however we propose three 267 broad applications of where and how the SSM may be used. The code and analyses described in 268 the following case studies are also included with our dataset. 269 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Discussion 190 In this case study, we provide an application that uses the SSM of the tibia- 274 fibula to create a sample set of tibia-fibula surfaces representing standardized variation across all 275 and/or a selection of the model components. The ‘simulatedPopulations.m’ function provided 276 with our dataset allows users to generate a select number of surfaces from a pre-loaded shape 277 model. While the example provided uses the tibia-fibula shape model, this process could be 278 applied to any of the shape models included in our dataset. The function is structured to allow 279 users to: (i) load the desired shape model; (ii) set the desired number of samples (i.e. n) to be 280 generated; (iii) select the components of the model to be perturbed in creating the surface 281 variations; (iv) set the magnitude of variation to perturb the model components by (in standard 282 deviations); and (v) set the output options. The function takes these user inputs and randomly 283 samples values for the chosen components within the standard deviation bounds provided to 284 reconstruct n samples. Users can select to output a visual representation of the surfaces with 285 heatmaps applied to highlight the areas of variation (see Figure 4), as well as outputting the 286 three-dimensional surfaces in STL format. 287 The SSM developed in our study represent the major sources of variation in the bone surfaces, and this model can be used to produce a representative sample of surfaces or a ‘simulated population.’ The ability to perturb or manipulate specific components of the shape models also allows for samples to be generated with isolated geometric variations of interest (e.g. specifically isolating variation in mid-shaft tibial thickness). . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Figure 4. Discussion 190 Simulated surface sample of 30 tibia-fibulas, this sample was created using the first 295 five and the seventh principal components, opting to ignore the sixth. Standard deviation bounds 296 were set at 1.5. Each surface is displayed with a corresponding heatmap, detailing the changes 297 that occurred with each sample. Warmer colours describe greater change occurring at that area of 298 tibia-fibula, respective to the mean shape model. 299 Figure 4. Simulated surface sample of 30 tibia-fibulas, this sample was 295 Figure 4. Simulated surface sample of 30 tibia-fibulas, this sample was created using the first 295 five and the seventh principal components, opting to ignore the sixth. Standard deviation bounds 296 were set at 1.5. Each surface is displayed with a corresponding heatmap, detailing the changes 297 that occurred with each sample. Warmer colours describe greater change occurring at that area of 298 tibia-fibula, respective to the mean shape model. 299 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Case Two: Predicting and Generating Trabecular Volumes 300 Finite element simulations of tibial loading typically include different parts and material 301 properties for the cortical and trabecular volumes of the bone (Edwards et al., 2010; Haider, 302 Baggaley & Edwards, 2020). Certain scenarios can make generating surface and volumetric 303 meshes of the trabecular more difficult or time consuming. Segmenting the trabecular surface 304 from CT scans is possible, yet more time-consuming given the need for greater manual 305 corrections. MRIs that are not optimized to detect the trabecular volume can also generate 306 difficulties in segmenting the trabecular area due to lacking clearly identifiable borders between 307 the cortical and trabecular areas. Other bone surface reconstruction methods (e.g. MAP-client 308 (Zhang et al., 2014b)) only provide estimates of external bone surface and not internal trabecular 309 volumes. Discussion 190 Given that performing finite element simulations with both trabecular and cortical bone 310 is more reflective of real-world scenarios, there are practical benefits to developing methods that 311 can predict and generate trabecular volumes based on the more easily obtainable outer bone 312 surface. 313 314 In this case study, we use the SSM of the tibia and trabecular to develop and evaluate a method 315 for estimating a correspondent trabecular surface. The theoretical hypothesis for this approach is 316 that the outer shape of the tibia can be effective in predicting the internal trabecular shape. The 317 ‘generateTrabecularFromSurface.m’ function provided with our dataset demonstrates the 318 development and evaluation of this method on the dataset used in the present study, followed by 319 the application of the method to a new set of tibial surfaces. Briefly, this code develops a set of 320 linear regression models (see supplementary file 4) using the retained principal component 321 scores from the tibia model to predict each of the retained principal component scores of the 322 trabecular model. The predicted scores can then be combined with the trabecular SSM to 323 reconstruct the trabecular surface. In developing this method we noted that it was common for 324 the reconstructed trabecular to project outside of the tibial surface (i.e. not fitting within the outer 325 tibia boundary). To combat this, we included a final step where points on the reconstructed 326 trabecular surface that protruded outside the tibia surface were translated to the closest point one 327 millimetre inside the boundary of the tibia. 328 329 We evaluated the accuracy of the trabecular reconstruction method on the surfaces used to create 330 the SSM using a leave-one-out approach. Specifically, the linear regression models were 331 iteratively developed while leaving out one of the participants surfaces – and the reconstruction 332 accuracy of the ‘left-out’ trabecular surface evaluated using the mean and peak error in surface 333 node position, alongside the Jaccard index. Mean and peak error in surface node position (in 334 mm), and the Jaccard index for the reconstructed trabecular surfaces using the prediction model 335 were 3.05 [2.64, 3.47 95% CIs], 8.38 [7.21, 9.54 95% CIs] and 0.756 [0.750, 0.762 95% CIs], 336 respectively (see Figure 5). Figure 7 presents an ‘average’ performing reconstruction (i.e. Discussion 190 surface 337 with Jaccard index closest to the mean) of the trabecular surface compared to the originally 338 segmented trabecular for this participant. 339 Case Two: Predicting and Generating Trabecular Volumes 300 Finite element simulations of tibial loading typically include different parts and material 301 properties for the cortical and trabecular volumes of the bone (Edwards et al., 2010; Haider, 302 Baggaley & Edwards, 2020). Certain scenarios can make generating surface and volumetric 303 meshes of the trabecular more difficult or time consuming. Segmenting the trabecular surface 304 from CT scans is possible, yet more time-consuming given the need for greater manual 305 corrections. MRIs that are not optimized to detect the trabecular volume can also generate 306 difficulties in segmenting the trabecular area due to lacking clearly identifiable borders between 307 the cortical and trabecular areas. Other bone surface reconstruction methods (e.g. MAP-client 308 (Zhang et al., 2014b)) only provide estimates of external bone surface and not internal trabecular 309 volumes. Given that performing finite element simulations with both trabecular and cortical bone 310 is more reflective of real-world scenarios, there are practical benefits to developing methods that 311 can predict and generate trabecular volumes based on the more easily obtainable outer bone 312 surface. 313 In this case study, we use the SSM of the tibia and trabecular to develop and evaluate a method 315 for estimating a correspondent trabecular surface. The theoretical hypothesis for this approach is 316 that the outer shape of the tibia can be effective in predicting the internal trabecular shape. The 317 ‘generateTrabecularFromSurface.m’ function provided with our dataset demonstrates the 318 development and evaluation of this method on the dataset used in the present study, followed by 319 the application of the method to a new set of tibial surfaces. Briefly, this code develops a set of 320 linear regression models (see supplementary file 4) using the retained principal component 321 scores from the tibia model to predict each of the retained principal component scores of the 322 trabecular model. The predicted scores can then be combined with the trabecular SSM to 323 reconstruct the trabecular surface. In developing this method we noted that it was common for 324 the reconstructed trabecular to project outside of the tibial surface (i.e. not fitting within the outer 325 tibia boundary). Discussion 190 To combat this, we included a final step where points on the reconstructed 326 trabecular surface that protruded outside the tibia surface were translated to the closest point one 327 millimetre inside the boundary of the tibia. 328 We evaluated the accuracy of the trabecular reconstruction method on the surfaces used to create 330 the SSM using a leave-one-out approach. Specifically, the linear regression models were 331 iteratively developed while leaving out one of the participants surfaces – and the reconstruction 332 accuracy of the ‘left-out’ trabecular surface evaluated using the mean and peak error in surface 333 node position, alongside the Jaccard index. Mean and peak error in surface node position (in 334 mm), and the Jaccard index for the reconstructed trabecular surfaces using the prediction model 335 were 3.05 [2.64, 3.47 95% CIs], 8.38 [7.21, 9.54 95% CIs] and 0.756 [0.750, 0.762 95% CIs], 336 respectively (see Figure 5). Figure 7 presents an ‘average’ performing reconstruction (i.e. surface 337 with Jaccard index closest to the mean) of the trabecular surface compared to the originally 338 segmented trabecular for this participant. 339 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint 340 341 Figure 5. Mean and peak error in surface node position, and the Jaccard index for the 342 reconstructed trabecular surfaces using the prediction model. Error variables are presented in 343 mean (dashed line) and 95% confidence intervals (dotted lines). 344 he in 340 340 341 Figure 5. Mean and peak error in surface node position, and the Jaccard index for the 342 reconstructed trabecular surfaces using the prediction model. Error variables are presented in 343 mean (dashed line) and 95% confidence intervals (dotted lines). 344 he in Figure 5. Mean and peak error in surface node position, and the Jaccard index for the 342 reconstructed trabecular surfaces using the prediction model. Error variables are presented in 343 mean (dashed line) and 95% confidence intervals (dotted lines). 344 he in Figure 5. Mean and peak error in surface node position, and the Jaccard index for the 342 reconstructed trabecular surfaces using the prediction model. Discussion 190 Error variables are presented in 343 mean (dashed line) and 95% confidence intervals (dotted lines). 344 he in 345 346 Figure 6. Comparison of a reconstructed trabecular surface (i.e. surface with Jaccard index 347 closest to the mean) compared to the originally segmented trabecular for this participant. The 348 transparent grey surface is the original segmentation and the colour map is the predicted 349 trabecular surface. Warmer colours represent greater point distance (mm) from the original 350 segmentation. 351 ex he ed al 346 Figure 6. Comparison of a reconstructed trabecular surface (i.e. surface with Jaccard index 347 closest to the mean) compared to the originally segmented trabecular for this participant. The 348 transparent grey surface is the original segmentation and the colour map is the predicted 349 trabecular surface. Warmer colours represent greater point distance (mm) from the original 350 segmentation. 351 ex he ed al Figure 6. Comparison of a reconstructed trabecular surface (i.e. surface with Jaccard index 347 closest to the mean) compared to the originally segmented trabecular for this participant. The 348 transparent grey surface is the original segmentation and the colour map is the predicted 349 trabecular surface. Warmer colours represent greater point distance (mm) from the original 350 segmentation. 351 ex he ed al . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint This case study also provides an example of how this method can be applied to estimate 352 trabecular volumes on newly acquired tibial surface (Nolte et al., 2016). The trabecular of a 353 randomly selected sample from the tibia-fibula surfaces provided by Nolte et al., (2016) was 354 predicted using the shape models from the present study (see Figure 7). The relative accuracy of 355 these specific trabecular reconstructions cannot be evaluated due to Nolte et al., (2016) only 356 providing the outer tibial surface meshes. Discussion 190 We used the same set of 378 palpable landmarks as Bruce et al., (2021) those being the tibial tuberosity, medial condyle, 379 lateral and medial malleoli, lateral aspect of the fibula head, anterior border of the tibia at 25%, 380 50% and 75% of the distance between the medial condyle and malleolus, and the lateral fibula 381 diaphysis at 25% of the distance from the lateral malleolus to the lateral point on the fibula head. 382 These landmarks were digitized on the mean surface from the tibia-fibula SSM, as well as on 35 383 surfaces from the new dataset (Nolte et al., 2020). We performed nodal correspondence and 384 registration of the new surfaces to the SSM mean using the coherent drift point algorithm and 385 Procrustes analysis, respectively. The principal component scores from the SSM were then 386 manipulated within an optimization that minimized the summed Euclidean distance between the 387 landmarks on the SSM mean surface and new surface. 388 389 The mean and summed error in landmark positions (in mm) were 4.29 [4.01, 4.58 95% CIs] and 390 38.65 [36.09, 41.22 95% CIs], respectively; while the Jaccard index values for the entire 391 reconstruction were 0.537 [0.517, 0.557 95% CIs] (see row 1 of Figure 8). Mean and peak error 392 in surface node position (in mm) for the tibia surface were 6.63 [5.88, 7.39 95% CIs] and 13.39 393 [12.12, 14.65 95% CIs], respectively. The Jaccard index for the reconstructed tibia surfaces was 394 0.638 [0.620, 0.657 95% CIs] (see row 2 of Figure 8). Mean and peak error in surface node 395 position (in mm) for the fibula, and the Jaccard index for the reconstructed fibula surfaces were 396 10.34 [8.85, 11.84 95% CIs], 20.82 [17.97, 23.67 95% CIs] and 0.271 [0.240, 0.303 95% CIs], 397 respectively (see row 3 of Figure 8). 398 399 Case Three: Generating Tibia-Fibula Surfaces from Palpable Landmarks 366 Providing methods to generate three-dimensional models of the tibia-fibula allow those who 367 cannot acquire medical images to use these models within their work or practice. Obtaining 368 spatial measurements from palpable landmarks on the tibia-fibula surface is a much more 369 accessible method and can be achieved through motion capture or video-based methods. Discussion 190 Past 370 work (Bruce et al., 2021) has demonstrated that relatively accurate reconstructions of tibia-fibula 371 surfaces can be obtained by fitting SSM to palpable landmarks via optimization procedures. 372 373 In this case study, we provide a similar application to that outlined by Bruce et al., (2021). We 374 combine our SSM of the tibia-fibula with a set of measured palpable landmarks to reconstruct 375 the entire surface. The ‘generateSurfaceFromLandmarks.m’ function provided with our dataset 376 demonstrates an optimization procedure which fits the tibia-fibula SSM to a set of palpable 377 landmarks on a new set of tibia-fibula surfaces (Nolte et al., 2020). We used the same set of 378 palpable landmarks as Bruce et al., (2021) those being the tibial tuberosity, medial condyle, 379 lateral and medial malleoli, lateral aspect of the fibula head, anterior border of the tibia at 25%, 380 50% and 75% of the distance between the medial condyle and malleolus, and the lateral fibula 381 diaphysis at 25% of the distance from the lateral malleolus to the lateral point on the fibula head. 382 These landmarks were digitized on the mean surface from the tibia-fibula SSM, as well as on 35 383 surfaces from the new dataset (Nolte et al., 2020). We performed nodal correspondence and 384 registration of the new surfaces to the SSM mean using the coherent drift point algorithm and 385 Procrustes analysis, respectively. The principal component scores from the SSM were then 386 manipulated within an optimization that minimized the summed Euclidean distance between the 387 landmarks on the SSM mean surface and new surface. 388 389 The mean and summed error in landmark positions (in mm) were 4.29 [4.01, 4.58 95% CIs] and 390 38.65 [36.09, 41.22 95% CIs], respectively; while the Jaccard index values for the entire 391 reconstruction were 0.537 [0.517, 0.557 95% CIs] (see row 1 of Figure 8). Mean and peak error 392 in surface node position (in mm) for the tibia surface were 6.63 [5.88, 7.39 95% CIs] and 13.39 393 [12.12, 14.65 95% CIs], respectively. The Jaccard index for the reconstructed tibia surfaces was 394 0.638 [0.620, 0.657 95% CIs] (see row 2 of Figure 8). Discussion 190 Qualitative evaluation of the predicted trabecular 357 surface from this new sample revealed potential quality issues towards the ends of the bone. 358 Nonetheless, the code is provided to demonstrate how this method can be applied to any tibial 359 surface data with the caveat that quality control checks following predictions are likely required. 360 361 362 Figure 7. Predicted trabecular surface (red) of a randomly selected tibia (transparent grey) from 363 Notle et al., (2016). 364 365 Figure 7. Predicted trabecular surface (red) of a randomly selected tibi 363 abecular surface (red) of a randomly selected tibia (transparent grey) from e 7. Predicted trabecular surface (red) of a randomly selected tibia (transparent gr . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Case Three: Generating Tibia-Fibula Surfaces from Palpable Landmarks 366 Providing methods to generate three-dimensional models of the tibia-fibula allow those who 367 cannot acquire medical images to use these models within their work or practice. Obtaining 368 spatial measurements from palpable landmarks on the tibia-fibula surface is a much more 369 accessible method and can be achieved through motion capture or video-based methods. Past 370 work (Bruce et al., 2021) has demonstrated that relatively accurate reconstructions of tibia-fibula 371 surfaces can be obtained by fitting SSM to palpable landmarks via optimization procedures. 372 373 In this case study, we provide a similar application to that outlined by Bruce et al., (2021). We 374 combine our SSM of the tibia-fibula with a set of measured palpable landmarks to reconstruct 375 the entire surface. The ‘generateSurfaceFromLandmarks.m’ function provided with our dataset 376 demonstrates an optimization procedure which fits the tibia-fibula SSM to a set of palpable 377 landmarks on a new set of tibia-fibula surfaces (Nolte et al., 2020). Discussion 190 Mean and peak error in surface node 395 position (in mm) for the fibula, and the Jaccard index for the reconstructed fibula surfaces were 396 10.34 [8.85, 11.84 95% CIs], 20.82 [17.97, 23.67 95% CIs] and 0.271 [0.240, 0.303 95% CIs], 397 respectively (see row 3 of Figure 8) 398 Case Three: Generating Tibia-Fibula Surfaces from Palpable Landmarks 366 Providing methods to generate three-dimensional models of the tibia-fibula allow those who 367 cannot acquire medical images to use these models within their work or practice. Obtaining 368 spatial measurements from palpable landmarks on the tibia-fibula surface is a much more 369 accessible method and can be achieved through motion capture or video-based methods. Past 370 work (Bruce et al., 2021) has demonstrated that relatively accurate reconstructions of tibia-fibula 371 surfaces can be obtained by fitting SSM to palpable landmarks via optimization procedures. 372 surfaces can be obtained by fitting SSM to palpable landmarks via optimization procedures. 372 373 In this case study, we provide a similar application to that outlined by Bruce et al., (2021). We 374 combine our SSM of the tibia-fibula with a set of measured palpable landmarks to reconstruct 375 the entire surface. The ‘generateSurfaceFromLandmarks.m’ function provided with our dataset 376 demonstrates an optimization procedure which fits the tibia-fibula SSM to a set of palpable 377 landmarks on a new set of tibia-fibula surfaces (Nolte et al., 2020). We used the same set of 378 palpable landmarks as Bruce et al., (2021) those being the tibial tuberosity, medial condyle, 379 lateral and medial malleoli, lateral aspect of the fibula head, anterior border of the tibia at 25%, 380 50% and 75% of the distance between the medial condyle and malleolus, and the lateral fibula 381 diaphysis at 25% of the distance from the lateral malleolus to the lateral point on the fibula head. 382 These landmarks were digitized on the mean surface from the tibia-fibula SSM, as well as on 35 383 surfaces from the new dataset (Nolte et al., 2020). We performed nodal correspondence and 384 registration of the new surfaces to the SSM mean using the coherent drift point algorithm and 385 Procrustes analysis, respectively. The principal component scores from the SSM were then 386 manipulated within an optimization that minimized the summed Euclidean distance between the 387 landmarks on the SSM mean surface and new surface. Discussion 190 388 The mean and summed error in landmark positions (in mm) were 4.29 [4.01, 4.58 95% CIs] and 390 38.65 [36.09, 41.22 95% CIs], respectively; while the Jaccard index values for the entire 391 reconstruction were 0.537 [0.517, 0.557 95% CIs] (see row 1 of Figure 8). Mean and peak error 392 in surface node position (in mm) for the tibia surface were 6.63 [5.88, 7.39 95% CIs] and 13.39 393 [12.12, 14.65 95% CIs], respectively. The Jaccard index for the reconstructed tibia surfaces was 394 0.638 [0.620, 0.657 95% CIs] (see row 2 of Figure 8). Mean and peak error in surface node 395 position (in mm) for the fibula, and the Jaccard index for the reconstructed fibula surfaces were 396 10.34 [8.85, 11.84 95% CIs], 20.82 [17.97, 23.67 95% CIs] and 0.271 [0.240, 0.303 95% CIs], 397 respectively (see row 3 of Figure 8). 398 399 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint 400 Figure 8. The mean and summed error, and Jaccard index for landmark positions (Row 1). Mean 401 and peak error of the surface node position, and the Jaccard index for the tibia surfaces (Row 2) 402 and fibula surface (Row 3) reconstructed using the palpable landmarks. Error variables are 403 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 404 an 2) re Figure 8. The mean and summed error, and Jaccard index for landmark positions (Row 1). Mean 01 and peak error of the surface node position, and the Jaccard index for the tibia surfaces (Row 2) 02 and fibula surface (Row 3) reconstructed using the palpable landmarks. Error variables are 03 presented in mean (dashed line) and 95% confidence intervals (dotted lines). 04 an 2) re . Discussion 190 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint performance was likely due to how well the original SSM captured the shape characteristics 418 present in the individual samples. 419 performance was likely due to how well the original SSM captured the shape characteristics 418 present in the individual samples. 419 420 421 422 Figure 9. Example of a ‘poor’ reconstruction of the fibula from a minimized set of palpable 423 landmarks. The transparent grey surface is the original segmentation, and the color map is the 424 reconstructed surface. Warmer colours represent greater point distance (mm) from the original 425 segmentation. 426 427 422 Figure 9. Example of a ‘poor’ reconstruction of the fibula from a minimized set of palpable 423 landmarks. The transparent grey surface is the original segmentation, and the color map is the 424 reconstructed surface. Warmer colours represent greater point distance (mm) from the original 425 segmentation. 426 Figure 9. Example of a ‘poor’ reconstruction of the fibula from a minimized set of palpable 423 landmarks. The transparent grey surface is the original segmentation, and the color map is the 424 reconstructed surface. Warmer colours represent greater point distance (mm) from the original 425 segmentation. 426 g 427 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint 428 Figure 10. Discussion 190 Example of a ‘good’ reconstruction of the tibia and fibula from a minimised set of 429 palpable landmarks. The transparent grey surface is the original segmentation, and the color map 430 is the reconstructed surface. Warmer colours represent greater point distance (mm) from the 431 original segmentation. 432 433 Figure 10. Example of a ‘good’ reconstruction of the tibia and fibula from a minimised set of 429 palpable landmarks. The transparent grey surface is the original segmentation, and the color map 430 is the reconstructed surface. Warmer colours represent greater point distance (mm) from the 431 original segmentation. 432 Limitations 434 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502722 doi: bioRxiv preprint Lastly, due to only using the right tibia-fibula, researchers would be unable to use the code and 449 SSM to reconstruct or perform analysis on a left tibia-fibula. 450 451 Lastly, due to only using the right tibia-fibula, researchers would be unable to use the code and 449 SSM to reconstruct or perform analysis on a left tibia-fibula. 450 SSM to reconstruct or perform analysis on a left tibia-fibula. 450 451 Conclusions 452 Across all three of our SSM the largest source of geometric variation was a general size 453 variation, with changes in both length and width observed. Important variations that could 454 increase the risk of tibial stress injury were observed in the tibia and tibia-fibula SSM. These 455 included general tibial thickness, midshaft thickness, tibial length, and medulla cavity diameter 456 (indicative of cortical thickness). Further research is warranted to better understand the effect of 457 these tibial and fibula shape characteristics on tibial stress, loading and injury risk. This SSM 458 and associated code has been provided in an opensource dataset for use within the research 459 community. The associated code includes three example applications, these include generation of 460 a random sample, reconstruction of trabecular surfaces and reconstruction from palpable 461 landmarks. In providing this dataset we hope to improve research skills in those individuals who 462 may not have access to the knowledge or training. Further, this dataset could help to improve 463 understanding of bone stress injuries, and potentially assist other research such as the 464 implementation of medical devices (i.e. plates and braces). The SSM and all available code will 465 be available for use at (https://simtk.org/projects/ssm_tibia.). Limitations 434 466 467 Acknowledgements 468 The authors would like to acknowledge and thank the creators of the New Mexico Decedent 469 Image database for providing the CT imagery used to conduct this research. 470 471 Conclusions 452 Across all three of our SSM the largest source of geometric variation was a general size 453 variation, with changes in both length and width observed. Important variations that could 454 increase the risk of tibial stress injury were observed in the tibia and tibia-fibula SSM. These 455 included general tibial thickness, midshaft thickness, tibial length, and medulla cavity diameter 456 (indicative of cortical thickness). Further research is warranted to better understand the effect of 457 these tibial and fibula shape characteristics on tibial stress, loading and injury risk. This SSM 458 and associated code has been provided in an opensource dataset for use within the research 459 community. The associated code includes three example applications, these include generation of 460 a random sample, reconstruction of trabecular surfaces and reconstruction from palpable 461 landmarks. In providing this dataset we hope to improve research skills in those individuals who 462 may not have access to the knowledge or training. Further, this dataset could help to improve 463 understanding of bone stress injuries, and potentially assist other research such as the 464 implementation of medical devices (i.e. plates and braces). The SSM and all available code will 465 be available for use at (https://simtk.org/projects/ssm_tibia.). 466 46 References 472 References 472 Beck BR. 1998. Tibial stress injuries. An aetiological review for the purposes of guiding 473 management. Sports Medicine 26:265–279. 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https://openalex.org/W3196500375	https://europepmc.org/articles/pmc8464692?pdf=render	English	null	Progressing towards Sustainable Machining of Steels: A Detailed Review	Materials	2,021	cc-by	28,540	Review Progressing towards Sustainable Machining of Steels: A Detailed Review Kashif Ishfaq 1,, Irfan Anjum 1, Catalin Iulian Pruncu 2, , Muhammad Amjad 3 , M. Saravana Kumar 4 and Muhammad Asad Maqsood 1 1 Department of Industrial and Manufacturing Engineering, University of Engineering & Technology, Lahore 548900, Pakistan; irfanhanjum@gmail.com (I.A.); 2016im11@student.uet.edu.pk (M.A.M.) 2 Design, Manufacturing & Engineering Management, University of Strathclyde, Glasgow G1 1XJ, Scotland, UK 3 Department of Mechanical, Mechatronics and Manufacturing Engineering, University of Engineering & Technology, Lahore 548900, Pakistan; amjad9002@uet.edu.pk 4 Department of Production Engineering, National Institute of Technology, Tiruchirappalli 620015, Tamil Nadu, India; saravana312@gmail.com * Correspondence: kashif.ishfaq@uet.edu.pk (K.I.); Catalin.pruncu@strath.ac.uk (C.I.P.) 1 Department of Industrial and Manufacturing Engineering, University of Engineering & Technology, 1 Department of Industrial and Manufacturing Engineering, University of Engineering & Technology, Lahore 548900, Pakistan; irfanhanjum@gmail.com (I.A.); 2016im11@student.uet.edu.pk (M.A.M.) 2 Design, Manufacturing & Engineering Management, University of Strathclyde, Glasgow G1 1XJ, Scotland, UK 3 1 Department of Industrial and Manufacturing Engineering, University of Engineering & Technology, Lahore 548900, Pakistan; irfanhanjum@gmail.com (I.A.); 2016im11@student.uet.edu.pk (M.A.M.) 2 Design, Manufacturing & Engineering Management, University of Strathclyde, Glasgow G1 1XJ, Scotland, UK 3 Department of Mechanical, Mechatronics and Manufacturing Engineering, University of Engineering & Technology, Lahore 548900, Pakistan; amjad9002@uet.edu.pk Lahore 548900, Pakistan; irfanhanjum@gmail.com (I.A.); 2016im11@student.uet.edu.pk (M.A.M.) 2 Design, Manufacturing & Engineering Management, University of Strathclyde, Glasgow G1 1XJ, Scotland, UK gy j p 4 Department of Production Engineering, National Institute of Technology, 4 Department of Production Engineering, National Institute of Technology, Tiruchirappalli 620015 Tamil Nadu India; saravana312@gmail com pp g * Correspondence: kashif.ishfaq@uet.edu.pk (K.I.); Catalin.pruncu@strath.ac.uk (C.I.P.) Abstract: Machining operations are very common for the production of auto parts, i.e., connecting rods, crankshafts, etc. In machining, the use of cutting oil is very necessary, but it leads to higher machining costs and environmental problems. About 17% of the cost of any product is associated with cutting ﬂuid, and about 80% of skin diseases are due to mist and fumes generated by cutting oils. Environmental legislation and operators’ safety demand the minimal use of cutting ﬂuid and proper disposal of used cutting oil. The disposal cost is huge, about two times higher than the machining cost. To improve occupational health and safety and the reduction of product costs, companies are moving towards sustainable manufacturing. Keywords: sustainable manufacturing; minimum quantity lubrication; cryogenic machining; solid lubricants; vegetable oils; steels Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Citation: Ishfaq, K.; Anjum, I.; Pruncu, C.I.; Amjad, M.; Kumar, M.S.; Maqsood, M.A. Progressing towards Sustainable Machining of Steels: A Detailed Review. Materials 2021, 14, 5162. https://doi.org/10.3390/ ma14185162 Review Progressing towards Sustainable Machining of Steels: A Detailed Review Therefore, this review article emphasizes the sustainable machining aspects of steel by employing techniques that require the minimal use of cutting oils, i.e., minimum quantity lubrication, and other efﬁcient techniques like cryogenic cooling, dry cutting, solid lubricants, air/vapor/gas cooling, and cryogenic treatment. Cryogenic treatment on tools and the use of vegetable oils or biodegradable oils instead of mineral oils are used as primary techniques to enhance the overall part quality, which leads to longer tool life with no negative impacts on the environment. To further help the manufacturing community in progressing towards industry 4.0 and obtaining net-zero emissions, in this paper, we present a comprehensive review of the recent, state of the art sustainable techniques used for machining steel materials/components by which the industry can massively improve their product quality and production. Citation: Ishfaq, K.; Anjum, I.; Pruncu, C.I.; Amjad, M.; Kumar, M.S.; Maqsood, M.A. Progressing towards Sustainable Machining of Steels: A Detailed Review. Materials 2021, 14, 5162. https://doi.org/10.3390/ ma14185162 Academic Editors: Stefano Guarino and Flaviana Tagliaferri Received: 31 July 2021 Accepted: 3 September 2021 Published: 8 September 2021 materials materials 1.1. Manufacturing Industries and Sustainable Manufacturing 1.1. Manufacturing Industries and Sustainable Manufacturing It is well noted that machining is widely used to produce automotive parts within the manufacturing industry sector. In all machining operations, cutting ﬂuids play a vital role in reducing the machining cost by increasing tool life. It was observed that 7–17% of the cost incurred in the machining of a part is associated with using cutting ﬂuids. Further, the tooling cost is about 2–4%, so it is necessary to improve the whole process. In addition, the use of cutting ﬂuids causes health diseases like skin problems, allergies, eyes problems, and cancer in workers. Here, skin problem is about 80% [6]. Lawal et al. [7] also witnessed that major skin problems, about 80% in quantity, are due to cutting ﬂuids. They also proposed that vegetable oil-based and metal working liquids have been proven to be environmentally sustainable in the dielectric regime. g Strict environmental regulations demand that cutting oil used during machining pro- cesses should be recycled or disposed of in such a way that it will not spoil the environment and will be harmless for all interested parties. These ﬂuids are extremely costly to dispose of or store. The cost is about double the machining cost depending on the cutting ﬂuid which is being used. Mineral oils used as cutting ﬂuids are difﬁcult to dispose of into the environment without any prior treatment [8–10]. 1. Introduction Manufacturing products while conserving natural resources and causing no negative environmental impacts is called sustainable manufacturing. Manufacturing industries create products for fulﬁlling human needs; however, this includes the consumption of huge amounts of raw resources and the generation of wastes which are increasing day by day and can be very detrimental for our environment. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The following three stages of product waste are primary factors for waste generation and the degradation of the environment: • In the manufacturing processes; • During usage of the product; • At the end of the life of the product. The production of metals triggers the consumption of natural resources and has created a harmful effect on humankind. To avoid using resources needed by future genera- https://www.mdpi.com/journal/materials Materials 2021, 14, 5162. https://doi.org/10.3390/ma14185162 Materials 2021, 14, 5162 2 of 47 2 of 47 tions, it is necessary to use fewer natural resources and reduce the negative environmental impact caused by manufacturing systems. That is why industries are now moving towards sustainable manufacturing. Early ideas about sustainable manufacturing ﬁrst appeared in the 1970s and 1980s [1–5]. 1.2. Need for Sustainable Manufacturing Jordi Oliver Solà, Chief Executive Ofﬁcer (CEO) of a circular economy consulting group, has demonstrated the importance of sustainability, not only from an ethical or environmental point of view, but also that it is needed for markets to be competitive and important for the survival of any sector [11]. Therefore, to save natural resources, sustainable manufacturing is very important [12,13]. The need for sustainable manufacturing techniques is also depicted by the three pillars of sustainable manufacturing, as shown below in Figure 1. One of these is the need for improvement from an economic, social, and environmental point of view. It brings balance between social, economic, and environmental aspects [14,15]. This technique mainly deals with the minimal usage of cutting ﬂuids. It does not mean just stoping the supply of cutting oils to make the environment better. Cutting oils serve many purposes like lubrication and temperature reduction in the cutting zones. Figure 1. Three Pillars of Sustainability, reprinted with permission from ref. [16]. Copyright 2017 BSP books Pvt Ltd. Figure 1. Three Pillars of Sustainability, reprinted with permission from ref. [16]. Copyright 2017 BSP books Pvt Ltd. Materials 2021, 14, 5162 3 of 47 3 of 47 Concerns about environmental impacts, climate change, occupational health and safety, and machining costs have forced companies to move towards sustainable tech- niques. As per the investigation of Jayal et al. [17], the selection of sustainability aspects occurs mainly because of factors like the increase in diseases in shop ﬂoor workers, inﬂex- ibility in government plans, and when targeted to minimize the cost of production. So, sustainable machining is highly recommended where traditional cutting methods became null. Currently, advanced technologies like cryogenic cooling, nano cutting ﬂuids, dry cutting, and minimum quantity lubrication (MQL), etc. are being used [18]. Several studies have been published that emphasize the importance of sustainability. For example, Zein et al. [19] presented certain resources which are associated with the manufacturing technologies, including production tools and methods that directly correlate with economic impact. They outlined that the sustainability of a ﬁrm can be affected by manufacturing approaches. Jayal et al. [17] established a case study on machining tech- niques by improving the model at the process, product, and system-level for sustainable manufacturing. Sarkis [20] found a relationship between environmental concerns and manufacturing activities. 1.2. Need for Sustainable Manufacturing The study concluded that sustainable machining not only deals with environmental initiatives but also included techniques that empower beneﬁts for hu- manity. Lu et al. [21] developed a metric to ensure the sustainability of the manufacturing process. They also established the interrelationship between the elements of metrics and studied the potential impact. The metrics broadly covered the given elements, i.e., social, environmental, and economic. Jawahir and Dillon [22] and Hegab et al. [23] highlighted six of the most important factors that alter the paradigm of sustainability in manufacturing processes. The factors included cost, energy, the safety of workers, personal health, environ- mental impact, and waste. The researchers said that out of the six aforementioned elements, waste, cost, and energy can be more easily computed than the rest of the elements. Waas et al. [24] made a framework for the sustainability of manufacturing sectors by taking the hierarchy of social needs and combinations. Then they used the Delphi technique to propose the metrics for each category. Some researchers suggested rules to achieve sustainability in the manufacturing ﬁrms, as demonstrated by Lovin et al. [25]. The rules are (1) minimal usage of energy and material, (2) usage of cleaner production, recycling and conversion techniques to reusable substance, (3) adoption of a solution based system (i.e., supply chain structure) rather than a proactive business model, and (4) reinvestment in natural substitutions that are available for distinct materials, such as investing in renewable resources instead of non-renewable substances. From the machining perspective, Diaz Elsayed et al. [26] discussed a detailed study about the combination of green and lean in the automotive organization. The purpose of their research was to determine the effect of green-lean in the manufacturing sector. They concluded that grouping of green-lean proved an effective way of improving manufacturing ﬁrms in terms of waste reduction, less resource utilization, and energy consumption. Thus, they stated that the use of green-lean is a sustainable manufacturing approach for different enterprises. Abdul Rashid et al. [27] also investigated environmental performance by employing sustainable manufacturing techniques. They proposed that the main environmental initiatives are entirely based on manufacturing practices. In the same vein, Rusinko [28] established the relationship between manufacturing activities and their results. The results revealed that manufacturing cost is decreased by preventing waste and unnecessary substances. According to Gimenez et al. 1.2. Need for Sustainable Manufacturing [29], organizations should improve their environmental, social, and economic be- havior to get a sustainable approved system. The aforementioned literature divulged the importance of sustainability in manufacturing or business ﬁrms. Therefore, the current study was conducted to scrutinize a systematic review of the sustainable machining of steel, as it is being used in scattered application areas including aerospace, automotive, nuclear power plants, and medical equipment, etc. Th b ﬁt i ﬁ i l i t l d f t hi h There are numerous beneﬁts, i.e., ﬁnancial, environmental, and safety, which are related to the three pillars as discussed before. The need to turn towards sustainable manu- facturing is due to many reasons like occupational health-related problems, environmental Materials 2021, 14, 5162 4 of 47 regulations, and unsafe or polluted environments for workers, but the largest is the waste cost in using too much cutting ﬂuid in metal cutting industries. In such industries, the costs associated with purchasing, maintaining, the makeup of cutting ﬂuid, cutting oil, and system cleaning are more prominent. The consideration of the following points allows companies to improve these three pillars: • Efﬁcient resource utilization (Energy, Material, Water, Labor, etc.); • Improvement in the application of metalworking ﬂuids; • Adopting other sustainable manufacturing techniques; • Lean Implementation; • Lean Implementation; p mprovement in the working environment by applying best machining practices; • Improvement in the working environment by applying best machining practices; Most important, training to all employees related to sustainable machining. • Most important, training to all employees related to sustainable machining. Figure 2 shows the basic objectives by which pressure was built on manufactures to change their way of working. It is clearly depicted that there is a need to change the whole scenario of conventional working in manufacturing industries to improve socially, economically, and environmentally. Technology revolution should be introduced in manufacturing industries to lower the cost per piece of product. Whereas Figure 3 shows the breakdown of the product cost, including cooling and lubricating costs, which are about 18%. Figure 2. Pressure to change the paradigms of the manufacturing industry, reprinted with permission from ref. [30]. Copyright 2010 Elsevier Ltd. Figure 2. Pressure to change the paradigms of the manufacturing industry, reprinted with permission from ref. [30]. Copyright 2010 Elsevier Ltd. Figure 3. Cooling and lubricating costs incorporated in the automotive sector, reprinted with permission from ref. [31]. Copyright 2010 Elsevier Ltd. 1.2. Need for Sustainable Manufacturing Figure 3. Cooling and lubricating costs incorporated in the automotive sector, reprinted with permission from ref. [31]. Copyright 2010 Elsevier Ltd. Manufacturing companies can improve their costs and tackle environmental issues by implementing sustainable principles. Implementation can be done by analyzing the current situation of the process or system in any industry. There is a need to adopt alternate Materials 2021, 14, 5162 5 of 47 5 of 47 technologies for redesigning systems for the effective realization of these principles in factories [30]. The key methods that provide a direct path to create a cleaner manufacturing sector are depicted in Figure 4. technologies for redesigning systems for the effective realization of these principles in factories [30]. The key methods that provide a direct path to create a cleaner manufacturing sector are depicted in Figure 4. Figure 4. Implementation of Clean Manufacturing process, reprinted with permission from ref. [31]. Copyright 2010 Elsevier Ltd. Figure 4. Implementation of Clean Manufacturing process, reprinted with permission from ref. [31]. Copyright 2010 Elsevier Ltd. Further, some key characteristics of sustainable machining are presented in Figure 5, which clearly shows that this technique justiﬁed three pillars of sustainable manufacturing. Figure 6 shows the evolution over time of sustainable manufacturing, which depicts the critical importance of embedding sustainable manufacturing by 2025. It is assumed that the industries will work on 6-reduction (6R) elements rather than 3-reduction (3R) entities which are used in the actual green manufacturing model [32]. Figure 5. Characteristics of Sustainable machining, reprinted with permission from ref. [17]. Copy- right 2010 CIRP, Published by Elsevier Ltd. Figure 5. Characteristics of Sustainable machining, reprinted with permission from ref. [17]. Copy- right 2010 CIRP, Published by Elsevier Ltd. Figure 5. Characteristics of Sustainable machining, reprinted with permission from ref. [17]. Copy- right 2010 CIRP, Published by Elsevier Ltd. 6 of 47 Materials 2021, 14, 5162 Figure 6. Evolution with the time of sustainable manufacturing, reprinted with permission from ref. [32]. Copyright 2010 CIRP, Published by Elsevier Ltd. Figure 6. Evolution with the time of sustainable manufacturing, reprinted with permission from ref. [32]. Copyright 2010 CIRP, Published by Elsevier Ltd. As per the given literature, it has been found that certain areas need to be reviewed. For example, material wastage and the amount of disposed-off material during the process may cause the cost of machining to be high. 1.2. Need for Sustainable Manufacturing Moreover, suspended particles enter the environment and damage the quality of the air. The disturbing air quality index inﬂu- ences human life, and thus sustainability is compromised. Therefore, this review has been developed to understand the conditions/parameters of different sustainable machining techniques which alter the cost, pollute the environment, and decrease the overall pro- ductivity. For this context, a PRISMA approach has been adopted to study the variants of sustainable manufacturing processes as far as steel material is considered. The current review is restricted to sustainable techniques used for the machining of steel in order to ensure cost-effective, environmentally stable, and eco-friendly processes. Even though this review provides a comprehensive discussion on sustainable man- ufacturing techniques, other elements may be added, like frostbite hazards in cryogenic machining and initial setup cost, which is difﬁcult to afford by any local industry. Therefore, a comprehensive investigation is required to mitigate the aforesaid issues of cryogenic machining by ensuring a controlled temperature environment. The mathematical modeling of the sustainable cutting mechanisms with respect to the cutting of steel is still an area that needs special focus. This paper is presented in the following order: (i) A brief introduction with mechanical properties of some steel grades is proposed in Section 2. (ii) The comprehensive method- ology is given in Section 3. (iii) Different sustainable techniques employed in a couple of manufacturing sectors are demonstrated in Section 4. It constitutes different subsections; each outlines the discussion, signiﬁcance, advantages, disadvantages, and limitations of each sustainable technique separately. (iv) The detailed discussion about the present work, along with comparisons between each sustainable technique, has been granted in Section 5. (v) Section 6 illustrates the multiple challenges faced with the implementation of sustain- able manufacturing. (vi) Fundamental issues associated with additive manufactured steel has been given in Section 7. (vii) The ﬁndings are summarized in Section 8. (viii) Finally, future implications have been revealed in Section 9. Materials 2021, 14, 5162 7 of 47 2. Steels’ Classiﬁcation, Properties, Machining Difﬁculties, and Sustainability Requirements in Steels’ Machining 2. Steels’ Classiﬁcation, Properties, Machining Difﬁculties, and Sustainability Requirements in Steels’ Machining 2. Steels’ Classiﬁcation, Properties, Machining Difﬁculties, and Sustainability Requirements in Steels’ Machining An alloy of iron (Fe) with minimal carbon content is referred to as steel. Carbon (C), generally up to 1.5%, is present in steel [33]. As per the literature, about 1808 million tons of steel were produced in 2018 worldwide. This is depicted in Figure 7, along with the emis- sions of CO2 [34]. If the machinability perspective of steel is under consideration, then up to 29% of steel is employed in machining, as given by Diva Metal Ltd. Company [35]. Figure 8 represents the division of steel in different applications. Steel exists in the form of different variants like structural steel, heat resistant steel, and tool steel, etc. Another important class of steel is named Alloy Steel, a standard form that constitutes various elements (i.e., nickel, magnesium, copper, titanium, vanadium, silicon, boron, and manganese, etc.) in different proportions that range from 1.0% to 50% by weight. Alloy steel can be categorized into low alloy steel (LAS) and high alloy steel (HAS). Usually, the phrase “alloy steel” is related to LAS. Nickel (Ni) is a prime element in LAS that has the ability to increase the strength and ductility of different engineering applications, including jet engines, spacecraft, and nuclear reactors. Interestingly, Ni also ampliﬁes the characteristics of ferrite steel, such as stability at low-temperature toughness, which allows them to be used in cryogenic applications [36]. For instance, steel with 9% Ni can be employed for liqueﬁed natural gas (LNG) handling and storage purposes. Moreover, it assists in nitriding, carburizing, and tool steel due to tremendous properties like good strength, high hardness, superior toughness, the ability to withstand elevated temperatures, excellent wear, and corrosion resistance. The other combination, such as an alloy of Fe with C, is known as the simplest alloy. The ferromagnetic feature of Fe permits the use of the simplest steel in magnetic applications like electric motors, transformers, etc. [37]. The details about some key classes of steel are presented in the forthcoming sections. Figure 7. Worldwide steel production in 2018, along with the CO2 emission, reprinted with permis- sion from ref. [34]. Copyright 2019 Elsevier Ltd. Figure 7. Worldwide steel production in 2018, along with the CO2 emission, reprinted with permis- sion from ref. [34]. Copyright 2019 Elsevier Ltd. 8 of 47 Materials 2021, 14, 5162 Figure 8. Division of steel usage in different applications. 2. Steels’ Classiﬁcation, Properties, Machining Difﬁculties, and Sustainability Requirements in Steels’ Machining Figure 8. Division of steel usage in different applications. 2.1. Structural Steel (SS) SS is a commonly used building material in the construction industry. The perfor- mance of SS is now predictable and depends on standards recognized by the American Institute of Steel Construction (AISC), which elaborate shapes, sizes, elemental composi- tion, as well as mechanical attributes. SS is 100% recyclable and has proven to be one of the most reprocessed materials in the world [38]. The fundamental classiﬁcation of structural steel is: i. Carbon-manganese steel; ii. High strength low alloy (HSLA) steel; iii. High strength quenched and tempered alloy steels. From the above-mentioned classes of structural steel, HSLA is important because it provides good mechanical properties, high resistance to rust, and high weldability with a carbon percentage between 0.05–0.25%. The other beneﬁts of HSLA steel are: (i) light in weight, (ii) good strength to wear ratio, (iii) and control over internal and external stresses. However, HSLA has limitations in terms of acquiring more power (>25–30%) as compared to carbon steel. Additionally, such steel has sensitivity in directional properties [39]. The chemical composition of HSLA-80 steel is presented in Table 1, whereas the mechanical properties are listed in Table 2. Table 1. Chemical composition of HSLA Steel, reprinted with permission from ref. [39]. Copyright 2018 Elsevier Ltd. Elements Cu Ni Cr Mn Si Mo C Nb S P wt. % 1.25 0.83 0.78 0.54 0.37 0.19 0.07 0.03 0.024 0.022 Table 2. Mechanical characteristics of HSLA steel, reprinted with permission from ref. [39]. Copyright 2018 Elsevier Ltd. Properties Units Values Yield Stress (MPa) 450 ± 32 Ultimate strength (MPa) 778 ± 17 Elastic Modulus (GPa) 203 ± 5 Total Strain % 21 ± 2 Table 1. Chemical composition of HSLA Steel, reprinted with permission from ref. [39]. Copyright 2018 Elsevier Ltd. Materials 2021, 14, 5162 9 of 47 2.2. Heat Resistant Steel 2.2. Heat Resistant Steel Heat resistant steels (HRS) are a unique class of steel alloys that can easily be operated at temperatures as high as 750 ◦C. To attain their speciﬁc properties, all HRS are composed of numerous elements, two of which are considered basic elements, i.e., Chromium (Cr) and Nickel (Ni). Cr is preferred for corrosion resistance, while Ni is useful to obtain high strength and ductility. The other elements (aluminum, cobalt, manganese, niobium, copper, zirconium, and phosphorous, etc.) are added to achieve high-temperature properties along with good weldability. Based on chemical stability, high strength, and superb corrosion resistance, HRS are divided into three types; (1) Low alloy steels, (2) Martensitic steels, (3) Austenitic steels [40]. Low allow steels are extensively used in pressure-based applications like steam boilers and thermal power plants due to unique characteristics such as mechanical strength, great toughness, and sufﬁcient rust resistance ability. Such steel alloys are mostly preferred in thick components such as headers, pipes, and control valves. The different grades of low alloy steel have applications in distinct areas. Grade 11 (1CrMoV) and 22 (2.25Cr1Mo) are used in power developing industries. The mechanical properties of these low alloy steels are tabulated in Table 3. In the same way, Fe-0.1C-xMn and Fe-0.1C-xNi, where x = 1.5, 3% by mass, are special kinds of low alloy steels used for cryogenic treatments to decrease the corrosion property as well as improve the microstructure. The chemical composition of all the said low alloy steels are mentioned in Table 4. Table 3. Mechanical features of low alloy steels (grade 11 & 22), reprinted with permission from ref. [41]. Copyright 2007 Elsevier B.V. Alloys Yield Stress (MPa) Ultimate Tensile Stress (MPa) Elongation in % 1CrMoV 205 415 30 2.25Cr1Mo 205 415 30 Table 4. Chemical composition of different grades of low alloy steels, reprinted with permission from refs. [41,42]. Copyright 2015 Elsevier Ltd. Table 3. Mechanical features of low alloy steels (grade 11 & 22), reprinted with permission from ref. [41]. Copyright 2007 Elsevier B.V. Table 4. Chemical composition of different grades of low alloy steels, reprinted with permission from refs. [41,42]. Copyright 2015 Elsevier Ltd. Table 4. Chemical composition of different grades of low alloy steels, reprinted with permission from refs. [41,42]. Copyright 2015 Elsevier Ltd. 2.2. Heat Resistant Steel There are different variants of tool steels, including cold working, hot working, high speed, vibration resistance, water hardening, and some unusual purposes. The selection of this group is based on cost, temperature, surface hardness, ductility, and toughness values. In severe circumstances, carbide tool steels are utilized. They have applications in cutting, drawing dies, pressing, cold extrusion dies, broaches, thread rolling, forming rolls, and coining of materials. Another important application of tool steel is in the injection molding process, where durability plays an integral role. The common scale of tool steel grade is AISI-SAE. The chemical composition of some tool steel is given in Table 8. tal composition of some austenitic alloys, reprinted with permission from refs. [36,46]. Copyright 2018 Table 7. Elemental composition of some austenitic alloys, reprinted with permission from refs. [36,46]. Copyright 2018 Elsevier Ltd. Alloys Fe C Mn Si Mo Co Cr Cu Ni Others ASS304L 70.78 0.025 1.140 0.410 0.360 0.210 18.40 0.180 8.190 0.305 ASS316L 67.69 0.018 1.28 0.38 2.42 0.21 16.63 0.21 10.85 0.312 AISI 304 Balance 0.06 3.97 0.49 0.008 0.11 17.61 1.17 8.85 0.076 AISI 201 Balance 0.04 7.38 0.588 0.008 0.072 17.40 2.17 3.13 0.292 2.3. Tool Steel Table 7. Elemental composition of some austenitic alloys, reprinted with permission from refs. [36,46]. Copyright 2018 Elsevier Ltd Table 7. Elemental composition of some austenitic alloys, reprinted with permission from refs. [36,46]. Elsevier Ltd. 2.3. Tool Steel Tool steels are alloy steels that are appropriate for the manufacture of tools due to their excellent properties like high hardness, low deformation, minimal abrasion, and no wear and tear, even at elevated temperatures. Apart from the mentioned properties, these steels have a high magnitude of tensile and compressive yield strength which tends to minimize the plastic deformations at the stress concentration points in the tooling [47]. There are different variants of tool steels, including cold working, hot working, high speed, vibration resistance, water hardening, and some unusual purposes. The selection of this group is based on cost, temperature, surface hardness, ductility, and toughness values. In severe circumstances, carbide tool steels are utilized. They have applications in cutting, drawing dies, pressing, cold extrusion dies, broaches, thread rolling, forming rolls, and coining of materials. Another important application of tool steel is in the injection molding process, where durability plays an integral role. The common scale of tool steel grade is AISI-SAE. 2.2. Heat Resistant Steel Alloys C Si Mn P S Ni Fe Fe-0.1C-1.5Mn 0.10 0.01 1.48 0.001 0.002 0.01 Balance Fe-0.1C-3Mn 0.10 0.02 2.96 0.001 0.002 0.01 Balance Fe-0.1C-1.5Ni 0.09 0.01 0.02 0.001 0.002 1.58 Balance Fe-0.1C-3Ni 0.09 0.01 0.02 0.001 0.002 3.16 Balance 1CrMoV 0.15 0.50 0.60 0.025 0.025 0.03 Balance 2.25Cr1Mo 0.082 0.23 0.41 0.051 0.0054 0.03 Balance Martensitic heat resistant steels contain medium and high chromium contents of about 5–9% Cr and 12%, respectively. They have been fabricated for power plant materials where the temperature is signiﬁcantly higher, such as 650 ◦C. The high percentage of Cr in such steel alloys enhances the creep strength and corrosion resistance of the materials because of a low coefﬁcient of thermal expansion and high thermal conductivities in contrast to austenitic steels. Moreover, the emission of hazardous fumes and gases and the efﬁciency of the power plant are also raised due to the application of Cr. 9Cr and 12Cr, a special series of martensitic alloy steels [40]. The elemental composition of some martensitic alloys is given in Table 5, and mechanical properties are elaborated in Table 6. Alloys C Si Mn P S Ni Fe Fe-0.1C-1.5Mn 0.10 0.01 1.48 0.001 0.002 0.01 Balance Fe-0.1C-3Mn 0.10 0.02 2.96 0.001 0.002 0.01 Balance Fe-0.1C-1.5Ni 0.09 0.01 0.02 0.001 0.002 1.58 Balance Fe-0.1C-3Ni 0.09 0.01 0.02 0.001 0.002 3.16 Balance 1CrMoV 0.15 0.50 0.60 0.025 0.025 0.03 Balance 2.25Cr1Mo 0.082 0.23 0.41 0.051 0.0054 0.03 Balance Martensitic heat resistant steels contain medium and high chromium contents of about 5–9% Cr and 12%, respectively. They have been fabricated for power plant materials where the temperature is signiﬁcantly higher, such as 650 ◦C. The high percentage of Cr in such steel alloys enhances the creep strength and corrosion resistance of the materials because of a low coefﬁcient of thermal expansion and high thermal conductivities in contrast to austenitic steels. Moreover, the emission of hazardous fumes and gases and the efﬁciency of the power plant are also raised due to the application of Cr. 9Cr and 12Cr, a special series of martensitic alloy steels [40]. The elemental composition of some martensitic alloys is given in Table 5, and mechanical properties are elaborated in Table 6. 10 of 47 Materials 2021, 14, 5162 Table 5. Elemental composition of some martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). 2.2. Heat Resistant Steel Alloys Cr Mo V Nb C Mn Cu Si N Ni P S W 9Cr-1Mo 8.55 0.88 0.21 0.08 0.1 0.51 0.18 0.32 0.035 0.15 0.012 0.005 - 9Cr-1MoVNb 8.44 0.89 0.24 0.08 0.086 0.37 0.03 0.16 0.054 0.11 0.012 0.003 - 9Cr-1MoVNb-2Ni 8.57 0.98 0.22 0.066 0.064 - - - 0.053 2.17 - - 0.01 12Cr-1MoVW 11.99 0.93 0.27 0.018 0.21 - - - 0.020 0.43 - - 0.54 Table 6. Mechanical characteristics of martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). Alloys Yield Strength (MPa) Ultimate Tensile Stress (MPa) Elongation in % 9Cr-1Mo 533 683 26.0 9Cr-1MoVNb 547 697 11.9 9Cr-1MoVNb-2Ni 148 171 19.1 12Cr-1MoVW 110 142 19.6 Austenitic steel is also called austenitic stainless steel, with a Cr percentage of about 13% by weight at room temperature. The high cost associated with these alloys is due to the high percentage of supplementary elements compared to other steel alloys. The applications of austenitic alloys are limited to those conditions where chances of corrosion are substantial, such as in boiler tubes. They have similar properties to martensitic steels, except high thermal loading can lead to wear and tear over the surface. The FeCrNi is the most commonly used austenitic steel. However, AISI (American Iron and Steel Institute) 302, 304, 321, 347, 316, 309, ASS304L, ASS316L, and other alloys are also employed in different application sectors. The elemental composition of few austenitic alloys is provided in Table 7. Table 7. Elemental composition of some austenitic alloys, reprinted with permission from refs. [36,46]. Copyright 2018 Elsevier Ltd. Table 5. Elemental composition of some martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). Alloys Cr Mo V Nb C Mn Cu Si N Ni P S W 9Cr-1Mo 8.55 0.88 0.21 0.08 0.1 0.51 0.18 0.32 0.035 0.15 0.012 0.005 - 9Cr-1MoVNb 8.44 0.89 0.24 0.08 0.086 0.37 0.03 0.16 0.054 0.11 0.012 0.003 - 9Cr-1MoVNb-2Ni 8.57 0.98 0.22 0.066 0.064 - - - 0.053 2.17 - - 0.01 12Cr-1MoVW 11.99 0.93 0.27 0.018 0.21 - - - 0.020 0.43 - - 0.54 Table 6. Mechanical characteristics of martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). 2.2. Heat Resistant Steel Alloys Yield Strength (MPa) Ultimate Tensile Stress (MPa) Elongation in % 9Cr-1Mo 533 683 26.0 9Cr-1MoVNb 547 697 11.9 9Cr-1MoVNb-2Ni 148 171 19.1 12Cr-1MoVW 110 142 19.6 Table 5. Elemental composition of some martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). Table 6. Mechanical characteristics of martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). Alloys Yield Strength (MPa) Ultimate Tensile Stress (MPa) Elongation in % 9Cr-1Mo 533 683 26.0 9Cr-1MoVNb 547 697 11.9 9Cr-1MoVNb-2Ni 148 171 19.1 12Cr-1MoVW 110 142 19.6 Table 6. Mechanical characteristics of martensitic alloys, reprinted with permission from refs. [43–45]. Copyright 2020 International Atomic Energy Agency (IAEA). Austenitic steel is also called austenitic stainless steel, with a Cr percentage of about 13% by weight at room temperature. The high cost associated with these alloys is due to the high percentage of supplementary elements compared to other steel alloys. The applications of austenitic alloys are limited to those conditions where chances of corrosion are substantial, such as in boiler tubes. They have similar properties to martensitic steels, except high thermal loading can lead to wear and tear over the surface. The FeCrNi is the most commonly used austenitic steel. However, AISI (American Iron and Steel Institute) 302, 304, 321, 347, 316, 309, ASS304L, ASS316L, and other alloys are also employed in different application sectors. The elemental composition of few austenitic alloys is provided in Table 7. Table 7. Elemental composition of some austenitic alloys, reprinted with permission from refs. [36,46]. Copyright 2018 Elsevier Ltd. Alloys Fe C Mn Si Mo Co Cr Cu Ni Others ASS304L 70.78 0.025 1.140 0.410 0.360 0.210 18.40 0.180 8.190 0.305 ASS316L 67.69 0.018 1.28 0.38 2.42 0.21 16.63 0.21 10.85 0.312 AISI 304 Balance 0.06 3.97 0.49 0.008 0.11 17.61 1.17 8.85 0.076 AISI 201 Balance 0.04 7.38 0.588 0.008 0.072 17.40 2.17 3.13 0.292 2.3. Tool Steel Tool steels are alloy steels that are appropriate for the manufacture of tools due to their excellent properties like high hardness, low deformation, minimal abrasion, and no wear and tear, even at elevated temperatures. Apart from the mentioned properties, these steels have a high magnitude of tensile and compressive yield strength which tends to minimize the plastic deformations at the stress concentration points in the tooling [47]. 2.2. Heat Resistant Steel Alloys C Si Mn P S Ni Cr Mo Cu V W AISI D2 1.56 0.24 0.25 0.025 0.001 0.175 11.31 0.83 0.14 0.25 - AISI M4 1.33 0.33 0.26 0.03 0.03 0.3 4.25 4.88 0.25 4.12 5.88 HWS 1.08 1.38 0.34 - - - 7.80 1.86 - 2.66 1.73 Table 8. Chemical composition of some tool steel alloys, reprinted with permission from ref. [48]. Copyright 2018 MDPI Metals. Different machining methods have been practiced in past investigations on the steel material, including milling, drilling, broaching, grinding, planing, and turning, etc. How- ever, some methods induced complications while machining steel materials. For instance, Nagy et al. [49] said that machining (turning operation) of super duplex stainless steel is highly challenging when cutting tool inserts made up of PVD coating are used. The difﬁculties may be due to continuous and long chips formation, which is often problematic in the context of chip handling. Furthermore, long chips rolled on the part, and accordingly, stimulates surface imperfections. Eventually, surface quality is compromised. A similar problem has engaged with the austenitic steel. Sunil Magadum et al. [50] claimed that high strength, greater toughness, large fatigue, and corrosion resistivity are the prime reasons behind the poor machinability of steel. All the stated factors cause build-up-edge, irregular electrode wear, early tool failure during cryogenic machining of SS304 steel. Ingle et al. [51] proposed that there are certain grades of steel that have a machinability rating of 40%. Those grades belong to the austenitic steel such as 302B, 309, 309S, 330, 384, and 314. The authors demonstrated that a rating of less than 100% refers to the difﬁculty of machining alloys. As long as a rating is going down, then the difﬁculty level raises accord- ingly. The issues attributed to the aforesaid grades of austenitic steel are characterized by high ductility, toughness, prolong work-hardening, and less thermal conductivity. The machinability issues of martensitic steel grades (414, 422, 431, 440A, 440B, and 440C) has also been discussed for the austenitic steel grades. Steel materials are the most used materials in designing and manufacturing automo- tive components and in several other industrial sectors. The growth of the manufacturing industry, together with the need for cleaner production, makes the integration of sustain- able techniques necessary. 2.2. Heat Resistant Steel The chemical composition of some tool steel is given in Table 8. Materials 2021, 14, 5162 11 of 47 Table 8. Chemical composition of some tool steel alloys, reprinted with permission from ref. [48]. Copyright 2018 MDPI Metals. Alloys C Si Mn P S Ni Cr Mo Cu V W AISI D2 1.56 0.24 0.25 0.025 0.001 0.175 11.31 0.83 0.14 0.25 - AISI M4 1.33 0.33 0.26 0.03 0.03 0.3 4.25 4.88 0.25 4.12 5.88 HWS 1.08 1.38 0.34 - - - 7.80 1.86 - 2.66 1.73 Different machining methods have been practiced in past investigations on the steel material, including milling, drilling, broaching, grinding, planing, and turning, etc. How- ever, some methods induced complications while machining steel materials. For instance, Nagy et al. [49] said that machining (turning operation) of super duplex stainless steel is highly challenging when cutting tool inserts made up of PVD coating are used. The difﬁculties may be due to continuous and long chips formation, which is often problematic in the context of chip handling. Furthermore, long chips rolled on the part, and accordingly, stimulates surface imperfections. Eventually, surface quality is compromised. A similar problem has engaged with the austenitic steel. Sunil Magadum et al. [50] claimed that high strength, greater toughness, large fatigue, and corrosion resistivity are the prime reasons behind the poor machinability of steel. All the stated factors cause build-up-edge, irregular electrode wear, early tool failure during cryogenic machining of SS304 steel. Ingle et al. [51] proposed that there are certain grades of steel that have a machinability rating of 40%. Those grades belong to the austenitic steel such as 302B, 309, 309S, 330, 384, and 314. The authors demonstrated that a rating of less than 100% refers to the difﬁculty of machining alloys. As long as a rating is going down, then the difﬁculty level raises accord- ingly. The issues attributed to the aforesaid grades of austenitic steel are characterized by high ductility, toughness, prolong work-hardening, and less thermal conductivity. The machinability issues of martensitic steel grades (414, 422, 431, 440A, 440B, and 440C) has also been discussed for the austenitic steel grades. Table 8. Chemical composition of some tool steel alloys, reprinted with permission from ref. [48]. Copyright 2018 MDPI Metals. 2.2. Heat Resistant Steel To help the manufacturing sector and research community ﬁnd the best option to meet these goals, we present in this work a detailed review of major sustainable techniques used in the manufacturing of steel materials. Further, the details presented in this review can act as a guide in selecting the best solution to be integrated towards achieving net-zero emissions in their manufacturing process. g g p Numerous studies have been presented on the various grades of steel. Laleh et al. [52] demonstrated the unexpected behavior of LPBF 316L in the context of erosion and corro- sion. They proposed that lower erosion and corrosion resistance of the selected austenitic stainless steel is due to its minimum repassivation through traditional techniques. Thomp- son [53] contrasted HSLA-80 steel with two alternatives of HSLA, i.e., HSLA-80/100 & HSLA-100, considering yield strength, fracture, and results of Charpy impact test. They found that outcomes of yield strength are enough to study the microstructure, as long as strength and toughness are concerned. Durmusoglu et al. [54] joined the HSLA-80 steel by employing gas metal arc welding based on the high strength of weld metal followed by the heat-affected zone (HAZ) and target metal. Furthermore, the author detected that martensite needle-like sand is looked up in the HAZ, whereas the weld metal has residual austenite. Rajbongshi et al. [55] analyzed the effect of the surface topology of AISI D2 steel at the ﬂank side using texturing and non-texturing coated carbide tools. Two responses (ﬂank wear and surface integrity) were evaluated against three factors, i.e., speed, feed, and depth of cut. The results predicted that texturing tools yield minimal ﬂank wear and less surface roughness (SR). Rath et al. [56] investigated the effect of dry machining on the newly developed grade AISI D3 steel using a mixed ceramic insert (Al2O3 + TiCN). Three control parameters (cutting speed, feed rate, and depth of cut) were used to evaluate the inﬂuence on cutting forces, SR, electrode wear, and chip thickness. They revealed that feed Materials 2021, 14, 5162 12 of 47 12 of 47 rate is the most dominant factor, which alters the magnitude of all the deﬁned responses magniﬁcently. Kajendirakumar et al. [57] also conducted a study on AISI D3 steel. They optimized the process parameters via the electric discharge machining (EDM) technique by utilizing grey relational analysis. Material removal rate (MRR) and SR were taken as output responses. 2.2. Heat Resistant Steel They said that optimum parameters were achieved at low pulse on time, high pulse off time, and a large value of current. Guo et al. [58] studied the microstructure and characteristics of heat resistant steel (2.25Cr1Mo0.25V) using the Wire-Arc AM (WAAM) process. They claimed that subtract after processing through the WAAM technique exhibit high quality, excellent metallurgical features, and defect-free surface. Baddoo [59] has proposed a review article about the challenges, applications, and opportunities of stainless steel in the construction sector. The author stated that stainless steel had been proven to be a good alternative in construction sectors because of its good mechanical strength and high ductility. However, these are also fundamental requirements of any architectural applications. Ramana et al. [60] depicted the inﬂuence of powder (Nickel) contained EDM on MRR, tool wear rate (TWR) using die steel material against copper electrode. They estimated that nickel in dielectric ﬂuid substantially improves both the said output when pulse-on/off time and current are considered as input variables. 3. Methodology This module describes a detailed methodology for the sustainable machining of steel that has undergone a comprehensive review procedure. A PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) approach was adopted, as displayed in Figure 9, to study the multiple intents of sustainable techniques [61]. The different aspects of sustainable machining techniques for the steel material comprise processing, beneﬁts, drawbacks, and limitations. Afterward, the three pillars of sustainability, such as social- environment -economic, are critically reviewed and highlighted during steel machining to ﬁnd out the research gaps and future implications. From this perspective, different literature has been studied from various Journals, including Science Direct, Tandfonline, MDPI, Springer, Hindawi, Wiley, Web of Science, etc. The iterative forward and backward strategy was practiced in the identiﬁcation process to collect the explicit information using the Keywords, Sustainable manufacturing, MQL, Cryogenic machining, Solid lubricants, Vegetable oils, and Steels. Figure 9. A PRISMA Methodology, reprinted with permission from ref. [61]. Copyright 2009 BMJ Publishing Group Ltd. Figure 9. A PRISMA Methodology, reprinted with permission from ref. [61]. Copyright 2009 BMJ Publishing Group Ltd. Materials 2021, 14, 5162 13 of 47 13 of 47 For the acquisition of Journal articles, books, reports, and web pages, the string of sustainable machining was utilized in each of the databases’ searches. Then a screening operation was performed to ﬁnd out the future implications in sustainability machining of steel by appraising the existing issues and tentative solutions in a contextual manner. For the acquisition of Journal articles, books, reports, and web pages, the string of sustainable machining was utilized in each of the databases’ searches. Then a screening operation was performed to ﬁnd out the future implications in sustainability machining of steel by appraising the existing issues and tentative solutions in a contextual manner. Each of the content of the research articles has been extensively examined while taking the sustainability viewpoint of steel into account. Based on the following established criteria, a large amount of information taken from published literature was systematically organized for assessing future research possibilities: y pp g g Each of the content of the research articles has been extensively examined while taking the sustainability viewpoint of steel into account. 3. Methodology Based on the following established criteria, a large amount of information taken from published literature was systematically organized for assessing future research possibilities: • Studies belonging to human health, environmental and economic impact on the machining of steel; • Investigations related to the mechanical and chemical characterization of the machin- ing under special cutting oils/ﬂuids; • Investigations related to the mechanical and chemical characterization of the machin i d i l tti il /ﬂid g ing under special cutting oils/ﬂuids; g ing under special cutting oils/ﬂuids; ing under special cutting oils/ﬂuids; • Articles linked with the MQL machining attributes of steel material plus the cryoge treatment cutting effect on the suitability of steel; • Articles linked with the MQL machining attributes of steel material plus the cryogenic treatment cutting effect on the suitability of steel; Q g p y g treatment cutting effect on the suitability of steel; treatment cutting effect on the suitability of steel; • Content afﬁliated to the behavior of dry machining of steel. The references have been cited within a broad time span from 1982 to 2021. Out of the complete list of references, about 43% of articles have been selected from the last six years (2015–2021). The fundamental information based on the sustainable machining techniques for steel was collected and organized, then sub-categorized as per the importance in the respective studies. A comprehensive revision of the research records was developed to examine the sustainability aspect of steel, keeping an eye on its machining attributes. In the present study, challenges to the sustainable machining of steel were also described, and the discussion of this research is summarized in the Conclusion. Finally, future directions and research limitations have been consolidated using the identiﬁed knowledge about the sustainable machining aspect of steel. 4. Sustainable Techniques Different sustainable techniques, i.e., cryogenic cooling, MQL, solid lubricants, and other techniques which are being used in the auto industry that fulﬁll the overall objectives of this review, are depicted in Figure 10 [8]. The techniques mentioned in Figure 10 have certain beneﬁts, as portrayed in Figure 11. Figure 10. Sustainable Techniques, reprinted with permission from ref. [8]. Copyright 2015 Else- vier Ltd. Figure 10. Sustainable Techniques, reprinted with permission from ref. [8]. Copyright 2015 Else- vier Ltd. Figure 10. Sustainable Techniques, reprinted with permission from ref. [8]. Copyright 2015 Else- vier Ltd. 14 of 47 Materials 2021, 14, 5162 Figure 11. Beneﬁts associated with sustainable machining. Figure 11. Beneﬁts associated with sustainable machining. 4.1. Cryogenic Cooling In cryogenic cooling, low temperature (below −150 ◦C) materials and medium are used for cooling purposes. Liquid nitrogen, whose boiling point is (−195.82 ◦C) and frozen carbon dioxide, whose sublimation point is (−78.5 ◦C), are two common media used in this process. Nitrogen is employed to cool down the temperature in the cutting zone because of exothermic conditions. The large amount of heat that is generated during machining causes tool failure and tends to alter the mechanical properties of the specimen. Therefore, to minimize the detrimental effects due to heat and elevated temperature, nitrogen is used, which decreases wear and tear as well as improves the build-up edge [62]. Cryogenic is an eco-friendly technique that shows better results at higher cutting speeds. It is best to control machining temperature along with enhanced tool life [63]. The schematic diagram of the cryogenic cooling setup is represented in Figure 12. Figure 12. Diagram of cryogenic cooling technique setup, reprinted with permission from ref. [64]. Copyright 2010 Elsevier Ltd. Figure 12. Diagram of cryogenic cooling technique setup, reprinted with permission from ref. [64]. Copyright 2010 Elsevier Ltd. Figure 12. Diagram of cryogenic cooling technique setup, reprinted with permission from ref. [64]. Copyright 2010 El i Ltd It was noted during the comparison of dry cutting, MQL, and cryogenic machining that the cryogenic technique is better in increasing tool life with the reduction of cutting temperature. With this product, life improved due to better surface quality [65]. Materials 2021, 14, 5162 15 of 47 15 of 47 The use of liquid nitrogen in hard turning caused the improvement in cutting speed, and higher productivity and greater tool life were achieved. All of the surface ﬁnishes also improved as it causes a decrease in machined surface temperature. Also, it is good for the environment and has no toxic properties [66]. Figure 13 shows the environmental impact of different cooling techniques in the machining of AISI 304. Wet cooling has a tremendous impact on the environment, like ozone depletion, etc. Cryo MQL-CO2 is best found in all these. Figure 13. The impact on the environment by different cooling techniques, reprinted with permission from ref. [67]. Copyright 2016 Elsevier Ltd. Figure 13. The impact on the environment by different cooling techniques, reprinted with permission from ref. [67]. Copyright 2016 Elsevier Ltd. 4.1. Cryogenic Cooling It was observed that cryogenic machining, which is suitable for environmental impact, may also have other beneﬁts in terms of lesser tool life and low power consumption as compared to dry cutting. Figure 14 shows a graphical representation of tool life in different cooling techniques, which clearly depicts that tool life is longer in CryoMQL-CO2 as compared to other techniques [67]. In the milling of hardened AISI D3 steel, the effect of cryogenic cooling (liquid nitrogen) was noted for tool life, surface roughness, and cutting forces. Cutting forces were reduced by 20% to 27%, and surface roughness was decreased up to 16 to 29% due to less cutting temperature at the tool chip interface. Tool life was increased up to 26% to 35% as compared to dry cutting conditions [68]. Figure 14. Comparison of Tool life between different cooling techniques, reprinted with permission from ref. [67]. Copyright 2016 Elsevier Ltd. Figure 14. Comparison of Tool life between different cooling techniques, reprinted with permission from ref. [67]. Copyright 2016 Elsevier Ltd. Materials 2021, 14, 5162 16 of 47 16 of 47 The cryo-cooling process consists of many input variables: cooling rate, soaking time and temperature, tempering temperature, and its required time [69]. Gill et al. [70] ﬂour- ished that three of above parameters (cooling rate, soaking time, and soaking temperature) have been extensively increased the tool life upto 98% by compromising the mechanical characteristics of it. Stratton [71] put forward that cooling rate must be low enough to avoid cracking and deforming in the material. Molinari et al. [72] reported about soaking time that must be less than 35 h. It also stated that tool fracture mainly because of insufﬁcient cooling rate, so the optimum value for cooling rate should be near to 30 ◦C/h. Barron [73] had observed the effect of soaking temperature (189.15 K and 77.15 K) on the wear resis- tance property of M2 Steel. Besides, many researchers witnessed that increase in hardness, toughness, improving stability and resistance to corrosion is enhanced the tool life [74,75]. Dhar and Kamruzzaman [76] have compared the dry, wet and cryogenic techniques for AISI-4037 Steel. They concluded that cryogenic has been proved as sustainable method followed by dry and wet method in terms of reduction in heat upto 673.15 K. SR is another important criterion to check whether the machining is sustainable or not. Rotella et al. 4.1. Cryogenic Cooling [77] carried out machining under dry, wet and cryogenic condition on Ti-6Al-4V. They noted that cryogenic machining is more prominent than dry and wet machining in term of getting high surface integrity. They also summarized that cryogenic machining has been proved as effective at high feed rates. Kumar and Dhananchezian [64] also demonstrated the similar consideration about SR in cryogenic machining of Ti-6Al-4V. The 35% improvement in SR magnitude has been observed in comparison to dry and wet processing. In the turning of 17-4 PH SS, different cooling techniques were used like cryogenic, MQL, and wet and dry turning. Different depth of cut (DOC) was used to check the optimum conditions for each technique. It was noted that the cryogenic technique was best in terms of cutting zone temperature decrement, improved surface integrity, and less tool wear. Chip thickness was also less, and also this technique was environmentally friendly. Figure 15 shows the surface morphology obtained after applying different cooling techniques. The surface was smoother in cryogenic as compared to dry machining [78]. Figure 15. Images of surface morphology under different cooling environments. (a) cryogenic, (b) wet, (c) MQL, (d) dry, reprinted with permission from ref. [78]. Copyright 2018 CIRP. Figure 15. Images of surface morphology under different cooling environments. (a) cryogenic, (b) wet, (c) MQL, (d) dry, reprinted with permission from ref. [78]. Copyright 2018 CIRP. Materials 2021, 14, 5162 17 of 47 Figure 16 shows the cutting temperature according to the depth of cut increment, which is lower in cryogenic machining than dry, wet, and MQL machining. In machining AISI 52100 Bearing steel, the effect of cryogenic coolant compared to dry cutting on surface integrity was observed. It was noted that residual stresses and white layer formation were less. This layer is non-recommended because it causes fatigue of the product and affects its life. It became evident that it enhances the surface integrity of hard components in many aspects [79]. In hard turning of 17-4 PH stainless steel, the effect of cryogenic machining was found to be positive. It reduced the cutting temperature by using liquid nitrogen as a cooling medium, and it is eco-friendly. This method can be effectively used in any type of hard material [80]. Figure 16. Effect of DOC on cutting temperature under different cooling techniques, reprinted with permission from ref. [78]. Copyright 2018 CIRP. Figure 16. 4.1. Cryogenic Cooling Effect of DOC on cutting temperature under different cooling techniques, reprinted with permission from ref. [78]. Copyright 2018 CIRP. Nitrogen is most commonly used as it is a safe, noncombustible, noncorrosive gas. The air we breathe has 78% nitrogen gas in it. Liquid nitrogen has the property of easy evaporation, so when it is used in cryogenic machining, it evaporates quickly, and no wastes remain on surfaces, tools, and machines, etc. It contributes to cost savings by avoiding disposal costs [81]. Currently, cryogenic turning is being used to achieve deformation- induced surface hardening. For such purposes, the powerful coolant CO2-snow is used due to its good wetting behavior [82]. g g In hard turning of ASP23 steel, CO2 cryogenic media was used with two types of inserts: one negative and one positive. Tool life was increased in the negative insert up to 19.96%, but in the positive insert, the value of improvement rose to 69.5%. The white layer was also checked. In the negative insert during CO2 cryogenic machining, it produced a minimal thickness of 2 micrometers. In the positive insert, this layer was not produced. In Figure 17, the microstructure of the material in which machining is done with negative insert using both techniques: dry turning and CO2 machining [83], is shown. In Figure 18, the microstructure is presented in which machining is done with positive insert using dry turning and CO2 machining. The white layer is not produced, which indicates good structure. 18 of 47 18 of 47 Materials 2021, 14, 5162 Figure 17. After turning with VNGA160408 (Negative). (a) Dry machining; (b) CO2 machining, reprinted with permission from ref. [83]. Copyright 2015 Elsevier Ltd. Figure 17. After turning with VNGA160408 (Negative). (a) Dry machining; (b) CO2 machining, reprinted with permission from ref. [83]. Copyright 2015 Elsevier Ltd. Figure 18. Microstructure after turning with VCGW160408 (positive). (a) Dry machining; (b) CO2 machining, reprinted with permission from ref. [83]. Copyright 2015 Elsevier Ltd. Figure 18. Microstructure after turning with VCGW160408 (positive). (a) Dry machining; (b) CO2 machining, reprinted with permission from ref. [83]. Copyright 2015 Elsevier Ltd. In hard turning of AISI 420 steel, the effect of cryogenic cooling was noted compared to nano ﬂuids. It was noted that tool life at a cutting speed of 75 m/min was increased by approximately 29%. This effect was increased as the speed was increased. 4.1. Cryogenic Cooling Also, the temperature is reduced as compared to nano ﬂuids. Chip morphology was better than nano ﬂuids. It was noted that tool wear was also less [84]. In the machining of AISI 4340, it was found that cutting powers are reduced in cryogenic (LN2) cooling as compared to other water-based cutting ﬂuids. Material removal rate (MRR) was increased with a decrement in surface roughness, which was 0.97 micrometers in cryogenic cooling [85]. The comparison is shown between conventional machining and cryogenic machining. In Figure 19, a conventional machining setup is shown in which cooling and lubricants are required, and waste is generated. Figure 19. Machining, reprinted from ref. [86]. Copyright 2014 Elsevier Ltd. Figure 19. Machining, reprinted from ref. [86]. Copyright 2014 Elsevier Ltd. Materials 2021, 14, 5162 19 of 47 In Figure 20, a cryogenic setup is shown. Unlike conventional machining, there is no need for lubricants, and no waste is generated, which is better for the environment and saves on the cost of the product. To observe the cryogenic effect in hard turning of AISI 4340, a setup was done on the shop ﬂoor of the CNC turning center. By this process, surface roughness was achieved up to 0.4 micrometers. Tool life of order was achieved 34 min. Cutting forces were reduced by 18%, and power consumption was decreased by about 320 W. Figure 20. Cryogenic Machining, reprinted from ref. [86]. Copyright 2014 Elsevier Ltd. Figure 20. Cryogenic Machining, reprinted from ref. [86]. Copyright 2014 Elsevier Ltd. In Figure 21, an SEM image was captured to check the ﬂank and rake area of the cutting insert after machining. The insert was chipped off when ﬂood cooling was used while in cryogenic machining abrasion type phenomenon observed at ﬂank face [87]. In Figure 21, an SEM image was captured to check the ﬂank and rake area of the cutting insert after machining. The insert was chipped off when ﬂood cooling was used while in cryogenic machining abrasion type phenomenon observed at ﬂank face [87]. Figure 21. Images of (a) wet cooling insert (b) cryogenic machining insert, reprinted with permission from ref. [87]. Copyright 2017 Elsevier Ltd. Figure 21. Images of (a) wet cooling insert (b) cryogenic machining insert, reprinted with permission from ref. [87]. Copyright 2017 Elsevier Ltd. It was noted that power consumption in terms of electricity creates about 99% environ- mental impacts, which need to be minimized. 4.1. Cryogenic Cooling It was done by choosing the optimal cutting conditions in terms of CO2 emission, which leads to better environmental impacts [88]. The effects of dry, MQL, ﬂood, and cryogenic machining were observed during turning of 15-5 PH SS, and it was noted that cryogenic machining performed well in terms of tool life which was about 44% of ﬂood and 68% of MQL cooling technique. Surface roughness was better in cryogenic and ﬂood cooling as compared to MQL and dry cutting. In Figure 22, SEM images show the smoothness in the wear pattern of the ﬂank face of the tool in cryogenic as compared to other techniques [89]. p The effects of dry, MQL, ﬂood, and cryogenic machining were observed during turning of 15-5 PH SS, and it was noted that cryogenic machining performed well in terms of tool life which was about 44% of ﬂood and 68% of MQL cooling technique. Surface roughness was better in cryogenic and ﬂood cooling as compared to MQL and dry cutting. In Figure 22, SEM images show the smoothness in the wear pattern of the ﬂank face of the tool in cryogenic as compared to other techniques [89]. Materials 2021, 14, 5162 20 of 47 Figure 22. Micrographs of the ﬂank face of cutting inserts under different cutting environments, reprinted with permission from ref. [89]. Copyright 2020 Elsevier Ltd. Figure 22. Micrographs of the ﬂank face of cutting inserts under different cutting environments, reprinted with permission from ref. [89]. Copyright 2020 Elsevier Ltd. The growth of global production and the increase of cutting ﬂuids application has caused intensive research concerning economic and environmental aspects of systems for cooling/lubricating the cutting zone. Thus, recently several cooling/lubrication techniques were developed in order to achieve sustainable manufacturing by reducing or eliminating cutting ﬂuids. Currently, the most widely used cooling/lubricating techniques with a low negative effect on the environment and human operator’s health are dry cutting, cryogenic cooling, and minimum quantity lubrication (MQL), etc. [90]. LN2 was found to be good in milling of P20 hardened steel as compared to dry and ﬂood machining. Tool wear was less, about 15%, compared to dry machining, while about 5% compared to wet cooling. Also, it was noted that due to temperature reduction, chip curl was less, which leads to good surface morphology [91]. 4.1. Cryogenic Cooling In the machining of AISI D6 tool steel, a comparison was made between LN2 machining, dry, and wet machining. LN2 was good in surface integrity, and tool life was good, but the production cost for cryogenic setup was more compared to dry machining. This cost varied as the ﬂow rate of LN2 increased [92]. In the milling of AISI D2, the impact of cryogenic cooling was noted compared to a dry and wet cutting environment. Cutting zone temperature was reduced up to 44% by dry and about 36% by wet machining, while cutting forces were reduced by about 40% by dry and about 29% by wet machining [93]. Materials 2021, 14, 5162 21 of 47 21 of 47 During the study, a comparison was done in the machining of normalized and hard- ened bearing steel AISI 52100. The response of cryogenic and conventional turning tech- niques like dry and ﬂood cooling was checked in terms of tool life, surface ﬁnish, and productivity. Productivity was higher in cryogenic cooling, and tool life was about 315% in normalized while 15% in the hardened workpiece compared to other techniques. No white layer was formed in cryogenic that are not recommended for machining part. Table 9 shows the MRR for both techniques, and it can be seen that it is about 23% more in cryogenic [94]. Table 9. Optimum productivity comparison through MRR, reprinted with permission from ref. [94]. Copyright 2012 Elsevier B.V. Material Conventional Turing (mm3/min) Cryogenic Turning (mm3/min) Normalized AISI 52100 61,600 (Flood) 75,600 Hardened AISI 52100 5606 (Dry) 6300 Table 9. Optimum productivity comparison through MRR, reprinted with permission from ref. [94]. Copyright 2012 Elsevier B.V. During machining of duplex stainless steel, a comparison was conducted between cryogenic cooling and dry cutting. The tool which was used in the machining was coated carbide. Reduction of cutting zone temperature was observed in the case of cryogenic by 53–58%. Required cutting forces were decreased by 30–43%; also, it was noted that surface ﬁnish was improved by 18% to 23%. These results were in comparison with dry cutting. Figure 23 shows the cutting temperature for cryogenic and dry machining, which is less in cryogenic machining [95]. Figure 23. Effect of two cooling techniques on cutting Temperature, reprinted with permission from ref. [95]. Copyright 2018 Elsevier Ltd. 4.1. Cryogenic Cooling In the hard turning of AISI 52100 bearing steel, the impact of Cryo MQL with two different media (LN2 and CO2) was evaluated against conventional and dry turning. Machining was done with two different inserts: one was conventional cubic boron nitride (CBN), and the other was a wiper geometry insert. Less ﬂank wear and crater wear were observed using MQL + CO2. It was due to the combined effect of minimum quantity lubrication with cryogenic cooling. The surface ﬁnish was better, and this technique was found to be eco-friendly. Figure 24 shows the wear pattern of the ﬂank face, which is more in dry cutting, while in Cryo MQL + CO2, better wear performance was observed, especially Figure 23. Effect of two cooling techniques on cutting Temperature, reprinted with permission from ref. [95]. Copyright 2018 Elsevier Ltd. Figure 23. Effect of two cooling techniques on cutting Temperature, reprinted with permission from ref. [95]. Copyright 2018 Elsevier Ltd. In the hard turning of AISI 52100 bearing steel, the impact of Cryo MQL with two different media (LN2 and CO2) was evaluated against conventional and dry turning. Machining was done with two different inserts: one was conventional cubic boron nitride (CBN), and the other was a wiper geometry insert. Less ﬂank wear and crater wear were observed using MQL + CO2. It was due to the combined effect of minimum quantity lubrication with cryogenic cooling. The surface ﬁnish was better, and this technique was found to be eco-friendly. Figure 24 shows the wear pattern of the ﬂank face, which is more in dry cutting, while in Cryo MQL + CO2, better wear performance was observed, especially Materials 2021, 14, 5162 22 of 47 by using the wiper geometry insert [96]. Cryogenic machining has major beneﬁts in the sense of environment and product quality, but some limitations like lack of lubrication and chip cleaning. Also, a drawback is the coldness effect for the operator due to high cooling generation during this process [97]. by using the wiper geometry insert [96]. Cryogenic machining has major beneﬁts in the sense of environment and product quality, but some limitations like lack of lubrication and chip cleaning. Also, a drawback is the coldness effect for the operator due to high cooling generation during this process [97]. Figure 24. 4.1. Cryogenic Cooling Wear values and SEM images of conventional and wiper CBN inserts under different cutting conditions, reprinted with permission from ref. [96]. Copyright 2020. The Society of Manufacturing Engineers. Published by Elsevier Ltd. Figure 24. Wear values and SEM images of conventional and wiper CBN inserts under different cutting conditions, reprinted with permission from ref. [96]. Copyright 2020. The Society of Manufacturing Engineers. Published by Elsevier Ltd. In a nutshell, the cryogenic cooling technique assists us in minimizing chip adherence on a tool. Its beneﬁts include reduction of wear and tear, increase in tool life, improved surface ﬁnish, and a decrease in the coefﬁcient of friction. Although some literature has stated that the cryogenic method is beneﬁcial in all aspects, as mentioned earlier, it has certain limitations, as ascertained by Tushar and Suprabhat in their work [98]. The drawbacks are: (1) cryogenic process demands extra control and monitoring over cooling process, (2) a large amount of machining cost belong to process, so any failure during operational hours lead to high maintenance expenses, (3) liquid nitrogen cannot be reused, (4) it is not acceptable for heat treatment processes, (5) and cryogenic ﬂuid, when operated at low temperature, becomes reactive; therefore, it damages the workpiece by directly contacting it. 4.2. Minimum Quantity Lubrication To avoid using a large amount of cutting ﬂuids, a technique called minimum quantity lubrication, or near dry machining [99], is used in which cutting ﬂuid is supplied at the rate of 100 mL/h. Lawal et al. [100] demonstrated that MQL is a highly competitive approach for a sustainable environment. They explained that minimum usage of cutting ﬂuid in MQL reduces environmental and occupational health hazards. It is well known that metal cutting ﬂuids cause environmental problems. In this case, the amount of cutting oils is greatly reduced which also reduces the environment problem. It was also pointed out that the use of vegetable oils improves the performance of the MQL process, especially in the machining of hard materials, by using water soluble oil in the presence of nano particles. There was no toxic effect generated by using this process which leads to sustainable machining process [101]. Materials 2021, 14, 5162 23 of 47 23 of 47 Normally, machining is done in dry mode, but the problem which we face is shorter tool life, and sometimes, surface integrity suffers. On the other hand, ﬂooded type coolant application has a higher cost. So, a tradeoff is required in the form of minimal application of lubricants (MQL) which will serve both purposes. In a comparison of wet and MQL, it was found that MQL had better results in tool wear, tool vibration, surface roughness, cutting forces, and cutting temperature during hard turning. About 1.3%, 6.7%, and 8.6% reduction were observed in surface roughness, tool wear, and tool vibration, respectively. Tool wear was less observed in the minimal cutting application as compared to others. Figure 25 shows the surface morphology of three types of cutting techniques in which hard turning with minimal ﬂuid (HTMF) produced the smoothest surface [102]. Figure 25. Morphology SEM images; (a) By dry turning, (b) conventional wet turning, (c) and HTMF application, adapted from ref. [102]. Figure 25. Morphology SEM images; (a) By dry turning, (b) conventional wet turning, (c) and HTMF application, adapted from ref. [102]. To avoid cutting ﬂuids, dry cutting can be adopted, but this results in shorter tool life at higher cutting parameters, so near dry machining is recommended. Using cutting oil at optimal speeds serves both economic and environmental [103] purposes. In turning of AISI H13 hardened steel under the MQL method, it was noted that the surface ﬁnish was improved. 4.2. Minimum Quantity Lubrication This method also has the beneﬁt of being environmentally friendly due to the minimal use of aerosols and cutting oils [104]. An experiment was conducted on heat- treated AISI 4340 steel with a hardness of 52–54 HRC in MQL and dry turning conditions using different bio-cutting oils. It was observed that surface roughness improved as compared to dry turning. At higher cutting speeds, more than 240 m/min, sudden tool failure was observed under MQL conditions [62]. In the machining of AISI 1045, it was found that the cutting temperature and cutting forces were reduced by 10–30% and 5–28%, respectively, in MQL compared to dry machin- ing (see details in Figure 26). This reduction of temperature leads to better tool life and contributes to sustainable manufacturing [105]. Materials 2021, 14, 5162 24 of 47 Figure 26. Effect on cutting forces by using Dry and MQL, reprinted with permission from ref. [105]. Copyright 2015 Elsevier B.V. Figure 26. Effect on cutting forces by using Dry and MQL, reprinted with permission from ref. [105]. Copyright 2015 Elsevier B.V. In turning of AISI D2 steel, the effect of the eco-friendly MQL system was observed compared to dry machining in terms of tool life, tool wear, and surface ﬁnish. Reduction of about 100 ◦C was noted in cutting zone temperature, and surface ﬁnish was improved up to 91% compared with dry machining. Tool wear was less and tool life was increased about 267% in chemical vapor deposition (CVD) coated tools [106]. p p In a study [107] to check the sustainability and effectiveness of different cooling and lubrication techniques, it was found that MQL nanoﬂuids and cryogenic were the best techniques in terms of keeping a balance between the sustainable environment and not compromising machinability efﬁciency. MQL technique is an efﬁcient process when we compare it with wet machining. About 15% was saved using this technique. It was noted that it has a better effect in the form of a good surface ﬁnish and longer tool life compared to dry machining. When we used biodegradable oils, the effectiveness of this technique increased towards the sustainable point of view. Cutting temperature was reduced by about 50%, which reduced the cutting forces as well [108]. Table 10 shows the different cost estimations of different techniques used in machining. MQL was found better in terms of initial setup and tool cost. 4.2. Minimum Quantity Lubrication Cleaning and disposal costs are comparable with other techniques [109]. Table 10. Qualitative Cost Estimation data for different cooling/lubricating techniques, reprinted with p ref. [109]. Copyright 2017 Elsevier Ltd. The symbols used to depict Very low (), Low (), Medium (), H High (**). ative Cost Estimation data for different cooling/lubricating techniques, reprinted with permission from ight 2017 Elsevier Ltd. The symbols used to depict Very low (), Low (), Medium (), High (**), Very Table 10. Qualitative Cost Estimation data for different cooling/lubricating techniques, reprinted with permission from ref. [109]. Copyright 2017 Elsevier Ltd. The symbols used to depict Very low (), Low (), Medium (), High (*), Very High (**). Table 10. Qualitative Cost Estimation data for different cooling/lubricating techniques, reprinted with permission from ref. [109]. Copyright 2017 Elsevier Ltd. The symbols used to depict Very low (), Low (), Medium (), High (*), Very High (*). Sr. No. Type Raw Material Cost Fluid Consumption Tool Cost Equipment Costs Cleaning Costs Disposal Cost 1 Cutting ﬂuid *** ** * *** 2 Dry Machining * * ***** * * * 3 MQL * 4 Cryogenic Cooling * * * ***** * * 5 Gaseous Cooling * * *** ** * * 6 Sustainable Cutting ﬂuid * ** * 7 Solid lubricant ** * * * * 8 Nanoﬂuids * * ** *** Materials 2021, 14, 5162 25 of 47 25 of 47 In an experiment performed on a transmission housing using MQL rather than wet machining, about 15% in savings were achieved. It was noted that due to the reduction of wastewater, it is a sustainable process. One problem is in MQL is the cleaning of chips during machining, especially of hard materials. Figure 27 shows the cost comparison of two types of machining processes, MQL, and wet machining. Operation and maintenance costs are less using MQL. Equipment costs are also less, and the overall cost is about 78% than in wet machining [110]. Figure 27. Up to 10 years life cycle of two types of machining techniques, reprinted with permission from ref. [83]. Copyright 2015 Elsevier Ltd. Figure 27. Up to 10 years life cycle of two types of machining techniques, reprinted with permission from ref. [83]. Copyright 2015 Elsevier Ltd. MQL can be applied in two types of application methods. 4.2. Minimum Quantity Lubrication Different types of MQL systems are shown in Figure 28. In the external application, a compressed air and oil mixture is fed through an external nozzle to the cutting area from a chamber. There are two types of this system. One has an ejector nozzle in which air and oil are supplied separately to the ejector, and mixing is done after the nozzle. In conventional mixing, it is done before the feeding at the cutting zone. In internal application, the mixture is sent through the spindle and tool to the cutting area of the part [111]. Figure 28. Different types of MQL systems, reprinted from ref. [108]. Copyright 2020 Elsevier Ltd. Figure 28. Different types of MQL systems, reprinted from ref. [108]. Copyright 2020 Elsevier Ltd. Materials 2021, 14, 5162 26 of 47 Using cutting ﬂuids at a very large scale in machining creates many environmental problems, so it is necessary to adopt a strategy that minimizes the use of these oils but serves the purpose of machining. Also, governments have imposed restrictions on the disposal of such ﬂuids as these cause damage to natural resources. To avoid environmental, regulatory, and health-related problems, MQL is a better technique that serves most of the purposes and also reduces costs [112]. In the context of industry 4.0, sustainable manufacturing is very important. Research was conducted to check the sustainable aspects of MQL on the machining of difﬁcult-to-cut materials, and it concluded that MQL is a tradeoff between ﬂood type and dry cutting. It has more advantages for the environment and is more cost-effective than other techniques. Skin problems created by metalworking ﬂuid (MWF) were reduced by using MQL [113]. In the machining of a mold of tile industry, the impact of sustainable machining was observed. MQL technique was used for such purpose, and it was noted that by using optimal cutting parameters, a major improvement was achieved in the context of a safe cutting environment. There was an approximate 67% reduction in kg CO2, and about 3357 liters of water were saved. Costs were reduced by about 60% [114]. Four types of cooling techniques (dry, MQL, ﬂood, and solid lubricants with com- pressed air) were investigated in the machining of AISI 1060 in terms of temperature and surface roughness. In all these, MQL was found to be the best from a sustainable point of view. 4.2. Minimum Quantity Lubrication This technique is responsible for lower manufacturing costs and fewer occupational health and safety problems [115]. Due to sustainability, some properties possessed by MQL are high lubricity, high stability and should be biodegradable. Low consumption of oil is very common in these [116,117]. y In addition to the above literature, some studies have also been carried out under vegetable oil mixed MQL conditions. Khan et al. [118] machined low alloy steel of grade AISI 9310 using vegetable oil emulsion. They studied the effect of the MQL process on SR, cutting temperature, chip development, and electrode erosion in different cutting environ- ments. They proposed that surface roughness and tool tip wear were extensively reduced under the MQL environment, and ﬂank wear promisingly improved when machining was treated in vegetable oil. Likewise, some investigations are compiled based on conventional machining in MQL conditions. For instance, Braga et al. [119] compared the results of two scenarios; one in MQL state and the second in the mixture of Al-Si (7% Si) alloy. They conducted a drilling process in both conditions and then measured the potency of each. The results yielded the same SR values in both of the aforementioned drilling conditions, which generally conﬁrms the sustainability of vegetable oil-based machining. In another work, Kishawy et al. [120] used Al alloy (Al-356) to examine the effect of high-speed face milling under dry, wet, and MQL setups at various cutting conditions such as speed of cutting up to 5225 m/min. They illustrated that high cutting forces were noted in the case of dry cutting while fewer cutting forces were observed in wet machining. Whereas in MQL, intermediate cutting forces were marked. MQL has some disadvantages: (1) removal of chips from the machining zone is not carried out properly, (2) MQL permits corrosion in the work parts or in the chips, (3) there must be great care taken in nozzle adjustment, as it should be more than 1 or 2 inches from the tool, (4) MQL is limited to chip heat removal only, it does not cool down the workpiece and tool, (5) mist creation is also one of the major drawbacks of MQL [121]. 4.3. Dry Cutting Sustainable manufacturing refers to the use of all available natural resources which reduce environmental pollution. Machining is one of these which is very much energy- intensive, and we have to bring improvements to reduce energy consumption. Using cutting ﬂuids is very common for this process but has tremendous impacts on the environ- ment as there are certain disposal costs associated with it. There is no convenient method to dispose of it after proper treating due to which skin diseases are common. To eliminate these problems, dry machining is used in which the need for cutting oils is eliminated. Dixit Materials 2021, 14, 5162 27 of 47 27 of 47 et al. [90] reported that the use of dry machining signiﬁcantly minimized air and water pollution. They called dry cutting an eco-friendly process. Eco-friendly refers to such tech- niques in which detrimental wastes and tiny particles are excluded. Schultheiss et al. [122] urged that dry machining can be performed without any ﬂuid; therefore, for sustainability, it is more appreciated to engage dry processing than the traditional machining approach where usually dielectric ﬂuid is used to compensate for the generated heat while operating on the work part. Dry cutting is more suitable for low-strength materials, and using coated tools is recommended, which can reduce heat generation [123–126]. In the machining of 15-5 PHSS hardened steel, an experiment was done to compare different cooling techniques like dry, wet, and cryogenic cooling. Sustainability assessments were conducted, and it was noted that dry cutting is more optimal towards sustainable assessment indicators, but when we talk about the combined effects of productivity and environment, cryogenic machining is good [127]. In the machining of stainless steel under dry cutting conditions, the effect of feed rate, cutting speed, and depth of cut was observed. The main objective was to reduce the energy cost and machining cost, which is the ultimate objective of sustainable machining. It was concluded that at a higher feed rate and cutting speed with a lower depth of cut, the energy consumption was reduced by 33.46% with a 17.81% reduction in machining cost [128]. Dry cutting is more useful in the context of the environment as there is no need to dispose of the water and metalworking cutting ﬂuids. Cost is also saved, but the problem is high cutting zone temperature and shorter tool life if the cutting parameters are high. 4.3. Dry Cutting Surface quality is better than wet cutting [129]. Although dry cutting is good to retain the sustainability factor while machining, it also has certain disadvantages. For example, Chetan et al. [130] and Rotella et al. [77] outlined that adhesion between the electrode and chips takes place in speciﬁc tool and workpiece materials. The said issue led to the reduction of the material erosion rate, and thus the quality of the machined surface is compromised. Moreover, there is the chance of a greater heat-affected zone over the surface, which decreases the strength and durability of the workpiece. Therefore, sustainability plays a prime role in the metal processing areas. 4.4. Cryogenic Treated Tools Tool life is very important to increase the productivity of any machining industry. It is necessary to use tools that have a long tool life without the use of cutting oils for environmental protection. What are the requirements for sustainable machining? Cutting tools without any treatment wear very rapidly due to heat generation on the cutting zone. Cryogenic treatment is done on cutting tools to compensate for this. Cryogenic treatment is an add-on process that is required to improve tool life. The ultimate goal is to improve the performance, which cuts down the machining cost. It is a subzero heat treatment process that affects the entire cross-section area of cutting tools. Life enhancement of tools is accomplished by microstructure changes of the tool during cryogenic treatment. Two types of treatments are used; one is shallow, and the other is deep cryogenic treatment. Shallow treatment: −80 ◦C to −145 ◦C. Deep cryogenic treatment: −145 ◦C or below. It was noted that the performance of deep cryogenic treatment is more effective than shallow treatment [131]. Cryogenic treatment is an advanced process for increasing tool life, reducing wear resistance, improving the strength and microstructure of the tool [132–134]. With the help of cryogenic treatment on the tool, productivity in terms of tool durability is escalated satisfactorily. Much past literature based on cryogenic treatment has been enlisted. For example, Ramji et al. [135] studied the effect of drilling processes on non-treated and cryogenically treated tools, and a combination of cryogenically treated and heat-treated carbide tipped drills on thrust, SR, and torque of drilled holes in diverse cutting conditions. They concluded that cutting forces, thrust, and torque were reduced when cryogenic treated and a combination of heat-treated carbide insert was used. Gill et al. [136] evaluated the effect of cryogenic treatment of tools on cooling rate. They demonstrated that when cooling Materials 2021, 14, 5162 28 of 47 28 of 47 and heating are performed at different rates (say 0.5 ◦C/min and 1 ◦C/min), then the wear resistance of the tool and micro-cracks on the surface was improved, respectively. Another study conducted by Silva et al. [74] reported the impact of cryogenic treatment of M2 HSS tools and said that 65–34.3% improvement was observed in the reduction of tool fracture while drilling on steel. Cryogenic treatment has numerous beneﬁts in traditional machining, including milling, drilling, and turning. 4.4. Cryogenic Treated Tools It has been extensively used outside the conventional machining zone for microstructure analysis and wear resistance tests for increasing tool life [136–140]. Furthermore, an experiment was done to check the impact of cryogenic treatment on Tungsten carbide inserts. It was noted that the inserts’ life was increased up to 36% with deep cryogenic treatment compared to non-treated inserts. Cutting forces were lesser, and performance was more consistent. Tool life was about 56% in deep cryogenic treatment than by non-treated insert at cutting speed of 110 m/min [92,93]. Cryogenic treatment has many beneﬁts due to its enhancement of cutting tool proper- ties by changing the austenite phase to the marten site phase by heat treatment. By doing this, the hardness and toughness of cutting tools improved [141–143]. In the machining of PHSS, cryogenic treat inserts were used. Due to lesser ﬂank wear, tool life was improved as compared to non-treated tools. These tools resulted in lesser cutting forces, enhanced surface ﬁnish with longer tool life [144]. In the machining of 15-5 PHSS cryo- treated inserts were used, and it was noted that cutting forces were reduced, and due to high hardness and strength, the wear of the tool was less as compared to conventional types of tools [145]. Deep cryogenic treatment in hard turning of AISI D2 steel with ceramic cutting tools improves the surface roughness by 32.97%, and improvement in tool life was observed 21.79% [146]. In the turning of C 45 steel, the impact of cryo-treated tungsten carbide inserts was noted compared to non-treated inserts. Treated inserts were found best in machinability and long tool life. Tool tip temperature was decreased due to higher thermal conductivity by cryogenic treatment. This treatment is limited to smooth turning [147]. Contrarily, in cryogenic treatment of cutting tools, machinability increases, and due to good thermal conductivity, cutting temperature decreased. These types of tools are not preferable for interrupted cutting due to breakage problems. This statement indirectly limits the use of cryogenic treatment of tools. 4.5. Solid Lubricants In the solid lubricant-assisted machining of hardened steel, it was found that this technique is suitable for an ecofriendly environment with less cost of production and helps in the reduction of waste as well as occupational health and safety. It was noted that as demand for sustainable machining is increasing day by day, so solid lubricant assisted machining is emerging as a sustainable alternative machining process [148]. It was noted in a review that the performance of solid lubricants at higher cutting parameters is high, which leads to enhanced productivity. Also, it was observed that there is no negative impact while using these, but the issue is selecting the right type of solid lubricant [136]. In the turning of hardened steel, the effect of solid lubricants was noted, and it was discovered that Molybdenum disulﬁde is better than graphite. It was observed that solid lubricants are better than dry or wet turning in terms of improved surface ﬁnish and from an environmental point of view. The good lubricating effect of these solid lubricants caused the reduction of cutting zone temperature and tool wear. This is becoming a good alternative to dry and wet turning [149]. In the turning of AISI 1040 steel, the impact of solid lubricants (MoS2) was noted in terms of toxic effect, surface ﬁnish, and machinability efﬁciency. It was concluded that the surface ﬁnish was improved by 5% to 30%. The chip thickness ratio was reduced. The friction was reduced in this process, so the material removal rate was high, which leads to high productivity. Also, not using cutting ﬂuids leads to better environmental impact [150]. Materials 2021, 14, 5162 29 of 47 29 of 47 In machining, the effect of SAE 40 oil with different percentages of graphite and boric acid was studied. It resulted that the boric acid (20%) in SAE 40 oil was performing well. The surface ﬁnish was improved, and less tool wear and lesser cutting forces were observed to boric acid lubricious ﬁlm formation, which lessens the friction forces and cutting temperature. Figure 29 shows the impact of boric acid and graphite on cutting temperature compared to dry and wet cooling. Boric acid and graphite were comparable, and with the passage of cutting time, the performance of Boric acid fond good [151]. Graphite was used in grinding, and it was found that it had numerous effects on the process. 4.5. Solid Lubricants The major difference was in the surface ﬁnish of the workpiece as in other conventional cutting oils, which were very much improved [152]. Figure 29. Tool temperature with time in different cutting techniques, reprinted with permission from ref. [151]. Copyright 2008 Elsevier Ltd. Figure 29. Tool temperature with time in different cutting techniques, reprinted with permission from ref. [151]. Copyright 2008 Elsevier Ltd. In the machining of AISI 1040, the effect of nanoparticles in cutting ﬂuid was noted, and it was found that thermal conductivity increased, and heat transfer rate increased about 6%, which increased tool life. It was found that about 1% addition of nanoparticles in cutting ﬂuids is optimal [153]. Different types of solid lubricants like MoS2, CuO, SiO2, and CaF2, etc., are useful due to the low strength of bonding between these shears off rapidly. They are also nontoxic and produce a good lubricity effect [154]. In the turning of bearing steel, the effect of Cu nano-ﬂuid with vegetable oil under minimum quantity lubrication was noted. It was found that surface roughness was improved by about 51% due to self-laminated ﬁlm formation between the tool and workpiece, which reduced the friction. Due to the better thermal conductivity of Cu nanoﬂuid, a reduction in cutting zone temperature was observed, about 21%, compared to vegetable oil machining [155]. Solid lubricant-assisted machining is an ecofriendly technique that contributes to improving the economical aspect of any industry. Improved tool life and higher productivity were observed in the machining of AISI 304 steel. Surface roughness was improved up to 39%, which was improved due to less wear of the tool tip [156]. All lubricants were supplied to the machining area with the help of a special feeding system, as shown in Figure 30 [157]. Solid lubricants have several drawbacks over other sustainable techniques such as (i) high wear rate with a high coefﬁcient of friction, (ii) some lubricants have poor heat dissipation due to low thermal conductivity, like polymers lubricants, (iii) comprises poor self-absorption of heat ability which disturbs the durability of lubricants [158]. 30 of 47 Materials 2021, 14, 5162 Figure 30. Lubricant feeding system, reprinted with permission from ref. [157]. Copyright 2005 Elsevier Ltd. gure 30. Lubricant feeding system, reprinted with permission from ref. [157]. Copyright 2005 Elsevier Ltd. 4.6. Alternative Cutting Fluids Soybean and sunﬂower oils were tested as metalworking ﬂuids, and it was noted that these had a good impact on the environment and were suitable for cutting and forming operations. These are the best alternative to cutting oils [161]. Table 12 shows the different advantages and disadvantages of vegetable oils. They are cost-efﬁcient and less toxic than mineral oils. The low rate of environmental pollution and high biodegradability make these safer for use. One drawback is low thermal stability [162–170]. Table 12. Different Advantages and disadvantages of vegetable oils used as lubricants. Table 12. Different Advantages and disadvantages of vegetable oils used as lubricants. Advantages Disadvantages High Biodegradability Poor Corrosion Protection Less environmental pollution Low Thermal Stability Low Volatility High Freezing Points Lesser production cost Oxidative Stability High Flash Points Low Toxicity High Viscosity indices Wide Production Possibilities Compatibility with other additives High Biodegradability Less environmental pollution Low Volatility Lesser production cost High Flash Points Low Toxicity High Viscosity indices Wide Production Possibilities Compatibility with other additives Poor Corrosion Protection Low Thermal Stability High Freezing Points Oxidative Stability Poor Corrosion Protection Low Thermal Stability High Freezing Points Oxidative Stability About 95% usage of vegetable-based oil in Brazil was reported in contrast to petroleum- based oils due to their biodegradable properties and ability to be extracted from natural resources. Soybean oil is the most commonly used in industrial applications [171]. In addition to their positive impact on the environment, it was reported that surface roughness was improved by 31.6% when vegetable oils were used in MQL [100]. In the turning of alloy steel ASIS 9310, vegetable oil performed excellently in terms of the material removal rate, which lead to high productivity. Machining performance was increased by using these oils by 117% in terms of tool life, and thrust forces were also reduced. Also, it has a less negative environmental impact [7]. Vegetable oils have a high boiling point and molecular weight, due to which the chances of vaporization are less than other neat oils. Less smoke is produced, so it is less hazardous for the working environment as well as for people. The product quality is improved by the effect of the lubricating ﬁlm. Friction and heat generation were lower [118]. In the turning of AISI 4340 stainless steel, three different oils, palm oil, sunﬂower oil, and coconut oil, were used. 4.6. Alternative Cutting Fluids The use of cutting oils/lubricants causes diseases in employees. To minimize the effect of these oils, some user-friendly oils like vegetable-based oils and other bio-degradable oils can be used. The usage of these oils improved the surface ﬁnish and enhanced the tool life. Due to less coefﬁcient of friction than other mineral oils, the machining efﬁciency improved, and cutting forces were reduced. These are less toxic than other mineral oils, etc. Table 11 shows the positive and negative impacts of vegetable-based oils on energy, cost, and environment. These are efﬁcient in terms of all these parameters like in enhancement of tool life, less requirement of energy due to reduction in forces and eco-friendly. However, there are some negative issues like fume generation and cleaning problems, as chips adhere to oil [159]. Table 11. A critical analysis of environmental aspects in bio-degradable oil aided machining, reprinted with permission from ref. [159]. Copyright 2019 Elsevier Ltd. Inﬂuence Performance Issues Energy Cost Environment Positive Improved Tool Wear proﬁle Lower speciﬁc cutting energy due to reduced force Improve performance of bio-based oils reduce overall cost Eco-friendly cooling lubricating agent Increased Tool life Reduced temperature Cost of recycling Recycling of oils can be done improved surface ﬁnish and less friction Energy consumption during production of bio-oils Cost of bio-oil coolant sometimes higher than conventional coolant Fluids from chips need to be separated before chip processing Negative Conventional application mode lacks penetration Mode of oil application determines the additional energy consumption Cost of additives if used MQL spray cause inhalation problem Adhere with chips-separation of oils from chips are required - - Fumes can cause problems to human cal analysis of environmental aspects in bio-degradable oil aided machining, reprinted with permission Copyright 2019 Elsevier Ltd. Materials 2021, 14, 5162 31 of 47 In experimental machining of AISI 304, two types of vegetable-based cutting oils were used. One was sunﬂower oil, and the other was canola oil. The comparison was made with the semi-synthetic mineral oil. It was noted that the above two oils performed well in the context of being environmentally friendly and in cost reduction. The surface ﬁnish was also improved. It was noted that the performance of canola oil with the additive was best in the overall scenario [160]. 4.6. Alternative Cutting Fluids Sunﬂower oil performed well in terms of surface ﬁnish and chip compression ratio. One drawback of vegetable oils is the generation of smoke due to a lower ﬂash point [172]. 4.7. Air/Gas/Vapor Cooling The use of cutting ﬂuids causes environmental damage and health-related issues. In order to avoid these issues, a green cutting environment is being created. In this environment, the use of water vapor plays a major role because there is no need for recycling or disposal, and it is non-toxic and environmentally friendly. The setup diagram is below in Figure 31. 32 of 47 Materials 2021, 14, 5162 Figure 31. Vapor generator device and feeding system, reprinted with permission from ref. [173]. Copyright 2004 Else- vier Ltd. r generator device and feeding system, reprinted with permission from ref. [173]. Copyright 2004 Else- Figure 31. Vapor generator device and feeding system, reprinted with permission from ref. [173]. Copyright 2004 Else- vier Ltd. Temperature reduction, cutting force reduction and improvement in the surface ﬁnish is a positive impact of this technology. Below, Figure 32 shows the temperature comparison between different modes of cutting lubrication techniques in which the use of water vapor is the best technique compared to dry cutting, compressed air, and oil-water emulsion [173]. Figure 32. Cutting Temperature Variation on different Depth of cut, reprinted with permission from ref. [173]. Copyright 2004 Elsevier Ltd. Figure 32. Cutting Temperature Variation on different Depth of cut, reprinted with permission from ref. [173]. Copyright 2004 Elsevier Ltd. Cold air cooling is best during machining as it mitigates the environmental and health issues caused due to use of coolants. Energy consumption increased by 20%, but coolant cost was reduced by 80%, which is an economically good impact [174]. In the literature, different gases have been exploited as a coolant for sustainable machining of steel, i.e., carbon dioxide (CO2), argon, water vapor, oxygen, and nitrogen, as depicted by Kim et al. [175] and Yamazaki et al. [176] in their investigations. Contrarily, it comprises some drawbacks; for example, rough turning is not appropriate for the gas/air cooling method. It also acquires an additional setup for the supplement of gas particles to the machining area. As a coolant, compressed air is not suitable for machining a superalloy like Inconel alloy. From an environmental perspective, CO2 as a gas is not compatible for greenhouse effect. The past studies warrant the use of air or gas as a coolant to sustain the process environmentally. Liu et al. 4.7. Air/Gas/Vapor Cooling [177] performed machining on ANSI 1045 steel against a P10 Materials 2021, 14, 5162 33 of 47 33 of 47 carbide tool under different concentrations of gases and oils. For instance, water vapors (WV), a mixture of CO2 and O2, a combination of WV and CO2, a grouping of WV and O2, dry machining, and wet machining under oil-H2O emulsion were prepared for processing. They deduced that cutting forces improved signiﬁcantly with increased tool life up to 4 to 5 times and 2 to 3 times with CO2 state and WV, respectively. Junyan et al. [178] collated the two different machining contexts; process under WV and state of dry machining. They evaluated the impact of the K20 carbide insert on the performance of ANSI 304 stainless steel in the aforementioned two machining situations. They extrapolated that better results were obtained with WV followed by dry machining in terms of improvement in tool life, a reduction in cutting forces of 25 to 30%, and modiﬁcation in surface integrity. g g y In the machining of AISI 1040, the comparison was carried out between gases appli- cations, wet and dry machining. Three gases were taken, oxygen, nitrogen, and carbon dioxide. It was found that gas application had better result in surface quality, cutting zone temperature, and cutting forces, etc. CO2 had a better cooling effect than other gases used, and the cutting forces and thrust forces were less using this gas compared to other gases. At lower feed, good surface quality was achieved with gas compared to wet machining, in which surface quality improved at a high feed. Figure 33 shows the relation of mean cutting force with feed in dry, wet, and different gases. CO2 was best in all other techniques [179]. Figure 33. Variation in cutting force by using different machining techniques, reprinted with permis- sion from ref. [179]. Copyright 2004 Elsevier B.V. Figure 33. Variation in cutting force by using different machining techniques, reprinted with permis- sion from ref. [179]. Copyright 2004 Elsevier B.V. 4.8. High-Pressure Coolant (HPC) This is another widely accepted technique in the manufacturing industry. Conven- tional machining mostly uses one mechanical mechanism, but HPC is usually comprised of three systems: mechanical, thermal, and tribological controls, which makes it impressive and valuable in high-speed machining [180]. High-speed machining is useful in the follow- ing conditions, (i) difﬁcult to machine materials, (ii) high speed and feed, (iii) deep-hole drilling, (iv) continuous chips production [181]. HPC generally provides high pressure to the coolant, which allows the deep ﬂow of the ﬂuid between the work-electrode spaces or contact regions of tools and chips, as speciﬁed in Figure 34 [182]. The effect of the above phenomenon improves tool life, decreases the consumption of cutting ﬂuid, and maintains the temperature of the work part [183]. It has been found from the literature that HPC not only offers less TWR but also gives superior cooling properties, which results in lessened contact distance as the force of coolant pressure lifts the chip away from tool faces [184]. Ezugwu et al. [185] investigated that boron nitride (BN) and ceramic tools are not ﬁt for high-speed processing of Ti-alloys with HPC supply because it begins the nose rupture and generates discontinuous chips which damage the cutting edges. 34 of 47 Materials 2021, 14, 5162 Figure 34. Position of the tool with respect to the workpiece, reprinted with permission from ref. [182]. Copyright 2006 Elsevier Ltd. Figure 34. Position of the tool with respect to the workpiece, reprinted with permission from ref. [182] Copyright 2006 Elsevier Ltd. It was mentioned earlier that an increase in coolant supply with greater pressure increases the tool life. A study conﬁrmed that tool life is raised by 740% when pressure and coolant speed are set at 203 bar and 50 m/min, respectively. In addition, chip forma- tion is also affected by varying the cutting conditions and coolant pressure at acceptable levels [186]. Kumar et al. [187] have evaluated the effect of HPC on the machining per- formance of ASSAB 718 steel. The improvement in tool wear, ﬂank wear, chip shape and thickness, and cutting forces are governed by HPC. Dhar et al. [188] assessed the consequence of HPC on chips, tool life, and roundness deviation while drilling of AISI 4340 steel. They investigated the outcomes under HPC drilling with the dry drilling process. 4.8. High-Pressure Coolant (HPC) The results summarized that small chip thickness, less roundness, and minimal tool wear were observed via HPC drilling. Thus, researchers called it a more beneﬁcial process than drilling under conventional coolant. Naves et al. [189] presented the machinability of AISI 316 austenitic steel by employing HPC. They used 5% and 10% vegetable oil with coolant at different ranges of pressure (100, 150, 200 bar) against carbide tool inserts. They concluded that ﬂank wear was signiﬁcantly lower when pressure up to 100 bars with a 10% concentration of ﬂuid was applied. The above literature successfully showed that HPC is a highly effective method for achieving long tool life, minimum chip size, and, most importantly, the consumption of ﬂuid is decreased by 50%. 5. Discussion This section broadly investigates the sustainability aspect of steel governed by different machining techniques, such as expressed in Figure 10. To build a state-of-the-art review, comprehensive literature has been studied regarding the sustainability point of view for the manufacture of steel. For this reason, the key letters and strings were treated to reveal the different studies relevant to the above-mentioned case for the literature survey. The literature survey comprises published work obtained from various sources of Journals, including Science Direct, Emerald, Springer, and other publishers. The last 25–30 years articles, 43% from the past ﬁve years, were cited in this study as presented in the graph shown in Figure 35. 35 of 47 Materials 2021, 14, 5162 Figure 35. Graphical representation of the number of cited articles in different year bands. Figure 35. Graphical representation of the number of cited articles in different year bands. Figure 35. Graphical representation of the number of cited articles in different year bands. It has been regarded that steel is the most popular material and is preferred widely in diverse application sectors, i.e., automotive, aerospace, and manufacturing industries, etc. However, advancement in manufacturing areas, together with the requirement of cleaner production, demands sustainable machining techniques. Therefore, this review article is contributing towards the sustainable methods needed for the machining of steel. In addi- tion to the above discussion, this study is also presented as a guide for the selection of the best technique that gives net zero-emission in their manufacturing products. The important published work corresponds to sustainable techniques employed for the machining of steel is described below. Numerous researchers claimed that MQL is the most suitable technique for achieving a sustainable machining environment followed by conventional processes such as drilling, milling, and grinding, etc. [190–193]. Najiha et al. [194] said that MQL is considered a cleaner production process due to its cost-effectiveness and ensuring the safety of both workers and the environment. This statement is also validated by other researchers; for instance, Boswell et al. [111] and Eltaggaz et al. [195] predicted that MQL consumed a minimum quantity of cutting liquid which directly reduces the emission of hazardous fumes, and thus the performance of MQL process was upgraded. Moreover, vegetable oil and non-natural esters are the most commonly used ﬂuids in MQL owing to superb biodegradability and non-toxicity, as stated by Boswell et al. in their study [111]. 5. Discussion Cutting lubricants improve the design attributes of the machining, but they are strictly avoided by some researchers due to the production of health-hazardous fumes and gases during processing which causes serious diseases for the workers. Researchers have prac- ticed the machining operation without any ﬂuid referred to as “dry machining (DM)”. Many manufacturing companies, especially those which produce metallic products, are adopting this technology owing to the freedom from environmental impacts. However, DM has certain drawbacks, which prevent its usage at a level as high as the rest of the tech- niques which used cutting ﬂuids. Gyanendra and Prabir [129] enlisted some disadvantages of DM, which are mentioned below: i. Excessively raise the temperature of the cutting zone, which results in poor TL; ii. Heat affected zones are enlarged, which tends to decrease the strength of the specimen; iii. Surface ﬁnish compromised at such elevated conditions; ii. Heat affected zones are enlarged, which tends to decrease the strength of the specimen; iv. Geometric accuracy and dimensional accuracy of the work part is signiﬁcantly altered; v. The DM has a challenge to machine the difﬁcult-to-cut materials; vi. In comparison to other sustainable machining techniques, DM has high costs and less productivity. There are numerous studies based on DM. For example, Servaraj et al. [200] assessed the effect of cutting speed (80–120 m/min) and feed rate (0.04–0.12 mm/rev) on cutting forces at a DOC of 0.5 mm under DM state. They have tested all experiments on the stainless steel (SS) material. They proposed that magnitude of cutting forces is signiﬁcantly remodeled with a small alteration in feed rate. Fernandez-Abia et al. [201] also performed experimentation under DM environment at a cutting speed of 37–870 m/min, feed rate of 0.2 mm/rev, and 0.1 mm DOC while turning AISI 303 SS. They have taken two responses viz cutting capabilities and chips geometry. They deduced that a cutting rate greater than 450 m/min yielded minimal cutting forces along with satisfactorily decreased chip thickness. Salem and Ahmad [202] optimized the design parameters, such that surface integrity and power consumption, of 316 SS under DM situation using Boron Nitride (BN) electrode. An RSM methodology was developed to validate the machining parameters. The results pointed toward the reduction in power consumption up to 6.8%, with an increase in the surface ﬁnish of about 13.9%. 5. Discussion Dhar and Khan [196] explained that some beneﬁts of the aforementioned ﬂuids over conventional metalworking lubricants are: i. They provide high MRR with a small cutting time; ii. The small electrode erosion rate; iii. They are good absorbers at high pressure; iv. Minimal vaporization and evaporation lead to being environmentally sustainable. iv. Minimal vaporization and evaporation lead to being environmentally sustainabl Synthetic ester, sometimes also known as vegetable oil, is also a promising ﬂuid in order to sustain the machining process due to its high boiling point, excellent ﬂashpoint, and low viscosity, as implied by Dixit et al. [117] in their investigation. Hence, both stated ﬂuids extensively used in the MQL process are the best alternatives in terms of suitability than other conventional liquids. Cryogenic is another fundamental sustainable approach used for the cutting of steel by manipulating cryogenic ﬂuid at optimum temperature. To keep the cutting temperature low, a coolant like nitrogen gas is used because of its non-corrosive and non-combustible nature. Pereira et al. [67] carried out turning operation on AISI 304 material and inferred that a 50% improvement in tool life with a 30% reduction in cutting speed was commemorated under cryogenic machining conditions. Pusavec et al. [197] evaluated the impact of cryogenic Materials 2021, 14, 5162 36 of 47 machining on Inconel 718 alloy by taking surface integrity as a responses parameter. They have used various mixtures of cryogenic liquids and found good surface asperities over the machined region with a cryogenic cutting procedure. Another study by Pusavec et al. [198] was written about the effect of various machining applications (such as cryogenic cooling, MQL, dry machining, cryo-lubricant machining) on the same alloy of Ni (Inconel 718) by constructing a response surface methodology (RSM) model. They validated the model with the support of an ANOVA study. The results were explicated that cryogenic cutting ﬂuid or lubrication signiﬁcantly improved the performance of machining while treating. Machai and Biermann [199] tested the tool life (TL) during machining of Ti-1023 at speciﬁc cutting conditions (Cutting rate = 50–150 m/min, Feed = 0.1 mm, machined depth = 0.3 mm, and stroke length = 50–250 m) under wet and CO2 blend. They summarized that TL is raised approximately by two times with the cryogenic machining as compared to wet operating conditions. Machining under emulsion state also generated large size craters than that of cryogenic condition. 5. Discussion Another prime technique used to ascertain the sustainability perspective of steel can be accomplished with the help of using various cutting ﬂuids. Many pieces of research claimed the ampliﬁcation of machining performance in terms of output responses like cutting rate, MRR, TWR, and SR when different cutting ﬂuids in the dielectric are exercised. For instance, Kashif et al. [203] comprehensively examined the dispersion of graphene nano-powder mixed in the dielectric medium onto the output parameters (i.e., MRR & TWR) of electric discharge machining (EDM) using three electrodes (copper, brass, and aluminum). They proposed that graphene particles in the dielectric disperse the sparking by raising the plasma channel, which leads to raise the MRR and reduce the TWR. Although there are numerous cutting ﬂuids, vegetable oil-based dielectric combinations are mostly Materials 2021, 14, 5162 37 of 47 37 of 47 preferred owing to being environmentally friendly, renewable, non-toxic, non-hazardous, and high biodegradability [125]. Vegetable oil also possesses a high freezing point, good corrosion resistance, and is thermally considered stable [204]. Cetin et al. [205] conducted a study to illustrate the impact of canola oil, sunﬂower oil, and mineral oils on the machining performance of an AISI 304L work part by noticing the SR, cutting forces, and feed forces. They suggested that both vegetable oils provide better surface asperities over the machined surface. Other than this, vegetable oil is outperformed in terms of less feed and cutting forces, followed by mineral oil. Hence, different powder mixed dielectric inﬂuences the machining processes and improved the results. g p p Table 13 shows the comparison in terms of some response parameters like surface ﬁnish and MRR, cutting temperature, tool life, and cutting forces. Tool life was best in cryogenic cooling and Cryo treatment of tools. Dry cutting was not found comparable with other techniques in terms of all these parameters, but from an environmental point of view, it was good. The surface ﬁnish was best in cryogenic cooling and solid lubricants technique. The material removal rate was observed to be greater in the solid lubricants technique. Table 13. Comparison between different sustainable Techniques. (× Bad ×× Good ××× Better ×××× Best). 5. Discussion Technique Tool Life Surface Finish MRR Cutting Temperature Cutting Forces Cryogenic Cooling ×××× ×××× ××× ×××× ××× MQL ××× ××× ×× ××× ××× Dry Cutting × ×× ×× × ×× Solid Lubricants ××× ×××× ×××× ×××× ××× Air/Vapor/Gas ×× ×× ××× ××× ××× Cryogenic treatment ×××× ××× ×× ××× ×××× Alternative cutting ﬂuids ××× ××× ××× ××× ××× Table 13. Comparison between different sustainable Techniques. (× Bad ×× Good ××× Bet mparison between different sustainable Techniques. (× Bad ×× Good ××× Better ×××× Best). After careful review of the presented literature, it has been inferred that sustainable techniques are environmental-friendly and manifest as non-hazardous for human beings. If the demand of manufacturers is to achieve a high MRR with a good surface ﬁnish, then the use of solid lubricants will be preferred. While, if the need is limited to high tool life with excellent surface asperities on the machined part, then cryogenic cooling will be favored. Similarly, sometimes studies are only restricted to minimum cutting forces, then dry machining would be used irrespective of the other machining attributes. The different challenges possessed by the sustainable techniques while their implementation is presented in the subsequent section. Then the paper is summarized in the conclusion section with the discussion of future directions. 6.1. Lack of Awareness Regarding These Techniques Most industries are not aware of these latest techniques, which can contribute con- siderably in terms of productivity improvement and a green and safe environment for workers and surroundings. They only trust conventional methods and consider these a technical requirement. Top management should be equipped with the latest knowledge of the world’s reforms in the ﬁeld of machining of parts. 6. Challenges in Sustainable Machining Different challenges faced for the implementation of sustainable techniques are following. 6.5. Fear of Losing Business during Adoption of These Techniques 6.5. Fear of Losing Business during Adoption of These Techniques It is a myth in conventional industries that whenever they try a new system, it will lead them towards loss of production. It seems that there is not enough time for the trial of any new technology, which is the wrong concept. Without taking a risk, no improvement will occur. 6.4. Usage of Old Technology In most industries, old and conventional machines are being used for the manufactur- ing of parts. There is very little or absence of provision for installation of equipment for working of these techniques. For installation, a huge cost is required for the replacement of the existing system. 6.3. High Equipment Cost No doubt these techniques are very helpful and of paramount importance to the industries for today and tomorrow, but some techniques like cryogenic machining need much attention because right now, their setup cost is high. Indeed, most industries do not know the payback of these, which makes them reluctant to implement. 7. Post-Processing Challenges of Additive Manufactured Steel Additive manufactured steel parts are widely used in different engineering applica- tions. However, geometric inaccuracy and poor surface integrity disallow the use of steel components after their manufacturing through additive manufacturing techniques. From this perspective, post-processing operations are performed to mitigate the above issues. Those post-processes include drilling, milling, grinding, blasting, and tapping, etc. The challenges inherent with the aforesaid traditional processes have been explained by the National Institute of Standards and Technology (NIST), as far as additive manufactured (AM) steel is concerned. The residual, tensile (outer surface), and compressive (inner surface) stresses still remain in the steel component after its production from AM, which hinder the use of such parts of steel via milling, drilling, and other conventional processes because it can be generated trust forces, vibrations, and high frequency that may decrease the tool life rapidly, as discussed by Brandon and Eric in their study [206]. They also reported that residual stress also altered the chips’ formation owing to high tensile forces at the outer surface when plastic deformation is reached. The fabrication of steel parts via direct energy deposition (DED) has inhomogeneities because of inappropriate cooling and porosity that tend to affect the surface ﬁnish of the desired steel parts when turning operation is carried out at certain input variables. Teo et al. [207] also studied the effect of the post-processing technique (sandblasting) on the AM 316L stainless steel part. The surface quality issue governed by the DED process has been successfully eliminated by the sandblasting process, but the introduction of surface damage and peel-off layer takes place. They reported that the peel-off layer could be removed either by electro-polishing technique or by adding abrasive particles; however, it leads to another problem of corrosion. Therefore, steel parts comprising limitations towards machining after their fabrication through AM process. 6.2. Lack of Management Commitment For the implementation of these techniques in any manufacturing industry, a change mindset is most important. Management should be willing to provide all needed resources for effectiveness, but in most industries, this commitment is not present. Materials 2021, 14, 5162 38 of 47 8. Conclusions Sustainable machining techniques became the need of the hour to fulﬁll environmental regulations and to improve operator’s safety. These techniques play an important role in any industry in terms of economic, social, and environmental beneﬁts. To maximize productivity and to make products market competitive, sustainable machining techniques should be adopted. The following main points are concluded for understanding and implementation: • Cryogenic machining is a very popular technique in terms of its excellent cooling impact, which leads to longer tool life and good surface integrity. It does not require Materials 2021, 14, 5162 39 of 47 39 of 47 residual cleaning as in conventional cutting, but some drawbacks are still there like, chips cleaning problem and frostbite hazard associated with the operator’s health. residual cleaning as in conventional cutting, but some drawbacks are still there like, chips cleaning problem and frostbite hazard associated with the operator’s health. • Dry machining is good in terms of an environmental point of view but leads to low surface integrity of the product and higher tooling cost. • Dry machining is good in terms of an environmental point of view but leads to low surface integrity of the product and higher tooling cost. • MQL technique is an intermittent solution between dry and cryogenic in terms of ma- chining cost and product quality. However, problem is chip evacuation is a problem. • In cryogenic treatment of cutting tools, machinability increases, and due to good thermal conductivity, the cutting temperature decreases. This type of tool is not suitable for interrupted cutting due to breakage problems. p g g p • Solid lubricants are found more effective in terms of surface integrity of parts, and tool life increases as the heat transfer rate increased. • Vegetable oils are found to be good from an environmental point of view as there is no need to dispose of these as compared to mineral oils. Due to low ﬂash points, smoke is produced during the turning of steel. Sometimes cleaning may be problematic as chips engaged with oil which is difﬁcult to separate. • Air-gas cooling is a good technique for the environment compared to conventional coolants, but energy costs increased about 20%. • Air-gas cooling is a good technique for the environment compared to conventional coolants, but energy costs increased about 20%. Numerous studies have been conducted on the sustainability of steel in various application conditions. 8. Conclusions As steel is emerging mostly in automotive sectors, along with other large setups like aerospace, nuclear power plant, marine areas, and biomedical equipment, etc. Organizations are conscious of environmental problems, which ultimately put the life of humans at risk. Therefore, it is much more necessary to examine the sustainability point of discussion on steel material. This review was compiled to investigate the sustainable machining techniques for the steel material. Further, the details presented in this review can act as a guide in selecting the best solution to be integrated towards achieving net zero-emission in their manufacturing products. 9. Future Implications This review highlights the major limitations like frostbite hazard in cryogenic machin- ing and initial setup cost, which is difﬁcult to afford by any local industry. A comprehensive investigation is required to mitigate the aforesaid issue of cryogenic machining by ensuring a controlled temperature environment. The mathematical modeling of the sustainable cutting mechanisms with respect to the cutting of steel is still an area that needs special attention. Author Contributions: All authors contributed equally to the data-curation, investigation, writing, and reviewing process. All authors have read and agreed to the published version of the manuscript. Funding: This study has not received any funding Author Contributions: All authors contributed equally to the data-curation, investigation, writing and reviewing process. All authors have read and agreed to the published version of the manuscript Funding: This study has not received any funding. Data Availability Statement: The raw/processed data required to reproduce these ﬁndings cannot be shared at this time as the data also forms part of an ongoing study. Conﬂicts of Interest: There is not any conﬂict of interest between parts. Conﬂicts of Interest: There is not any conﬂict of interest between parts. Compliance with Ethical Standards: There is not any potential conﬂict of interest. Compliance with Ethical Standards: There is not any potential conﬂict of interest. Consent to Publish: The authors provide their consent to publish this work. Consent to Publish: The authors provide their consent to publish this work. References 1. Rao, P.N. Sustainable Manufacturing: Principles, Applications and Directions. Efﬁc. Manuf. 2013, 28, 1 1. Rao, P.N. Sustainable Manufacturing: Principles, Applications and Directions. Efﬁc. Manuf. 2013, 28, 16. 2. Siva Rama Krishna, L.; Srikanth, P.J. Evaluation of environmental impact of additive and subtractive manufacturing processes for sustainable manufacturing. Mater. Today Proc. 2021, 45, 3054–3060. [CrossRef] 2. Siva Rama Krishna, L.; Srikanth, P.J. 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Nomenclature This section describes the nomenclature of various abbreviations used in this study: MQL Minimum Quantity Lubrication CEO Chief executive ofﬁcer OEM Original Equipment Manufacturers 40 of 47 Materials 2021, 14, 5162 LAS Low Alloy Steel HAS High Alloy Steel LNG Liqueﬁed Natural Gas AISC American Institute of Steel Construction HSLA High Strength Low Alloy MPa Mega Pascal GPa Giga Pascal SS Structural Steel HRS Heat Resistant Steels AISI American Iron and Steel Institute PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analysis SR Surface Roughness MRR Material Removal Rate TWR Tool Wear Rate CBN Cubic Boron Nitride HTMF Hard Turning with Minimal Fluid SEM Scanning Electron Microscope HRC Hardness Rockwell C Scale CVD Chemical Vapour Deposition MWF Metalworking Fluid MoS2 Molybdenum disulﬁde DOC Depth of Cut DM Dry Machining TL Tool Life EDM Electric Discharge Machining LOF Lack-of-fusion LAS Low Alloy Steel HAS High Alloy Steel LNG Liqueﬁed Natural Gas AISC American Institute of Steel Construction HSLA High Strength Low Alloy MPa Mega Pascal GPa Giga Pascal SS Structural Steel HRS Heat Resistant Steels AISI American Iron and Steel Institute PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analysis SR Surface Roughness MRR Material Removal Rate TWR Tool Wear Rate CBN Cubic Boron Nitride HTMF Hard Turning with Minimal Fluid SEM Scanning Electron Microscope HRC Hardness Rockwell C Scale CVD Chemical Vapour Deposition MWF Metalworking Fluid MoS2 Molybdenum disulﬁde DOC Depth of Cut DM Dry Machining TL Tool Life EDM Electric Discharge Machining LOF Lack-of-fusion References Development of novel sustainable neat-oil metal working ﬂuids for stainless steel and titanium alloy machining. 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https://openalex.org/W4390608700	https://www.bio-conferences.org/articles/bioconf/pdf/2024/03/bioconf_aquaculture2024_02002.pdf	English	null	Methods of automated detection of travel points when training a collaborative robot	Bio web of conferences/BIO web of conferences	2,024	cc-by	4,234	* Corresponding author: allochka@rambler.ru https://doi.org/10.1051/bioconf/20248402002 https://doi.org/10.1051/bioconf/20248402002 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). Methods of automated detection of travel points when training a collaborative robot N.A. Evstifeeva1, S.A. Gurdiumov1, A.A. Kleimenov1, and A.A. Gerasimova1* 1National University of Science and Technology MISiS, Leninskiy Prospekt, 4, 119049 Moscow, Russia N.A. Evstifeeva1, S.A. Gurdiumov1, A.A. Kleimenov1, and A.A. Gerasimova1* 1National University of Science and Technology MISiS, Leninskiy Prospekt, 4, 119049 Moscow, Russia Abstract. An algorithm has been developed and implemented in this paper, which allows automating the process of forming and controlling scenarios for the movement of a collaborative robot (“Cobot”) through a database of points without specific interfaces, services, and software tools characteristic of each Cobot model. The unification of the developed single graphical interface is achieved by automating the work with Cobot controllers through specialised structured file formats and Robot Operation System (ROS), and by automatically detecting marks as movement points in the image received from the stereo camera using neural network-based models and image processing techniques. Research based on a series of experiments ensured the selection of the most effective image processing method and neural network model in terms of accuracy, speed, resource consumption. The approach formalised in the paper and the graphical interface allowed to implement a classical set of industrial tasks of Cobot motion control. 1 Introduction The use of Cobots in production automation is aimed at reducing the cost of the final product, improving product quality, increasing productivity, and reducing scrap rates. However, when installing and configuring it is necessary to take into account all information input flows, possible work scenarios, and factors affecting the execution of functions and movements of Cobot. Whereas all Cobot actions must be performed with high precision, delicacy, and safety. One of the main advantages of Cobots compared to industrial Cobots is the ability to work together with humans on a production line, which formed the basis for the hypothesis of applying a unified approach to training Cobots regardless of their manufacturer and model. The ability of Cobots to perform tasks in close proximity to humans allows humans in Cobot training to directly indicate movement points by physically placing markers in the Cobot's workspace. Table 1 presents qualitative evaluations on 6 criteria showing the advantages of Cobots compared to classical industrial robots. * Corresponding author: allochka@rambler.ru https://doi.org/10.1051/bioconf/20248402002 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 AQUACULTURE 2023 Table 1. System of criteria for evaluating the efficiency of Cobots automation and industria robots Comparison criterion Industrial robots Cobots Implementation in the production process Sophisticated integration Fast and easy installation Management and training High programming level Overall simpler programming up to learning without skills Mobility Fixed installation location Portable and modular Dimensions of the working area Substantial space requirement Minimum installation dimensions Requirements for safety devices Requires safety guards and protection system Operates without guardrails and with a human nearby Additional costs High Low Table 1. System of criteria for evaluating the efficiency of Cobots automation and industrial robots As can be seen from Table 1, in cases where the payload capacity that a particular Cobot model has meets the requirements of an industrial task, automation with Cobots generally has advantages over industrial robots. Cobots are considered flexible enough in case of frequent changes of gripper devices are required, which in turn can be categorised into two types mechanical and pneumatic: 1. A mechanical claw of various tip shapes gripping the object directly. 1. A mechanical claw of various tip shapes gripping the object directly. 2 V i i h f f i 2. Vacuum gripper in the form of suction cups or pumps. 3. Pneumatic gripper with various tip shapes up to silicone tips Connecting the gripper to the manipulator requires positioning a point called Tool Central Point (TCP) in the correct position. The TCP is located at the centre of the tool attached to the end axis of the Cobot. Depending on the size of the tool, the TCP changes its coordinates accordingly, and therefore Cobot must take its new location into account when moving [1]. In the factory default settings, the TCP is specified for Cobot as a point on the flange of its end axis. Therefore, as soon as the tool is installed, it is required to change the value for TCP to the tool end point in the Cartesian coordinate system in the three axes X, Y, and Z. Correct calculation of the TCP point ensures that all coordinates of the Cobot movement points are correctly calculated. * Corresponding author: allochka@rambler.ru Thus, at present, three groups of Cobots learning can be distinguished: 2 2 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 https://doi.org/10.1051/bioconf/20248402002 1. With general-purpose proficiency in high-level programming languages not specialised with respect to tasks. 1. With general-purpose proficiency in high-level programming languages not specialised with respect to tasks. 2. With proficiency in special-purpose programming languages. 3. Without proficiency in programming languages: 3.1. Training on a touch screen tablet in intuitive non-unique software. 3.2. Training in the FreeDrive mode, i.e., bringing the manipulator to a g The undoubted advantages of training Cobots implemented in groups 2 and 3 are the individuality of the provided software, which takes into account all the capabilities and features of each specific Cobot model. However, there is also an obvious disadvantage, which leads to non-uniformity of interfaces and operators-commands, which forces the expert in training and control to master all variants of software used in production depending on the Cobot model. At the same time, group 1, which requires good skills in programming languages, such as Python, imposes restrictions on the management and training of Cobot by enterprise employees without additional professional development. * Corresponding author: allochka@rambler.ru y Cobots currently support two movement strategies: - Joint (from position to position with the geometry of this type of movement resembling an arc or curve); - Joint (from position to position with the geometry of this type of movement resembling an arc or curve); ) - Liner (along a line, the geometry of this type of movement is a straight line). The main feature of the Joint movement strategy is that there is no possibility to control the tool trajectory, which changes during the movement process. The Joint motion strategy is programmed at the target point and set in angular values for each axis of the Cobot. Also, the actual travel speed depends on the slowest axis. That is, the set speed is transferred to the axis that has to travel the longest distance to the target point during the movement process. Accordingly, the other axes move at the calculated speed so that they finish the movement simultaneously in time with the axis that has travelled the longest distance. The main feature of the Joint movement strategy is that there is no possibility to control the tool trajectory, which changes during the movement process. The Joint motion strategy is programmed at the target point and set in angular values for each axis of the Cobot. Also, the actual travel speed depends on the slowest axis. That is, the set speed is transferred to the axis that has to travel the longest distance to the target point during the movement process. Accordingly, the other axes move at the calculated speed so that they finish the movement simultaneously in time with the axis that has travelled the longest distance. The Liner strategy is programmed at the target point and is specified by coordinates in a Cartesian coordinate system. An important feature of the Liner motion strategy is the location of the point, which has coordinates (0,0,0), relative to the Cobot. In contrast to the Joint strategy, the Liner strategy specifies the overall velocity for the Cobot. The tool moves along a controlled trajectory. When developing and implementing the software algorithm to automate Cobot training, the Liner strategy is a necessary and sufficient condition. In general, the process of Cobot control and training tends to be combined. visual perception with direct motor embodiment of the received tasks [2]. 2 Research and development methods 3 3 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 https://doi.org/10.1051/bioconf/20248402002 , 02002 (2024) BIO Web of Conferences 84 AQUACULTURE 2023 Point description method using a neural network Point description method using picture processing Manual point input method Add, edit, delete a point Add, edit, delete Cobot’s linear motion function D B Manual point input method Point description method using picture processing Point description method using a neural k Add, edit, delete a point Add, edit, delete Cobot’s linear motion function Fig. 1. Units for execution for forming the database of movement points Fig. 1. Units for execution for forming the database of movement points Fig. 1 shows that mark detection and calculation of points coordinates is performed by three blocks of the module: - Manual input. The X, Y, Z coordinate values for a point are entered into the fields of the graphical interface with strict validity checks at the moment of input; - Neural network detection. A pre-trained Single Shot MultiBox Detector (SSD) neural network is used to detect the label and determine the coordinates of the point from the parameters of the stereo camera paired via ROS with the manipulator; - Detection using image analysis and processing methods The approach includes work in HSV colour space, contour analysis and Hough transform. Communication with the manipulator is done through a unified protocol. - Detection using image analysis and processing methods The approach includes work in HSV colour space, contour analysis and Hough transform. Communication with the manipulator is done through a unified protocol. All three blocks determine the X, Y, Z coordinates of a point in metres relative to the stereo camera. However, to determine the Cartesian coordinates of the movement points, a recalculation is required, taking into account the TCP point located on the flange of the Cobot itself, taking into account the offset of the TCP relative to the applied gripping tool and the general position of the zero point of the Cartesian coordinate system relative to the Cobot. The functional diagram of the second module presented in Fig. 2 shows that the purpose of this module is to execute the scenario directly on the Cobot controller. In fact, the task of the module is the sequential generation of commands for the execution of scenarios in an accessible format for reading by the Cobot controller. 2 Research and development methods Software has been developed in this paper that provides control and training of Cobot without special programming knowledge in a unified interface that connects the merits of groups 1 and 3 with methods of intelligent image analysis and filter-based image processing for automatic determination of coordinates of Cobot movement points. The software developed as an input information flow receives real-time data in the format of frames generated by the Intel RealSeanse D435 stereo camera, installed stationary with a full one-step capture of the entire working area of Cobot. The stereo camera of this model gives the image frame stream in RGB format as well as the depth of the scene [3]. In fact, the video stream received from the camera is used in a combination of depth and colour data, allowing for more reliable detection of objects in the scene. The choice of Intel RealSeanse D435 camera is primarily due to its multi-platform support, which facilitates its integration into information systems and applications including ROS. However, it should be considered that it is sensitive to lighting conditions, which will require additional customisation at the place of its installation. Also, although the camera provides relatively high resolution, it may be limited compared to some other high-resolution cameras, which in turn will affect the accuracy of detection of small objects or details. It is the task of detecting a mark, the Cartesian coordinate centre of which is actually the point for the Cobot to move, that is central to automating the development of industrial Cobot task scenarios. Physically, the mark is a red plastic circle with a diameter of 3 cm, located in the centre of a white plastic rectangle with sides of 5 and 7 cm. The developed software consists of two modules: 1. Cobot automated training; 2. Execution of movement scenarios directly by Cobot. The purpose of module 1, the information flows of which are presented in the scheme in Fig. 1, is to solve the problem of detecting the mark and calculating the coordinates of the movement point by its centre. Three tasks are allocated in the course of realisation of the goal, which include: formation of Cobot positions by points using several methods, storage, and correction of the database (DB) of points. 2 Research and development methods When implementing the executing unit, the ROS is enabled if a neural network model was used to detect movement points and for all other variants of mark detection, an algorithm is performed to convert the code of the executable script to binary form and serialise the structured data in the form of binary strings into Python objects that connect the work of the corresponding libraries and execute their commands. 4 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 , 02002 (2024) BIO Web of Conferences 84 https://doi.org/10.1051/bioconf/20248402002 AQUACULTURE 2023 Fig. 2. Units of execution for forming Cobot movement scripts Add, edit, delete a script DB function point JSON Analyzer Cobot Executing Unit Add, edit, delete a script JSON Analyzer Fig. 2. Units of execution for forming Cobot movement scripts In this case ROS is used in a standard way as an open-source environment for robot software development that provides services for creating, managing and distributing software code for controlling robotic systems, currently supported by Open Robotics [4]. In other words, the prepared nodes in ROS, which are independent processes executed at different stages of the scenario but interacting with each other through ROS protocols and services connect the control of the Cobot controller. It is the ROS when mediated by the nodes that undertakes the task of providing a convenient mechanism for message passing and service invocation between the node-nodes, which facilitates communication and integration of the different components of the robot. The software development used a simulation model of a 6-axis manipulator, which is dependent on the physical parameters of the Cobot, which will entail additional training of the model on the motion of the manipulator as a whole when they change. To ensure that the basic linear motion functions of the Cobot model can be compared and matched with the points and scenarios, it is necessary to specify the conjugation of the functions and check the occupancy of the mandatory parameters. Fig. 3 shows a part of the database scheme for storing data on points, scriptss, functions and parameters of Cobot motion. 5 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 https://doi.org/10.1051/bioconf/20248402002 AQUACULTURE 2023 Fig. 3. Partial diagram of the database of points, scripts, and motion functions Fig. 3. Partial diagram of the database of points, scripts, and motion functions In addition to the data set shown in Fig. 2 Research and development methods 3, the database stores information for the operation of the verification algorithm of Cobot movements from the point of view of not colliding with obstacles in the working area: the layout of the room, the area of safe movement of Cobot and unambiguously allowed ranges of correction in the offset relative to TCP of the applied capture tools. Verification takes place in several levels of safe movement checks with a minimum timeout of 10 ms and in case of erroneous coordinates, an immediate command to "freeze" the Cobot's movement takes place. 3 Results Detection based on the neural network model is implemented using the SSD neural network. Since SSD uses convolutional layers with different filter sizes, it has a higher accuracy compared to, for example, Yolo. Fig. 4 shows the result of label detection by the models based on the pretrained SSD and Yolov8m neural networks, respectively. Tests performed on the detection rate showed a clear advantage of the SSD network over Yolov8m. The higher detection rate is also confirmed by the more frequent use of SSD for real-time object detection tasks. 6 https://doi.org/10.1051/bioconf/20248402002 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 Fig. 4. a) Yolov8m detection result; b) SSD detection result Fig. 4. a) Yolov8m detection result; b) SSD detection result As a result of the comparative study, a pre-trained neural network SSD is selected for implementation in the developed software. The main objective of SSD is to simultaneously detect objects at different scales and abstraction levels of the input image. That is ensured by an architecture using convolution layers, association layers, and additional layers that generate predictions about object boundaries and classes. In this article, additional training is performed on a data volume of 500 detectable label images, which increased the average label detection accuracy by 12% to reach 94%. Fig. 5 shows the interaction scheme of the SSD neural network detection algorithm with the node, implemented using Real-Time Streaming Protocol (RTSP). Fig. 5. Detection and control of the manipulator with neural network model SSD Detection Camera Tag Action node Fig. 5. Detection and control of the manipulator with neural network model RTSP establishes and manages media sessions between client and server devices, which allows clients to control data streams such as playback, pause, rewind and other functions, 7 7 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 https://doi.org/10.1051/bioconf/20248402002 and supports streaming video processing [4]. It supports real-time video streaming, which allows neural network models to process video data as it arrives. and supports streaming video processing [4]. It supports real-time video streaming, which allows neural network models to process video data as it arrives. To write the programme in ROS, the MoveIt library was used, the main task of which is robot control. It provides services for motion planning, motion execution, interaction with the scene and control of dynamic properties of Cobot [5, 6]. The MoveIt is used to plan the motion trajectory of the manipulator given the constraints of its configuration and the scene and execute the planned motion trajectory of the manipulator given the given constraints. Calculation of the distance to the centre of the detected marker is performed using the depth map D based on the generated set of pixel values 𝑑𝑒𝑝𝑡ℎ_𝑠𝑒𝑡= {𝐷(𝑥, 𝑦) \| 𝐷(𝑥, 𝑦) 𝑖𝑛 𝑐𝑖𝑟𝑐𝑙𝑒}, which lie in the inner region of the detected marker. To determine the desired distance to the mark using formula 2, the median value is calculated using formula 1. 𝑑𝑒𝑝𝑡ℎ_𝑚𝑒𝑑𝑖𝑎𝑛= 𝑚𝑒𝑑𝑖𝑎𝑛(𝑑𝑒𝑝𝑡ℎ_𝑠𝑒𝑡) (1) 𝐷𝑖𝑠𝑡𝐿= 𝑑𝑚𝑖𝑛+ 1 255(𝑑𝑚𝑎𝑥−𝑑𝑚𝑖𝑛) ⋅𝑑𝑒𝑝𝑡h_𝑚𝑒𝑑𝑖𝑎𝑛 (2) 𝑑𝑒𝑝𝑡ℎ_𝑚𝑒𝑑𝑖𝑎𝑛= 𝑚𝑒𝑑𝑖𝑎𝑛(𝑑𝑒𝑝𝑡ℎ_𝑠𝑒𝑡) (1) 𝑑𝑒𝑝𝑡ℎ_𝑚𝑒𝑑𝑖𝑎𝑛= 𝑚𝑒𝑑𝑖𝑎𝑛(𝑑𝑒𝑝𝑡ℎ_𝑠𝑒𝑡) (1) 𝐷𝑖𝑠𝑡𝐿= 𝑑𝑚𝑖𝑛+ 1 255(𝑑𝑚𝑎𝑥−𝑑𝑚𝑖𝑛) ⋅𝑑𝑒𝑝𝑡h_𝑚𝑒𝑑𝑖𝑎𝑛 (2) (1) 𝐷𝑖𝑠𝑡𝐿= 𝑑𝑚𝑖𝑛+ 1 255(𝑑𝑚𝑎𝑥−𝑑𝑚𝑖𝑛) ⋅𝑑𝑒𝑝𝑡h_𝑚𝑒𝑑𝑖𝑎𝑛 (2) (2) As soon as the desired distance to the mark 𝐷𝑖𝑠𝑡𝐿 is obtained to determine specific Cartesian coordinate values 𝑋𝐿, 𝑌𝐿, 𝑍𝐿 of the marking centre, it is necessary to use the system of equations presented in formula 3. { 𝑋𝐿= 2𝑥−𝑊 𝑊 ⋅𝑡𝑔( 𝐹𝑜𝑉ℎ 2 ) ⋅𝑍𝐿 𝑌𝐿= 2𝑦−𝐻 𝐻 ⋅𝑡𝑔( 𝐹𝑜𝑉𝑣 2 ) ⋅𝑍𝐿 𝑋𝐿 2 + 𝑌𝐿 2 + 𝑍𝐿 2 = 𝐷𝑖𝑠𝑡𝐿 2 , (3) (3) and here:  𝑥, 𝑦 determine the centre of the found circle in pixels;  𝑥, 𝑦 determine the centre of the found circle in pixels;  𝑊, 𝐻 – image width and height in pixels;  𝑊, 𝐻 – image width and height in pixels;  𝐹𝑜𝑉ℎ, 𝐹𝑜𝑉𝑣 – the horizontal and vertical camera viewing angles specified in the stereo camera model documentation. 4 Conclusions Thus, the study has shown that both methods are comparable in terms of applicability in automating the training of Cobots, and the effectiveness of a particular method should rather be determined by the readiness of further application of points in motion functions, namely the presence or absence of tested solutions through the ROS system. Thus, when solving a problem in the absence of ready nodes for the manipulator in the ROS system or the impossibility of efficient adaptation of the ready solution to the current model, it is more reasonable to apply the interface module without intelligent data processing and vice versa. 7 Calculation of the distance to the centre of the detected marker is performed according to the identical algorithm, i.e., as in the case of the neural network model the distance is Calculation of the distance to the centre of the detected marker is performed according the identical algorithm, i.e., as in the case of the neural network model the distance 8 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 https://doi.org/10.1051/bioconf/20248402002 calculated 𝐷𝑖𝑠𝑡𝐿 by formula 2 using 𝑑𝑒𝑝𝑡ℎ_𝑚𝑒𝑑𝑖𝑎𝑛, derived from a set of pixel values 𝑑𝑒𝑝𝑡ℎ_𝑠𝑒𝑡 in the detected region. The result of the algorithm is shown in Fig. 6. calculated 𝐷𝑖𝑠𝑡𝐿 by formula 2 using 𝑑𝑒𝑝𝑡ℎ_𝑚𝑒𝑑𝑖𝑎𝑛, derived from a set of pixel values 𝑑𝑒𝑝𝑡ℎ_𝑠𝑒𝑡 in the detected region. The result of the algorithm is shown in Fig. 6. Fig. 6. Visualisation of the calculated distance to the detected marker Fig. 6. Visualisation of the calculated distance to the detected marker The task of tag detection to determine the coordinates of the movement point was solved by two methods: using a pre-trained SSD neural network and based on image processing without an intelligent component. As a result of research conducted on more than 300 experiments with Rozum Robotic model, Cobots revealed average comparative characteristics shown in Table 2. Table 2. Study result on tag detection methods effectiveness with and without intelligence Table 2. Study result on tag detection methods effectiveness with and without intelligence Feature Neural network and ROS model HSV model and Hough method Speed of finding the tag 0.15 sec 0.03 sec Tag detection accuracy 94% 95.7% Centre accuracy 99% 97.8% Distance accuracy ±3mm ±6𝑚𝑚 7 The search for a circle and determination of its parameters is performed using the HoughCircles function [7] of the computer vision library OpenCV with parameters: 𝑚𝑖𝑛𝐷𝑖𝑠𝑡= 0.25 ∗ℎ𝑒𝑖𝑔ℎ𝑡(𝐼𝑅𝐺𝐵), 𝑝𝑎𝑟𝑎𝑚1 = 200, 𝑝𝑎𝑟𝑎𝑚2 = 15, 𝑚𝑖𝑛𝑅𝑎𝑑𝑖𝑢𝑠= 20, 𝑚𝑎𝑥𝑅𝑎𝑑𝑖𝑢𝑠= 100. The method is run on a single channel image 𝐼𝑅𝐺𝐵,𝑖𝑛, formed according to formula 4. 𝐼𝑅𝐺𝐵,𝑖𝑛(𝑥, 𝑦) = { 𝐼𝑅𝐺𝐵,𝑔𝑟𝑎𝑦(𝑥, 𝑦), 𝐼𝑅𝐺𝐵,𝑔𝑟𝑎𝑦(𝑥, 𝑦) 𝑖𝑛 𝑐𝑜𝑢𝑛𝑡𝑜𝑢𝑟 0, 𝐼𝑅𝐺𝐵,𝑔𝑟𝑎𝑦(𝑥, 𝑦) 𝑜𝑢𝑡 𝑐𝑜𝑢𝑛𝑡𝑜𝑢𝑟 (4) The HoughCircles function mathematically uses the Hough method to find circles [8,9]. The Hough method uses a voting scheme, where each point (pixel) votes for all possible models to which it corresponds. In the accumulation space, these votes form local maxima, which are used to determine the specific values of the most significant models. The HoughCircles function mathematically uses the Hough method to find circles [8,9]. The Hough method uses a voting scheme, where each point (pixel) votes for all possible models to which it corresponds. In the accumulation space, these votes form local maxima, which are used to determine the specific values of the most significant models. In this case, the model is a circle described by the formula (𝑥−𝑎)2 + (𝑦−𝑏)2 = 𝑟2 where (𝑎, 𝑏) are the circle centre coordinates, 𝑟 is its radius. Thus, the circle is uniquely defined by three parameters < (𝑎, 𝑏), 𝑟> which form the feature space of the Hough method. q y defined by three parameters < (𝑎, 𝑏), 𝑟> which form the feature space of the Hough method. The set of centres of all possible circles of radius 𝑟 passing through a particular point forms a circle of the same radius. y p ( ) p g The set of centres of all possible circles of radius 𝑟 passing through a particular point forms a circle of the same radius. The result of circle detection by the Hough method are specific parameters ( ) The result of circle detection by the Hough method are specific parameters < (𝑎, 𝑏), 𝑟>. < (𝑎, 𝑏), 𝑟>. References 1. J. Kober, J. A. Bagnell, J. Peters, Reinforcement learning in robotics: A survey, The International Journal of Robotics Research, 32(11), 1238-1274 (2013) 2. S. Levine, et al., End-to-end training of deep visuomotor policies, The Journal of Machine Learning Research, 17(1), 1334-1373 (2016) 3. V. Tadic, et al., Application of Intel RealSense Cameras for Depth Image Generation in Robotics, WSAS Transactions on Computers, 18, 107-112 (2019) 4. K. M. Lynch, F. C. Park, Modern robotics (Cambridge University Press, 2017) 9 9 , 02002 (2024) BIO Web of Conferences AQUACULTURE 2023 84 https://doi.org/10.1051/bioconf/20248402002 5. M. Intisar, et al., Computer Vision Based Robotic Arm Controlled Using Interactive GUI, Intelligent Automation & Soft Computing, 27, 533-550 (2021) 6. A. Butterworth, et al., Leveraging Multi-modal Sensing for Robotic Insertion Tasks in R&D Laboratories, IEEE Conference on Automation Science and Engineering (CASE) (2023) 7. A. R. M. Khairudin, et al., Design and Control of an Articulated Robotic Arm for Archery, IEEE 5th International Symposium in Robotics and Manufacturing Automation (ROMA), 1-5 (2022) 8. A. Gerasimova, O. Mishedchenko, V. Devyatiarova, Determination of temperature conditions for steel plate rolling at Vyksa Steel Works (AO VMZ), IOP Conference Series: Materials Science and Engineering, 709(2), 022016, (2020) doi: 10.1088/1757- 899X/709/2/022016 9. M.A. Kudrina, Using the Hough transform to detect straight lines and circles in the image, Izvestia of Samara Scientific Centre of the Russian Academy of Sciences, 16, 4(2), 476-478 (2014) 10
https://openalex.org/W3042229206	https://www.frontiersin.org/articles/10.3389/fncir.2020.00043/pdf	English	null	Cholinergic Projections From the Pedunculopontine Tegmental Nucleus Contact Excitatory and Inhibitory Neurons in the Inferior Colliculus	Frontiers in neural circuits	2,020	cc-by	12,764	Cholinergic Projections From the Pedunculopontine Tegmental Nucleus Contact Excitatory and Inhibitory Neurons in the Inferior Colliculus William A. Noftz 1,2, Nichole L. Beebe 2, Jeffrey G. Mellott 2 and Brett R. Schoﬁeld 1,2* 1School of Biomedical Sciences, Kent State University, Kent, OH, United States, 2Department of Anatomy and Neurobiology, Hearing Research Group, Northeast Ohio Medical University, Rootstown, OH, United States The inferior colliculus processes nearly all ascending auditory information. Most collicular cells respond to sound, and for a majority of these cells, the responses can be modulated by acetylcholine (ACh). The cholinergic effects are varied and, for the most part, the underlying mechanisms are unknown. The major source of cholinergic input to the inferior colliculus is the pedunculopontine tegmental nucleus (PPT), part of the pontomesencephalic tegmentum known for projections to the thalamus and roles in arousal and the sleep-wake cycle. Characterization of PPT inputs to the inferior colliculus has been complicated by the mixed neurotransmitter population within the PPT. Using selective viral-tract tracing techniques in a ChAT-Cre Long Evans rat, the present study characterizes the distribution and targets of cholinergic projections from PPT to the inferior colliculus. Following the deposit of viral vector in one PPT, cholinergic axons studded with boutons were present bilaterally in the inferior colliculus, with the greater density of axons and boutons ipsilateral to the injection site. On both sides, cholinergic axons were present throughout the inferior colliculus, distributing boutons to the central nucleus, lateral cortex, and dorsal cortex. In each inferior colliculus (IC) subdivision, the cholinergic PPT axons appear to contact both GABAergic and glutamatergic neurons. These ﬁndings suggest cholinergic projections from the PPT have a widespread inﬂuence over the IC, likely affecting many aspects of midbrain auditory processing. Moreover, the effects are likely to be mediated by direct cholinergic actions on both excitatory and inhibitory circuits in the inferior colliculus. Keywords: acetylcholine, auditory, choline acetyltransferase, midbrain, viral tracing, hearing, neuromodulation, arousal Edited by: Edited by: Conny Kopp-Scheinpﬂug, Ludwig Maximilian University of Munich, Germany Reviewed by: Susanne Schmid, University of Western Ontario, Canada Troy Hackett, Vanderbilt University, United States Correspondence: Brett R. Schoﬁeld bschoﬁe@neomed.edu Received: 22 April 2020 Accepted: 19 June 2020 Published: 16 July 2020 Citation: Noftz WA, Beebe NL, Mellott JG and Schoﬁeld BR (2020) Cholinergic Projections From the Pedunculopontine Tegmental Nucleus Contact Excitatory and Inhibitory Neurons in the Inferior Colliculus. Front. Neural Circuits 14:43. doi: 10.3389/fncir.2020.00043 Edited by: Conny Kopp-Scheinpﬂug, Ludwig Maximilian University of Munich, Germany Conny Kopp-Scheinpﬂug, Ludwig Maximilian University of Munich, Germany Reviewed by: Susanne Schmid, University of Western Ontario, Canada Troy Hackett, Vanderbilt University, United States Correspondence: Brett R. Schoﬁeld bschoﬁe@neomed.edu ORIGINAL RESEARCH published: 16 July 2020 doi: 10.3389/fncir.2020.00043 INTRODUCTION Acetylcholine (ACh) plays a wide range of roles in normal auditory function, exerting influence from the cochlea to the auditory cortex. In the forebrain, ACh contributes to memory, learning, and attention (Hasselmo and Sarter, 2011; Leach et al., 2013). In the thalamus, ACh differentially influences the efficacy of inputs to a cell, affecting the gating of information flow and possibly biasing a cell toward top-down vs. bottom-up modulation (Sottile et al., 2017). At many levels July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 1 Cholinergic Inputs to Inferior Colliculus Noftz et al. of the auditory system, ACh can increase spontaneous activity and excitability of auditory neurons and can alter tuning profiles of cells (Farley et al., 1983; Sarter and Bruno, 1997; Ji et al., 2001; Metherate, 2011; Suga, 2012). Finally, ACh is a primary neurotransmitter in the olivocochlear system and plays a critical role in the cochlear amplifier (Dallos et al., 1996; Ryugo et al., 2011). Although much of the previous research on ACh in the auditory system has been done at the levels of forebrain and cochlea, the evidence is accumulating for widespread and varied effects of ACh in the inferior colliculus (IC), a midbrain hub for both ascending and descending auditory pathways (Winer and Schreiner, 2005; Schofield and Beebe, 2019). showed that both GABAergic and glutamatergic IC cells can express nicotinic receptors, but their methods did not provide information on the subcellular localization of those receptors (in fact, their study was focused on cholinergic effects on the axon terminals of IC cells that project to the thalamus). An understanding of cholinergic effects in the IC will require identification of the cell types that receive direct cholinergic inputs. The major source of cholinergic input into the IC is from the pontomesencephalic tegmentum (PMT; Motts and Schofield, 2009, 2011; Schofield et al., 2011). The PMT is the primary source of cholinergic innervation of the thalamus and brainstem and is closely associated with the sleep-wake cycle, sensory gating and attention (Reese et al., 1995a,b,c; Jones, 2017; Cissé et al., 2018). It comprises two groups of neurons: the laterodorsal tegmental nucleus (LDT) which is situated largely within the periaqueductal gray (PAG), and the pedunculopontine tegmental nucleus (PPT). Of the two components, the PPT is the predominant source of cholinergic inputs to the IC (Motts and Schofield, 2009). INTRODUCTION At its caudal end, the PPT is ventrolateral to the PAG and surrounds the superior cerebellar peduncle. The PPT extends rostro-ventrally from this location almost as far as the substantia nigra in the rostral and ventral midbrain. Nearly half of the neurons in the PPT region respond to sound, and the cholinergic neurons have been implicated in acoustic startle and tone-specific plasticity (e.g., Reese et al., 1995a,b,c; Xiong et al., 2009; Suga, 2012; Azzopardi et al., 2018). Application of ACh to the IC affects the responses to auditory stimuli of a majority of IC neurons (Watanabe and Simada, 1973; Farley et al., 1983; Habbicht and Vater, 1996). Such effects can modify temporal processing and forward masking (Felix et al., 2019). Supporting the idea of widespread effects of ACh in the IC, both nicotinic and muscarinic ACh receptors are present throughout the IC, as is acetylcholinesterase, the enzyme that degrades ACh (Shute and Lewis, 1967; Cortes et al., 1984; Glendenning and Baker, 1988; Henderson and Sherriff, 1991; Happe and Morley, 2004). This is relevant because the physiological studies described above focused on cells in the central nucleus of the IC (ICc), the main lemniscal division of the IC. The dorsal cortex (ICd) and the lateral cortex (IClc) are extralemniscal subdivisions that give rise to parallel ascending pathways that terminate in different parts of the thalamus and serve a variety of functions. These three subdivisions vary concerning cholinergic innervation; in fact, the extralemniscal divisions typically exhibit the highest levels of cholinergic receptors. Staining for β4 nicotinic cholinergic receptor subunits is heaviest in layer 2 of the IClc, with moderate expression in the ICc and less in the ICd (Gahring et al., 2004). These receptors have recently been reported to aid in the modulation of spike timing and forward masking in the IC (Felix et al., 2019). The IClc has also been noted for its comparatively high levels of the α7 nicotinic receptor subunit and high levels of acetylcholinesterase (Happe and Morley, 2004; Dillingham et al., 2017). Muscarinic receptors also stained differentially in the IC, with an expression of m2 receptors highest in the IClc and ICd and less so in the ICc (Hamada et al., 2010). Frontiers in Neural Circuits \| www.frontiersin.org INTRODUCTION All of this points to a diverse effect of ACh onto several different regions of IC which are known to participate in different parallel ascending auditory and multisensory pathways (Calford and Aitkin, 1983; Rouiller, 1997; Mellott et al., 2014). Here we take advantage of viral vectors and a transgenic rat line to allow for the selective tracing of cholinergic projections into the IC. This is important because the PMT contains a mixed population of neuronal neurotransmitter phenotypes, including cholinergic, GABAergic, and glutamatergic cells (Wang and Morales, 2009). Traditional tract-tracing methods rely on axonal transport of tracers without regard for neurotransmitter phenotype, making it difficult to identify the neurotransmitter associated with any particular axon. We used viral vectors that express fluorescent protein only in cells that contain Cre-recombinase. The vectors were injected into the PPT in ChAT-Cre rats, in which Cre-recombinase is expressed only in cholinergic cells. We then use antibodies against glutamic acid decarboxylase (GAD), a specific marker of GABAergic neurons, to distinguish GABAergic from glutamatergic IC neurons. Our analyses focus on the central nucleus (ICc), the lateral cortex (IClc), and the dorsal cortex (ICd), three of the largest IC subdivisions, and the focus of most previous studies of cholinergic effects in the IC. We observed cholinergic axons from the PPT throughout the IC ipsilateral and contralateral to the labeled PPT cholinergic neurons. The axons typically possessed many boutons, including ones in close apposition to GAD-immunopositive (GAD+) and GAD-immunonegative (GAD−) neurons in all the IC subdivisions examined. These results suggest that cholinergic axons from the PPT directly contact glutamatergic and GABAergic IC neurons and thus could modulate both excitatory and inhibitory circuits that arise from these cells. Despite the numerous studies of cholinergic receptors in the IC, there is very little information about the identity of IC cells that are directly targeted by the cholinergic inputs. Neurons of the IC are glutamatergic or GABAergic, with GABAergic neurons constituting 20–40% of this population (Oliver et al., 1994; Winer et al., 1996; Merchán et al., 2005; Mellott et al., 2014). Both glutamatergic and GABAergic IC cells likely receive direct cholinergic inputs. Yigit et al. (2003) provided evidence that cholinergic inputs directly activate GABAergic IC cells during development. Sottile et al. (2017) July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 2 Cholinergic Inputs to Inferior Colliculus Noftz et al. Surgery h Each rat was deeply anesthetized with isoflurane in oxygen (3.5%–5% isoflurane for induction; 1.75%–3% for maintenance). The rat’s head was shaved and disinfected with Betadine (Perdue Products L.P., Stamford, CT, USA). Atropine sulfate (0.08 mg/kg, i.m.) was given to minimize respiratory secretions and Ketofen (ketoprofen; 5 mg/kg, s.c.; Henry Schein, Melville, NY, USA) or Meloxicam SR (1.5 mg/kg, s.c.; ZooPharm, Laramie, WY, USA) was given for pain management. Moisture Eyes PM ophthalmic ointment (Bausch and Lomb, Rochester, NY, USA) was applied to each eye to protect the cornea. The animal’s head was positioned in a stereotaxic frame with a mouth bar positioned 3.5 mm ventral to the horizontal plane through interaural zero. Body temperature was maintained with a feedback-controlled heating pad. Sterile instruments and aseptic techniques were used for all surgical procedures. An incision was made in the scalp and the surrounding skin was injected with 0.5% bupivacaine (Hospira, Inc., Lake Forest, IL, USA), a long-lasting local anesthetic. A craniotomy was made using a dental drill. A 1 µl Hamilton microsyringe was mounted in a manipulator that was rotated caudally in the sagittal plane so that the syringe came in at a 30◦angle above the horizontal axis. Following viral injection, Gelfoam (Harvard Apparatus, Holliston, MA, USA) was placed in the craniotomy and the scalp sutured. The animal was then removed from the stereotaxic frame and placed in a clean cage. The animal was monitored until it could walk, eat, and drink without difficulty. Perfusion and Tissue Processing Perfusion and Tissue Processing Four weeks after surgery, the animal was deeply anesthetized with isoflurane and perfused transcardially with Tyrode’s solution, followed by 250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 and then by 250 ml of the same fixative with 10% sucrose. The brain was removed and stored at 4◦C in fixative with 25–30% sucrose for cryoprotection. The following day, the brain was prepared for processing by removing the cerebellum and cortex and blocking the remaining piece with transverse cuts posterior to the cochlear nucleus and anterior to the medial geniculate body. The tissue was frozen and cut on a sliding microtome into 40 µm thick transverse sections, collected in six sets. Before staining, sections were permeablized in 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 30 min at room temperature, then blocked in 10% normal goat serum in 0.2% Triton X-100 and PBS for 1 h, also at room temperature. Sections were then processed for markers as described below. EYFP label was amplified using an antibody against the green fluorescent protein (GFP, 1:400, Molecular Probes A10262; RRID: AB_2534023; note this antibody cross-reacts with EYFP) in combination with a Tyramide Signal Amplification Kit (Molecular Probes). In two cases, an antibody against ChAT (Chemicon AB144P 1:100; RRID: AB_2079751) was used to verify that viral expression was limited to cholinergic neurons. Putative GABAergic cells were stained with an antibody against GAD67 (1:400; Millipore MAB5406; RRID: AB_2278725). Neurons were counterstained with an antibody against the neuronal nuclear protein (NeuN; 1:500; Millipore ABN78; RRID: AB_10807945). In cases where the ChAT antibody was used, a biotinylated anti-goat antibody (1:100; Vector BA-5000; AB_2336126) was used followed by an AF546 Streptavidin tag (1:100; Thermo Fisher Scientific Cat# S-11225; RRID: AB_2532130) to label ChAT-positive neurons in the PPT. In all other cases, a mixture of an AF488 streptavidin tag (1:100; Thermo Fisher Scientific Cat# S11223; RRID: AB_2336881), an AF564 conjugated anti-mouse secondary (1:100; Thermo Fisher Scientific Cat# A10036, RRID: AB_2534012), and an AF750 conjugated anti-rabbit secondary (1:100; Thermo Fisher Scientific Cat# A21039; RRID: AB_10375716) were used to label GFP, GAD67, and NeuN, respectively. Stained sections were mounted on gelatin-coated slides, allowed to dry and coverslipped with DPX. Viral Tracing Long Evans LE tg (ChAT-Cre) 5.1 Deis rats were obtained from the Rat Resource and Research Center (University of Missouri). Cre-recombinase is expressed in nearly all cholinergic neurons in these animals (Witten et al., 2011). Two viral vectors were used. Each vector delivers a gene for the expression of fluorescent protein (EYFP or mCherry). The gene is in double-inverted orientation (DIO), so it is expressed only in neurons that contain Cre-recombinase (i.e., in cholinergic neurons). rAAV2/EF1a-DIO-EYFP (titer: 4.6 × 1012; UNC Vector Core) or rAAV2/EF1a-DIO-mCherry (titer: 3.2 × 1012; UNC Vector Core) was injected in the right PPT of each animal. Coordinates for the injections were chosen to target the caudal PPT, where the cholinergic cells that project to the IC are concentrated (Motts and Schofield, 2009). In two animals, 50 nl vectors were deposited over 2 min at a single site. In the remaining animals, 300–400 nl was delivered over 10 min MATERIALS AND METHODS at one site (12 animals) or each of two sites (four animals). In the latter cases, the syringe was inserted twice, so that one deposit was positioned 0.4–0.5 mm dorsal to the other. After each deposit, the syringe was left in place for 2 min before being withdrawn. All procedures were conducted following the Northeast Ohio Medical University Institutional Animal Care and Use Committee and National Institutes of Health guidelines. Eighteen Long Evans LE tg (ChAT-Cre) 5.1 Deis rats (Rat Resource and Research Center, University of Missouri; 12 female; six male) received injections of the vector into the PPT. Efforts were made to minimize the number of animals and their suffering. A list of all key resources used in this study are presented in Table 1. Frontiers in Neural Circuits \| www.frontiersin.org Data Analysis Cholinergic neurons stained with anti-ChAT or labeled by the viral vector injections were used to identify the cholinergic nuclei according to previously published criteria (Motts et al., 2008). In the original study describing the generation of the ChAT-Cre rats, the authors tested the specificity of labeling after injection of a viral vector carrying a gene for Cre-dependent July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 3 Cholinergic Inputs to Inferior Colliculus Noftz et al. TABLE 1 \| Key resources. Reagent type (species) or resource Designation Source or Reference Identiﬁers Additional Information Genetic reagent (Rattus norvegicus) LE-Tg(Chat-Cre)5.1Deis Rat Resource and Research Center Donor: Dr. Karl Deisseroth (Stanford) RRRC:00658 RRID: RGD_10401204 Recombinant DNA reagent rAAV2/EF1a-DIO-EYFP UNC Vector Core Donor: Dr. Karl Deisseroth (Stanford) N/A titer: 4.6 × 1012 http://www.everyvector.com/ sequences/show_public/8791 Recombinant DNA reagent rAAV2/EF1a-DIO-mCherry UNC Vector Core Donor: Dr. Karl Deisseroth (Stanford) N/A titer: 3.2 × 1012 http://www.everyvector.com/ sequences/show_public/4897 Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientiﬁc Cat#: A10262 RRID: AB_2534023 IHC (1:400) Antibody anti-ChAT (goat polyclonal) Millipore Cat#: AB144P RRID: AB_2079751 IHC (1:100) Antibody anti-GAD67 (mouse monoclonal) Millipore Cat#: MAB5406 RRID: AB_2278725 IHC (1:400) Antibody anti-NeuN (rabbit polyclonal) Millipore Cat#: ABN78 RRID: AB_10807945 IHC (1:500) Antibody biotinylated anti-goat (rabbit) Vector Cat#: BA-5000 RRID: AB_2336126 IHC (1:100) Antibody AF546 streptavidin Thermo Fisher Scientiﬁc Cat#: S11225 RRID: AB_2532130 IHC (1:100) Antibody AF488 streptavidin Thermo Fisher Scientiﬁc Cat#: S11223 RRID: AB_2336881 IHC (1:100) Antibody AF564 anti-mouse (donkey polyclonal) Thermo Fisher Scientiﬁc Cat#: A10036 RRID: AB_2534012 IHC (1:100) Antibody AF750 anti-rabbit (goat) Thermo Fisher Scientiﬁc Cat#: A21039 RRID: AB_10375716 IHC (1:100) Software Neurolucida MBF Bioscience RRID: SCR_001775 expression of YFP (Witten et al., 2011). Their results showed that over 90% of the YFP-labeled neurons were immunoreactive for ChAT, a specific marker of cholinergic neurons. To ensure that subsequent mutations had not interfered with that specificity, we immunostained sections from two animals with anti-ChAT. IC subdivisions were also identified based on previous criteria (Coote and Rees, 2008; Beebe et al., 2016). Plots and analyses were performed using a Neurolucida system (MBF Bioscience; RRID: SCR_001775) attached to a Zeiss AxioImager Z2 fluorescence microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). every sixth section through the ipsilateral and contralateral IC. The overall pattern of labeling was consistent across cases, with variations appearing to relate generally to the relative size of the injections (i.e., the relative number of labeled cells in the PMT). Frontiers in Neural Circuits \| www.frontiersin.org Injection of Viral Vector Into PPT Labels Cholinergic Neurons g Injection of either AAV-EF1a-DIO-mCherry or AAV-EF1a- DIO-EYFP into the PPT of Long Evans ChAT-Cre transgenic rats yielded expression of fluorescent protein in neurons associated with the PPT (Figure 1). The number of labeled cells varied across cases, leading to quantitative differences, but qualitatively the results were similar with the two vectors and across sexes. In one case, we saw labeled neurons in the adjacent LDT, the other component nucleus of PMT (not shown). Results, in this case, did not differ from cases in which the label was confined to PPT. In some cases, additional labeled cells were present in the parabigeminal nucleus, a nucleus on the lateral edge of the rostral midbrain that includes a dense cluster of cholinergic cells. These cases were excluded from the analysis in the present study. In the remaining 18 cases, all produced labeled axons in the IC. Twelve of these cases had substantial labeling (‘‘good to very good’’) while the remaining cases had fewer labeled axons that served to support and confirm the conclusions. By plotting the labeled cells in every 6th section, we could assess both the viral spread and the efficacy of the labeling. In the twelve better cases, the labeled cell bodies were located across 1–5 sections, indicating that the injection site extended rostrocaudally from a minimum of less than 240 µm to a maximum of ∼1,200 µm. The number of fluorescent cells in these cases ranged from 7 (in a single section) to 328 across five sections. Interestingly, the cases that yielded the most labeled axons in the IC were not those with the most labeled PMT cells; we believe this reflects the fact that only a subset of PMT cells projects to the IC, and these cells are interspersed with those that project to other targets (see Motts and Schofield, 2009; Motts and Schofield, 2010). Within the PPT, our injections labeled cells mostly in the caudal portion of the nucleus, surrounding the superior cerebellar peduncle at the same rostrocaudal levels as the IC. An example of a large injection site is shown in Figure 1C, where each green marker represents a single EYFP-labeled PPT neuron. Cholinergic neurons are not as densely packed in more rostral regions of the nucleus (which extends as far as the substantia nigra in the ventral midbrain; Mesulam et al., 1983). RESULTS FIGURE 1 \| Injection of viral vector into the PPT of ChAT-Cre transgenic rats labeled neurons at the site of injection. Photomicrographs show virally-expressed ﬂuorescent protein in PPT neurons. (A) A representative example of mCherry expression in neurons of the left PPT following injection of rAAV2/EF1a-DIO-mCherry. Transverse section; lateral is left, dorsal is up. Scale bar = 500 µm. (B) A representative example of EYFP expression in neurons of the right PPT following injection of rAAV2/EF1a-DIO-EYFP. Transverse section; lateral is right, dorsal is up. Scale bar = 500 µm. (C) A representative three-dimensional reconstruction showing the extent of labeled neurons in the PPT through six transverse sections (spacing: 240 µm between sections). The image on the left shows the six sections stacked, with each EYFP-labeled cell indicated by a green circle. The image on the right shows the stack rotated for a lateral view. The “cloud” of labeled cells shifts ventrally moving from caudal to rostral, reﬂecting the orientation of the PPT. Aq, cerebral aqueduct; D, dorsal; IC, inferior colliculus; LL, lateral lemniscus; M, medial; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus; R, rostral, scp, superior cerebellar peduncle. Injection of Viral Vector Into PPT Labels Cholinergic Neurons Our cases contained few or no labeled cells in these rostral regions, so we may have missed a portion of the cholinergic projections to the IC. If so, it is likely to be a very small component because the majority of PPT cells that project to the IC are concentrated in the caudal PPT (Motts and Schofield, 2009). FIGURE 1 \| Injection of viral vector into the PPT of ChAT-Cre transgenic rats labeled neurons at the site of injection. Photomicrographs show virally-expressed ﬂuorescent protein in PPT neurons. (A) A representative example of mCherry expression in neurons of the left PPT following injection of rAAV2/EF1a-DIO-mCherry. Transverse section; lateral is left, dorsal is up. Scale bar = 500 µm. (B) A representative example of EYFP expression in neurons of the right PPT following injection of rAAV2/EF1a-DIO-EYFP. Transverse section; lateral is right, dorsal is up. Scale bar = 500 µm. (C) A representative three-dimensional reconstruction showing the extent of labeled neurons in the PPT through six transverse sections (spacing: 240 µm between sections). The image on the left shows the six sections stacked, with each EYFP-labeled cell indicated by a green circle. The image on the right shows the stack rotated for a lateral view. The “cloud” of labeled cells shifts ventrally moving from caudal to rostral, reﬂecting the orientation of the PPT. Aq, cerebral aqueduct; D, dorsal; IC, inferior colliculus; LL, lateral lemniscus; M, medial; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus; R, rostral, scp, superior cerebellar peduncle. FIGURE 1 \| Injection of viral vector into the PPT of ChAT-Cre transgenic rats labeled neurons at the site of injection. Photomicrographs show virally-expressed ﬂuorescent protein in PPT neurons. (A) A representative example of mCherry expression in neurons of the left PPT following injection of rAAV2/EF1a-DIO-mCherry. Transverse section; lateral is left, dorsal is up. Scale bar = 500 µm. (B) A representative example of EYFP expression in neurons of the right PPT following injection of rAAV2/EF1a-DIO-EYFP. Transverse section; lateral is right, dorsal is up. Scale bar = 500 µm. (C) A representative three-dimensional reconstruction showing the extent of labeled neurons in the PPT through six transverse sections (spacing: 240 µm between sections). The image on the left shows the six sections stacked, with each EYFP-labeled cell indicated by a green circle. The image on the right shows the stack rotated for a lateral view. Data Analysis For a quantitative description of the labeled boutons, we selected two cases with a large number of labeled axons. We then plotted the labeled boutons, identified as distinct swellings along a labeled axon, in every sixth section through the IC. A substantial number of labeled boutons appeared to be in close apposition to an IC neuron that was labeled with anti-NeuN and, in some cases, anti-GAD67. Such contacts were apparent across animals; two with a large number of labeled axons and robust immunostaining for both NeuN and GAD were chosen for quantitative analysis. Sections spaced 240 µm apart were chosen to include large parts of the major IC subdivisions in each animal (two sections from one case and three from the second case). The IC in each section was examined at high magnification (63× objective, NA 1.4) and the location of each neuron that appeared to be contacted by a cholinergic axon was plotted with a symbol indicating whether the neuron was GAD− or GAD+. For each experiment, fluorescent neurons in the PMT were plotted to visualize the extent of the injection site. The IC was then examined at high magnification in every sixth section through its entire rostral-caudal extent (typically 4–6 sections per animal). The amount of labeling varied across animals. In some cases, especially those with fewer labeled cells in the PPT, there were no labeled axons in the IC; this was not unexpected, given that PPT cholinergic cells projects to many structures throughout the brainstem and thalamus. Those cases were excluded from the present study. Labeled axons were examined to assess possible routes from the PPT to the IC. The results were consistent across cases and suggested several possible routes to both ipsilateral and contralateral IC. To illustrate routes to and within the IC, a representative case with many labeled axons was chosen for detailed plotting. The labeled axons were drawn in every sixth section through the midbrain. The distribution of boutons within the IC was also documented by visual examination of Photomicrographs were taken with a Zeiss AxioImager Z2 fluorescence microscope with either an AxioCam HRm camera (Zeiss) or an Orca Flash 4.0 camera (Hamamatsu). Additions of color, scale bars, and arrows as well as, cropping and global adjustment of levels were done in Adobe Photoshop (Adobe Systems). Data Analysis July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 4 Cholinergic Inputs to Inferior Colliculus Noftz et al. Frontiers in Neural Circuits \| www.frontiersin.org Injection of Viral Vector Into PPT Labels Cholinergic Neurons The “cloud” of labeled cells shifts ventrally moving from caudal to rostral, reﬂecting the orientation of the PPT. Aq, cerebral aqueduct; D, dorsal; IC, inferior colliculus; LL, lateral lemniscus; M, medial; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus; R, rostral, scp, superior cerebellar peduncle. We stained sections with antibodies against ChAT to determine whether the expression of the fluorescent protein was limited to cholinergic (i.e., ChAT+) cells (Figure 2). In these sections, all virally-labeled PPT neurons were co-labeled with the ChAT antibody (Figure 2, green arrows), indicating that the viral vector is selective for cholinergic neurons. However, it was common to see ChAT+ neurons that were not labeled by the viral gene, even though adjacent neurons were so labeled (Figure 2). It is impossible to determine whether this July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 5 Noftz et al. Cholinergic Inputs to Inferior Colliculus FIGURE 2 \| Viral injection selectively labeled ChAT-positive neurons in the PPT. (A) EYFP-expressing PPT neurons (green arrows) following AAV injection. (B) Same region as shown in (A), but imaged to show immunostaining for ChAT. (C) Merged image showing colocalization of ChAT antibody and viral EYFP expression (green arrows). Scale = 100 µm. FIGURE 2 \| Viral injection selectively labeled ChAT-positive neurons in the PPT. (A) EYFP-expressing PPT neurons (green arrows) following AAV injection. (B) Same region as shown in (A), but imaged to show immunostaining for ChAT. (C) Merged image showing colocalization of ChAT antibody and viral EYFP expression (green arrows). Scale = 100 µm. they could represent a projection from one PPT through the ipsilateral IC to the contralateral IC, or a recurrent loop from the PPT to the contralateral IC and then back to the ipsilateral side. was a failure of the transgene (i.e., Cre-recombinase was not expressed in the cholinergic neuron) or a failure of viral uptake of fluorescent gene expression by the presumptive cholinergic neurons. We chose three cases that produced the most labeled axons in the IC and counted both the virally- labeled cells and the ChAT+ PPT cells in the same sections. On average, 18% of the ChAT+ cells were labeled by the viral vector (range: 14–23%), suggesting that the efficacy of viral labeling is limited. We completed a similar analysis for a fourth case that had fewer labeled axons in the IC despite having many more labeled cells in the PPT. Cholinergic Boutons Are Present in all IC Subdivisions Labeled axons typically were thin and studded with en passant and terminal boutons. Figure 4 shows results from a case that had substantial labeling of axons in the IC. Cases with fewer labeled axons had fewer boutons but otherwise were similar to one another. In all cases, more boutons were present ipsilaterally than contralaterally and typically were present in all the IC subdivisions on both sides. Individual axons were observed to cross any of the borders between IC subdivisions, with boutons clearly visible in each subdivision; Figure 4G (asterisks) shows examples of axons crossing the ICc/IClc border. Within a subdivision, there was no obvious relationship between the axons or boutons and other features of the subdivision architecture. In the ICc, labeled axons were oriented in several directions without any clear relationship to the orientation of fibrodendritic laminae. In the shell areas (ICd and IClc), the labeled axons showed no particular relationship to borders between layers or between the ‘‘GABA modules’’ and the extramodular domains (see Chernock et al., 2004). Injection of Viral Vector Into PPT Labels Cholinergic Neurons This case had 155 virally-labeled cells in five sections, which constituted 72% of the ChAT+ cells. Clearly, some cases had greater efficacy of labeling, although we never observed 100% labeling. We conclude that the labeled axons and boutons that we observed in the IC are cholinergic and likely underrepresent the PPT-to-IC cholinergic pathway. Contralateral IC From the PPT, cholinergic axons travel to many regions of the brainstem and thalamus. Labeled axons were present in the IC bilaterally, with more axons present on the ipsilateral side (Figure 3). Axons coursing toward the IC take multiple routes. Axons leave the PPT dorsally and dorsolaterally to enter the ICc through its ventral border. Axons reaching the ipsilateral IClc do so either through the ventrolateral border of the IC or by coursing through the ICc and turning laterally. Axons traveling to the ICd travel first through either the ICc or the IClc. As described above, the labeled axons in the IC were typically studded with boutons, suggesting many sites of ACh release. Figure 5 plots the distribution of labeled boutons (green circles), demonstrating their wide distribution throughout the ipsilateral and contralateral IC. Consistent with the axonal pattern, the boutons were more numerous on the ipsilateral side than on the contralateral side. Also, labeled boutons were more numerous in the caudal part of the IC, even within a subdivision (compare Figure 5C vs. Figure 5A). The organization of labeled axons suggests several possible routes from the PPT to the contralateral IC. First, axon fragments could be followed from the PPT across the midline, traveling ventral to the PAG or even through the ventral PAG to the ventral border of the contralateral IC. At this point, the axons entered the contralateral IC all along its ventral border and were distributed to each major subdivision in a pattern similar to that in the ipsilateral IC. Also, labeled axons were present in the IC commissure. The directionality of these axons could not be determined, so Frontiers in Neural Circuits \| www.frontiersin.org Cholinergic PPT Axons Contact GAD+ and GAD−Neurons in the IC A majority of the labeled boutons were located in the neuropil between the labeled neuronal cell bodies, but a substantial July 2020 \| Volume 14 \| Article 43 6 Cholinergic Inputs to Inferior Colliculus Noftz et al. FIGURE 3 \| Cholinergic axons travel through the tegmentum to reach ipsilateral and contralateral IC. Fluorescent-labeled axons (magenta lines) were observed throughout transverse IC sections after labeling cholinergic cells in the left PPT (magenta squares). The plots show the axons in a series of transverse sections through the IC at caudal (A), middle (B), and rostral levels (C). On both sides, labeled axons appear to enter the IC all along its ventral border. Sections are 40 µm thick and 240 µm apart. Scale bar = 1 mm. number of boutons were in close apposition to the cell bodies, suggesting possible synaptic contacts. By staining the tissue of the labeled cholinergic boutons to presumptive GABAergic cells. Figure 6 shows examples of virally-labeled cholinergic boutons (green) in close contact (arrows) with GAD+ IC neurons (magenta). We also stained the tissue with a neuron-specific marker (NeuN), allowing us to distinguish GAD+ cells from GAD−cells (presumptive glutamatergic neurons). A neuron that is NeuN+ and GAD−located close to GAD+ profiles is considered to be non-GABAergic. GAD−neurons are likely glutamatergic neurons as IC neurons are either GABAergic or glutamatergic (reviewed by Schofield and Beebe, 2019). Glutamatergic neurons make up the majority of IC neurons and were frequently contacted by labeled cholinergic PPT boutons (Figures 7A,B, arrows). We observed cholinergic contacts onto neurons in both the ipsilateral and contralateral IC. Figure 8 shows the distribution of contacted cell bodies in two sections through the IC in a representative case. Several points are clear. First, contacts occur bilaterally in all three IC subdivisions, with more contacts ipsilateral than contralateral. Second, contacts occurred on both GAD−cells (presumptive glutamatergic cells, Figure 8, cyan symbols) and GAD+ cells (presumptive GABAergic cells, Figure 8, magenta symbols). Thus, cholinergic axons from a single PPT appear to contact GAD+ and GAD−neurons in each major IC subdivision both ipsilateral and contralateral to the injected PPT. Individual Cholinergic Axons Can Contact Both GAD+ and GAD−Neurons Individual sections often contained relatively long segments of labeled axons that allowed several observations. In each of the IC subdivisions, individual axons appeared to contact multiple IC neurons. Figure 7 includes examples of single cholinergic axons that appear to contact multiple GAD−neurons. Figure 9 shows additional patterns of multiple targets, including multiple GAD+ neurons (Figure 9B) and axons that contact both GAD+ and GAD−neurons (Figures 9A,B). Each pattern of contact—onto multiple GAD+ neurons, multiple GAD−neurons, or both GAD+ and GAD−neurons—was observed ipsilateral and, less often, contralateral to the injection site. DISCUSSION We used selective viral tract-tracing of cholinergic PPT neurons to identify cholinergic projections to the IC. The PPT is a prominent source of cholinergic innervation of the thalamus and brainstem and is involved in wide-ranging functions such as arousal, sensory gating, sleep-wake cycle, and plasticity (reviewed by Schofield et al., 2011; Schofield and Hurley, 2018). The present results demonstrate that cholinergic axons from the PPT terminate in the three largest IC subdivisions: the ICc, ICd, and IClc (Figure 10). These subdivisions contribute to different aspects of hearing, each of which may be affected by cholinergic modulation. The cholinergic axons typically contain many boutons and can cross borders between IC subdivisions as well as within subdivisions (e.g., the laminar boundaries in the IClc). Cholinergic axons contact IC cells that are GAD+ (presumptive GABAergic) and GAD−(presumptive FIGURE 3 \| Cholinergic axons travel through the tegmentum to reach ipsilateral and contralateral IC. Fluorescent-labeled axons (magenta lines) were observed throughout transverse IC sections after labeling cholinergic cells in the left PPT (magenta squares). The plots show the axons in a series of transverse sections through the IC at caudal (A), middle (B), and rostral levels (C). On both sides, labeled axons appear to enter the IC all along its ventral border. Sections are 40 µm thick and 240 µm apart. Scale bar = 1 mm. number of boutons were in close apposition to the cell bodies, suggesting possible synaptic contacts. By staining the tissue with an antibody to GAD, we could examine the relationship July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 7 Noftz et al. Cholinergic Inputs to Inferior Colliculus 4 \| Labeled cholinergic axons with many boutons were present in each subdivision of the IC. (A–C) Expression of mCherry in axons from PPT in (A) ICc, nd (C) ICd. (D–F) Expression of EYFP in axons from PPT in (D) ICc, (E) IClc, and (F) ICd. Scale bar in (F) = 25 µm and applies to (A–F). (G) Montage EYFP-labeled cholinergic axons in the ICc and IClc (separated by the dashed line), including individual axons that cross the border (asterisks), providing o both subdivisions. Scale = 25 µm. FIGURE 4 \| Labeled cholinergic axons with many boutons were present in each subdivision of the IC. (A–C) Expression of mCherry in axons from PPT in (A) ICc, (B) IClc, and (C) ICd. DISCUSSION (D–F) Expression of EYFP in axons from PPT in (D) ICc, (E) IClc, and (F) ICd. Scale bar in (F) = 25 µm and applies to (A–F). (G) Montage showing EYFP-labeled cholinergic axons in the ICc and IClc (separated by the dashed line), including individual axons that cross the border (asterisks), providing boutons to both subdivisions. Scale = 25 µm. FIGURE 4 \| Labeled cholinergic axons with many boutons were present in each subdivision of the IC. (A–C) Expression of mCherry in axons from PPT in (A) ICc, (B) IClc, and (C) ICd. (D–F) Expression of EYFP in axons from PPT in (D) ICc, (E) IClc, and (F) ICd. Scale bar in (F) = 25 µm and applies to (A–F). (G) Montage showing EYFP-labeled cholinergic axons in the ICc and IClc (separated by the dashed line), including individual axons that cross the border (asterisks), providing boutons to both subdivisions. Scale = 25 µm. glutamatergic), suggesting that ACh acts on both excitatory and inhibitory IC circuits. Moreover, an individual cholinergic axon can contact cells of both neurotransmitter phenotypes. Taken together, PPT cholinergic neurons appear to contact many excitatory and inhibitory cells across multiple IC subdivisions, suggesting wide-ranging effects of ACh in the IC. glutamatergic), suggesting that ACh acts on both excitatory and inhibitory IC circuits. Moreover, an individual cholinergic axon can contact cells of both neurotransmitter phenotypes. July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 8 Cholinergic Inputs to Inferior Colliculus Noftz et al. FIGURE 5 \| Cholinergic boutons are present in multiple IC subdivisions. Plots of transverse sections through the IC (the same sections illustrated in Figure 2) showing the distribution of labeled boutons (green circles) in the IC after ﬂuorescent labeling of cholinergic cells (magenta squares) in the left PPT. Cholinergic boutons were found in (A) caudal, (B) mid-IC, and (C) rostral sections of IC, where they terminated in the three major subdivisions: dorsal cortex (ICd), lateral cortex (IClc) and central nucleus (ICc). Scale bar = 1 mm. Technical Issues anti-ChAT, we found that fluorescent protein expression was limited to ChAT+ cells. We conclude that the labeled axons were cholinergic. The same analysis showed numerous ChAT+ cells that did not express fluorescent protein despite being among other cells that were so labeled. DISCUSSION It is unclear whether this reflects a failure of Cre expression or failure of viral uptake and subsequent expression of the fluorescent label. The two vectors used here were serotype 2, selected because of relatively high efficiency in anterograde labeling of neuronal pathways (Aschauer et al., 2013; Salegio et al., 2013). However, possibly a different serotype would label some of the cholinergic cells that were unlabeled here. We conclude that our experiments probably labeled <100% of the pathway of interest, and cannot rule out the possibility that a specific subtype of cholinergic cell failed to express the fluorescent proteins. p There are disparate views on volume vs. synaptic cholinergic transmission (Descarries et al., 1997; Zoli et al., 1999; Parikh et al., 2007; Lendvai and Vizi, 2008; Sarter et al., 2009; Muñoz and Rudy, 2014; Takács et al., 2018), with no data to argue strongly for one mode or the other in the IC. Of course, the specificity of cholinergic action also depends on the nature and location of cholinergic receptors, with opportunities for both presynaptic and postsynaptic effects. As discussed above (Introduction), the IC contains a variety of nicotinic and muscarinic receptor types, but as yet little is known about the specific cells and circuits associated with these receptors. By using light microscopy, we have been able to assess the distribution of cholinergic axons and likely release sites over a large area. The results suggest that a single PPT releases ACh across a wide expanse of both ipsilateral and contralateral IC. Although a majority of boutons were located in the neuropil, many of the cholinergic boutons were in close apposition to neuronal (NeuN+) cell bodies, allowing us to assess some of the cell types most likely affected by ACh. By adding immunostain for NeuN and GAD67, we were able to conclude that ACh is likely to have direct effects on both GAD+ (likely GABAergic) and GAD− neurons. Given that IC neurons are mostly either GABAergic or glutamatergic, the GAD−neurons are likely to be glutamatergic cells. Our conclusion that ACh affects both glutamatergic and GABAergic IC cells is consistent with previous studies showing physiologic activation of GABAergic cells (Yigit et al., 2003) and the presence of cholinergic receptor mRNA in both cell types (e.g., Sottile et al., 2017). Ultimately, electron microscopy will be needed to identify synaptic release sites for ACh. Functional Implications The present results extend the conclusions reached via retrograde tracing experiments that identified the PPT as the largest source of cholinergic input to the IC (Motts and Schofield, 2009). Those experiments were based on large injections of tracer that typically encroached on multiple IC subdivisions. The present results show that cholinergic PPT axons innervate each of the large IC subdivisions: ICc, ICd, and IClc. Also, the retrograde tracing studies DISCUSSION The present results indicate that such studies will be needed in each of the IC subdivisions. FIGURE 5 \| Cholinergic boutons are present in multiple IC subdivisions. Plots of transverse sections through the IC (the same sections illustrated in Figure 2) showing the distribution of labeled boutons (green circles) in the IC after ﬂuorescent labeling of cholinergic cells (magenta squares) in the left PPT. Cholinergic boutons were found in (A) caudal, (B) mid-IC, and (C) rostral sections of IC, where they terminated in the three major subdivisions: dorsal cortex (ICd), lateral cortex (IClc) and central nucleus (ICc). Scale bar = 1 mm. Technical Issues The combination of Cre-expressing cholinergic cells in transgenic animals and Cre-dependent expression of fluorescent proteins delivered via viral vectors provides an opportunity for highly selective labeling of a cholinergic pathway (e.g., Stornetta et al., 2013). Following immunostaining with July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 9 Noftz et al. Cholinergic Inputs to Inferior Colliculus FIGURE 6 \| Cholinergic axons contact GAD+ neurons in the IC. The upper panel in each column shows ﬂuorescent-labeled cholinergic axons in the IC (green). The lower panel in each column shows the same axons overlaid with an image of the GAD stain (magenta). Arrows show points of close contact between labeled boutons and the GAD+ cells. Images (A,C,D) are from the IC central nucleus; (B) is from IC lateral cortex. Scale bar = 20 µm. FIGURE 6 \| Cholinergic axons contact GAD+ neurons in the IC. The upper panel in each column shows ﬂuorescent-labeled cholinergic axons in the IC (green). The lower panel in each column shows the same axons overlaid with an image of the GAD stain (magenta). Arrows show points of close contact between labeled boutons and the GAD+ cells. Images (A,C,D) are from the IC central nucleus; (B) is from IC lateral cortex. Scale bar = 20 µm. FIGURE 7 \| (A,B) Cholinergic axons contact GAD−neurons in the IC. The upper panel in each column shows ﬂuorescent-labeled cholinergic axons (green) and NeuN+ neurons (cyan). Labeled boutons are in close contact (arrows) with several of the NeuN+ cells (). That these contacted cells are non-GABAergic is shown by the lower panel, which shows GAD immunostain in magenta. None of the asterisk-marked cells are GAD+, despite the presence of GAD+ cells nearby. IC, lateral cortex. Scale bar = 20 µm. FIGURE 7 \| (A,B) Cholinergic axons contact GAD−neurons in the IC. The upper panel in each column shows ﬂuorescent-labeled cholinergic axons (green) and NeuN+ neurons (cyan). Labeled boutons are in close contact (arrows) with several of the NeuN+ cells (). That these contacted cells are non-GABAergic is shown by the lower panel, which shows GAD immunostain in magenta. None of the asterisk-marked cells are GAD+, despite the presence of GAD+ cells nearby. IC, lateral cortex. Scale bar = 20 µm. indicated that, overall, more PPT cells project to the ipsilateral IC and fewer project to the contralateral IC. Technical Issues Scale bar = 1 mm. The origin of the inputs from the PPT may provide additional insight into cholinergic functions. Within the context of auditory processing, the PPT has been associated with arousal, plasticity (especially driven by top-down circuits) and sensory gating (Xiong et al., 2009; Schofield et al., 2011; Schofield and Hurley, 2018). The PPT projections to the auditory thalamus have been implicated in the enhancement of hearing in a noisy environment, in part by cholinergic enhancement of ascending inhibitory pathways as well as enhancement of top-down modulation (via effects on corticothalamic circuits; Sottile et al., 2017). Gut and Winn (2016) proposed that the PPT, particularly the caudal part (as studied here), is especially important for an organism’s ability to act quickly in response to sensory stimuli. This proposal ties together the sensory aspects of the PPT with its well-known ties to the basal ganglia. Many cells in the PPT respond to acoustic stimuli, so it is not surprising that they would contribute to auditory function (Reese et al., 1995a,b,c). Slee and David (2015) have shown that IC cells respond differently depending on whether the subject is performing a task. They concluded that arousal associated with task performance likely accounted for part of the difference in responses. Cholinergic projections as demonstrated in the present study may contribute to such responses. Kuenzel and colleagues (Goyer et al., 2016; Gillet et al., 2018; Kuenzel, 2019) have suggested a similar role for projections from the PPT to the cochlear nucleus, where ACh can modulate neuronal sensitivity in accord with an animal’s behavioral state and level of arousal. FIGURE 8 \| GAD-positive and GAD-negative IC neurons are contacted throughout the IC. Plots of transverse sections through the caudal (A) and rostral (B) IC show the distribution of neurons contacted by cholinergic boutons after labeling cholinergic cells in the left PPT and laterodorsal tegmental nucleus (LDT; green squares). Contacted cells included both GAD+ neurons (magenta triangles) and GAD−neurons (cyan triangles) in each of the major IC subdivisions. Contacted IC neurons were more numerous on the ipsilateral side than the contralateral side. Contacted GAD−neurons outnumbered contacted GAD+ neurons. Scale bar = 1 mm. The widespread distribution of PPT cholinergic axons is consistent with studies of cholinergic receptors, which describe nicotinic and muscarinic receptors throughout the IC (Schwartz, 1986; Glendenning and Baker, 1988; Morley and Happe, 2000; Gahring et al., 2004; Happe and Morley, 2004). Technical Issues The present results indicate that the axonal distribution reflects a similar indicated that, overall, more PPT cells project to the ipsilateral IC and fewer project to the contralateral IC. The present results indicate that the axonal distribution reflects a similar pattern, with denser projections to the ipsilateral IC than contralateral IC. Ipsilateral dominance applies to each of the IC subdivisions. July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 10 Cholinergic Inputs to Inferior Colliculus Noftz et al. cholinergic agents affect the firing rate but not the temporal response patterns of IC neuronal responses to sounds (Farley et al., 1983; Habbicht and Vater, 1996). They suggested that ACh acted through nicotinic and muscarinic receptors to set the level of neuronal activity in the IC, likely modulating neuronal sensitivity as well as gain. Cholinergic effects in other subcortical auditory nuclei have led to similar conclusions. Oertel and colleagues (Fujino and Oertel, 2001; Oertel and Fujino, 2001; Oertel et al., 2011) suggested that ACh enhances responses of T stellate cells in the cochlear nucleus, perhaps to enhance coding of spectral peaks and thus to improve interpretation of sounds in noise. Caspary and colleagues have suggested that cholinergic effects in auditory thalamus similarly support hearing in a noisy environment, and further that deterioration of the cholinergic system with aging could relate to presbycusis (Sottile et al., 2017). It is worth noting that the PPT is a source of cholinergic innervation to the cochlear nucleus and auditory thalamus, as well as the inferior colliculus. Moreover, individual PPT cells can send branching axons to innervate two or more of these target structures (e.g., one IC as well as left and right auditory thalamus; Motts and Schofield, 2011). Such divergent projections are typical of many modulatory systems and may reflect a common effect across auditory nuclei (reviewed by Schofield and Hurley, 2018). FIGURE 8 \| GAD-positive and GAD-negative IC neurons are contacted throughout the IC. Plots of transverse sections through the caudal (A) and rostral (B) IC show the distribution of neurons contacted by cholinergic boutons after labeling cholinergic cells in the left PPT and laterodorsal tegmental nucleus (LDT; green squares). Contacted cells included both GAD+ neurons (magenta triangles) and GAD−neurons (cyan triangles) in each of the major IC subdivisions. Contacted IC neurons were more numerous on the ipsilateral side than the contralateral side. Contacted GAD−neurons outnumbered contacted GAD+ neurons. Technical Issues Given that cholinergic axons are distributed widely in the IC, one would expect that a majority of IC cells are affected by cholinergic inputs. The available studies are consistent with such a view, but the data are limited to studies in which the location of recorded units was not related to IC subdivision (Curtis and Koizumi, 1961; Watanabe and Simada, 1973; Farley et al., 1983), or to studies in which the units were restricted to the ICc (Habbicht and Vater, 1996; Ji et al., 2001; Yigit et al., 2003). The present results suggest that direct cholinergic effects would be observed upon recording neurons in the ICd and the IClc. Suga and colleagues have shown evidence for a cholinergic role in corticofugal-driven plasticity of subcortical auditory nuclei, including plasticity in the IC (Xiong et al., 2009; Suga, 2012). Stimulation of the auditory cortex can lead to the retuning of an IC cell’s response selectivity. Such plasticity can involve a variety of stimulus parameters, such as shifting the Cholinergic Effects in the ICc Cholinergic Effects in the ICc Information on cholinergic effects within specific IC subdivisions has been limited to the ICc. Previous studies showed that Frontiers in Neural Circuits \| www.frontiersin.org July 2020 \| Volume 14 \| Article 43 11 Noftz et al. Cholinergic Inputs to Inferior Colliculus FIGURE 9 \| Single cholinergic axons contact both GAD+ and GAD−IC neurons. The upper panel in each column shows ﬂuorescent-labeled cholinergic axons (green) overlaid with an image of GAD staining (magenta). The lower panel in each column shows the same image overlaid with an image of the NeuN stain (cyan). (A) An axon segment in the IC lateral cortex appears to contact one GAD+ neuron (magenta arrowheads) and two different GAD−neurons (cyan arrowheads). (B) shows an axon segment from the IC central nucleus that appears to contact two GAD+ neurons (magenta arrowheads) and a nearby GAD−neuron (cyan arrowheads). Scale bar = 20 µm. FIGURE 9 \| Single cholinergic axons contact both GAD+ and GAD−IC neurons. The upper panel in each column shows ﬂuorescent-labeled cholinergic axons (green) overlaid with an image of GAD staining (magenta). The lower panel in each column shows the same image overlaid with an image of the NeuN stain (cyan). (A) An axon segment in the IC lateral cortex appears to contact one GAD+ neuron (magenta arrowheads) and two different GAD−neurons (cyan arrowheads). (B) shows an axon segment from the IC central nucleus that appears to contact two GAD+ neurons (magenta arrowheads) and a nearby GAD−neuron (cyan arrowheads). Scale bar = 20 µm. identify the effects of ACh on responses of IC cells outside the central nucleus, particularly given these non-central regions being the prime targets of cortical inputs as well as centers for multisensory processing. frequency to which the IC cell responds most readily. The retuning of IC responses can outlast the period of cortical stimulation by minutes to hours as a result of plasticity in both cortical and subcortical circuits. Significantly, that plasticity is dependent on ACh in the IC. We believe that the details of the retuning (e.g., whether a given cell retunes to a higher or a lower acoustic frequency) is determined by the massive direct projections from the auditory cortex to the IC, with cortical axons activating both excitatory and inhibitory circuits within the IC. ACh, on the other hand, provides a permissive signal that allows synaptic plasticity to sustain the effects of the cortical stimulation. Cholinergic Effects in the ICc Such a permissive signal could: (1) be elicited by direct auditory cortical projections to cholinergic PPT cells that project to the IC (Schofield and Motts, 2009; Schofield, 2010); and (2) affect multiple IC cells via highly divergent cholinergic axons, including axons that contact both glutamatergic and GABAergic IC cells (current results). Frontiers in Neural Circuits \| www.frontiersin.org Cholinergic Axons Target Both GABAergic and Glutamatergic IC Neurons postsynaptic effects. FIGURE 10 \| Cholinergic PPT cells project throughout the IC and contact GABAergic and presumptive glutamatergic cells. Green arrows show cholinergic projections from PPT to all three subdivisions of the IC. Cyan stars represent cholinergic-contacted glutamatergic neurons and magenta stars represent cholinergic-contacted GABAergic neurons. Aq, cerebral aqueduct; ICc, d, lc, inferior colliculus central nucleus, dorsal cortex, and lateral cortex; ICcomm, commissure of the IC; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus. FIGURE 10 \| Cholinergic PPT cells project throughout the IC and contact GABAergic and presumptive glutamatergic cells. Green arrows show cholinergic projections from PPT to all three subdivisions of the IC. Cyan stars represent cholinergic-contacted glutamatergic neurons and magenta stars represent cholinergic-contacted GABAergic neurons. Aq, cerebral aqueduct; ICc, d, lc, inferior colliculus central nucleus, dorsal cortex, and lateral cortex; ICcomm, commissure of the IC; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus. with projections from ICc, converge in the medial division of the medial geniculate nucleus, which then projects widely to auditory cortical areas and other forebrain targets to form the polysensory pathway (De Ribaupierre, 1997). Finally, the diffuse pathway originates from the ICd, which projects to the dorsal medial geniculate nucleus and then on to secondary and temporal auditory cortical areas. The ICd has long stood out as unique among IC subdivisions, with descending inputs from the auditory cortex appearing to dominate over ascending inputs (Ehret, 1997). ICd cells are typically broadly-tuned and respond only weakly to simple auditory stimuli, but respond more robustly to complex contextual stimuli (such as the cries of isolated offspring; De Ribaupierre, 1997). Early lesion studies suggested that ICd plays a particularly important role in auditory attention (Jane et al., 1965). The present results show that cholinergic axons from the PPT terminate broadly in each of the large IC subdivisions and are in a position to modulate the activity in each of the ascending parallel pathways. At the level of the thalamus (another target of PPT cholinergic projections), ACh can have different effects on lemniscal vs. extra-lemniscal cells (Mooney et al., 2004). The differences are thought to be mediated through different cholinergic receptors. In the IC, Gahring et al. (2004) noted higher levels of the nicotinic receptor subunit β4 in the IClc compared to other IC subdivisions. Happe and Morley (2004) noted high levels of the nicotinic receptor α7 subunits in the IClc. Cholinergic Axons Target Both GABAergic and Glutamatergic IC Neurons FIGURE 10 \| Cholinergic PPT cells project throughout the IC and contact GABAergic and presumptive glutamatergic cells. Green arrows show cholinergic projections from PPT to all three subdivisions of the IC. Cyan stars represent cholinergic-contacted glutamatergic neurons and magenta stars represent cholinergic-contacted GABAergic neurons. Aq, cerebral aqueduct; ICc, d, lc, inferior colliculus central nucleus, dorsal cortex, and lateral cortex; ICcomm, commissure of the IC; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus. Several studies have shown that ACh affects the majority of IC cells’ response to sound (Watanabe and Simada, 1973; Farley et al., 1983; Habbicht and Vater, 1996). The effects can be mediated via nicotinic and muscarinic receptors. In general, cholinergic agents can affect response rate but appear to have little effect on the temporal patterns of a neuron’s response to a sound, or on the selectivity for specific stimulus parameters. These studies could not discern direct from indirect cholinergic effects on the recorded cell, so the underlying mechanisms remain unclear. The present study suggests that the cholinergic effects are mediated by direct actions on both excitatory and inhibitory IC cells. We showed that PPT cholinergic axons are closely associated with both glutamatergic and GABAergic IC neurons. This sets the stage for postsynaptic effects on the closely apposed cell bodies or dendrites (without ruling out presynaptic effects on nearby axon terminals that contact the same postsynaptic cell). Evidence for cholinergic effects on identified GABAergic or glutamatergic IC cells is limited. Sottile et al. (2017) showed that GABAergic and presumptive glutamatergic IC cells express mRNA for nicotinic receptor subunits. These results do not indicate where the receptors are expressed on the cells [indeed, Sottile et al. (2017) were examining cholinergic effects on the collicular axon terminals in the thalamus]. In vitro recordings from the IC provide additional support. Yigit et al. (2003) provided physiological evidence that GABAergic IC cells are activated via muscarinic receptors. Those experiments were conducted in young animals and could potentially reflect mechanisms that disappear after the system has developed (see Morley and Happe, 2000 for a discussion of separate cholinergic roles during and after the development of the auditory system). In a preliminary study, Rivera-Perez et al. (2020) demonstrated direct nicotinic depolarization of VIP-expressing IC cells (which are known to be glutamatergic; Goyer et al., 2019). Additional experiments will be needed to identify the receptor types on glutamatergic and GABAergic IC cells and to differentiate presynaptic vs. Cholinergic PPT Axons Likely Modulate Lemniscal and Extralemniscal Auditory Pathways The termination of cholinergic axons across IC subdivisions is significant when we consider that each IC subdivision contributes differentially to three parallel auditory pathways: a lemniscal pathway, a polysensory pathway, and a diffuse pathway (Calford and Aitkin, 1983; Rouiller, 1997; Mellott et al., 2014). Each pathway is thought to serve different aspects of hearing. The lemniscal pathway is tonotopically organized and provides the primary-like representation of sound. It encompasses projections from the ICc to the ventral medial geniculate nucleus and on to tonotopically organized regions of the auditory cortex. The polysensory pathway incorporates inputs from other sensory systems, especially the somatosensory system. These inputs terminate heavily in the IClc. Projections from the IClc, along Interestingly, the work by Suga (2012) like that cited earlier regarding cholinergic effects on IC cells, focused on responses of cells in the ICc. It will be of interest in future studies to July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 12 Noftz et al. Cholinergic Inputs to Inferior Colliculus Cholinergic Axons Target Both GABAergic and Glutamatergic IC Neurons To the best of our knowledge, none of these receptors have been associated with specific output pathways from the IClc or ICd. Nonetheless, the current data, along with the prominent contributions of each IC subdivision to the parallel ascending pathways, suggests that PPT cholinergic modulation could affect a wide range of auditory functions. Frontiers in Neural Circuits \| www.frontiersin.org REFERENCES doi: 10.1523/eneuro.0270-18.2018 Goyer, D., Kurth, S., Gillet, C., Keine, C., Rübsamen, R., and Kuenzel, T. (2016). Slow cholinergic modulation of spike probability in ultra-fast time-coding sensory neurons. eNeuro 3:ENEURO.0186–16.2016. doi: 10.1523/eneuro.0186- 16.2016 Coote, E. J., and Rees, A. (2008). The distribution of nitric oxide synthase in the inferior colliculus of guinea pig. 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Hear. Res. 188, 12–20. doi: 10.1016/s0378-5955(03)00340-x Glendenning, K. K., and Baker, B. N. (1988). Neuroanatomical distribution of receptors for three potential inhibitory neurotransmitters in the brainstem auditory nuclei of the cat. J. Comp. Neurol. 275, 288–308. doi: 10.1002/cne. 902750210 Cissé, Y., Toossi, H., Ishibashi, M., Mainville, L., Leonard, C. S., Adamantidis, A., et al. (2018). Discharge and role of acetylcholine pontomesencephalic neurons in cortical activity and sleep-wake states examined by optogenetics and juxtacellular recording in mice. eNeuro 5:ENEURO.0270–18.2018. CONCLUSION The PPT, the largest source of cholinergic projections to the IC, sends axons to terminate bilaterally throughout the three major subdivisions of the IC. This termination pattern suggests that ACh modulates auditory processing associated with all three parallel ascending pathways to the thalamus—the tonotopic, multisensory, and diffuse pathways—and thus affects most aspects of auditory processing. Cholinergic boutons are found in close association with both glutamatergic and GABAergic IC cells, suggesting that ACh modulates both excitatory and inhibitory IC circuits. Overall, the PPT is likely to set the sensitivity of IC cells, modulating neuronal responses according to behavioral state and level of arousal. Further, by providing a permissive signal for plasticity in IC cells driven by top-down (corticofugal) mechanisms, ACh is likely to have both long-term as well as short-term effects on midbrain auditory processing. July 2020 \| Volume 14 \| Article 43 Frontiers in Neural Circuits \| www.frontiersin.org 13 Cholinergic Inputs to Inferior Colliculus Noftz et al. FUNDING This work was supported by the National Institutes of Health Grant R01 DC004391. This work was supported by the National Institutes of Health Grant R01 DC004391. ACKNOWLEDGMENTS We acknowledge Dr. Karl Deisseroth (Stanford University) as the donor of the transgenic rat model and the viral vectors used in the study, and also the Rat Resource and Research Center for providing the transgenic rat model. We gratefully acknowledge Colleen Sowick for technical support throughout the process. AUTHOR CONTRIBUTIONS All authors contributed to the conception and design of the study and data collection. WN analyzed the data and wrote the first ETHICS STATEMENT The animal study was reviewed and approved by Northeast Ohio Medical University Institutional Animal Care and Use Committee. The animal study was reviewed and approved by Northeast Ohio Medical University Institutional Animal Care and Use Committee. DATA AVAILABILITY STATEMENT draft of the manuscript. 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https://openalex.org/W2789594841	https://journalofinequalitiesandapplications.springeropen.com/track/pdf/10.1186/s13660-018-1654-6	English	null	Positive periodic solution for p-Laplacian neutral Rayleigh equation with singularity of attractive type	Journal of inequalities and applications	2,018	cc-by	5,512	© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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. R ES EA RCH Open Access Positive periodic solution for p-Laplacian neutral Rayleigh equation with singularity of attractive type Positive periodic solution for p-Laplacian neutral Rayleigh equation with singularity of attractive type Yun Xin1, Hongmin Liu1* and Zhibo Cheng2,3 Correspondence: hongminliu_1982@126.com 1College of Computer Science and Technology, Henan Polytechnic University, Jiaozuo, China Full list of author information is available at the end of the article Correspondence: hongminliu_1982@126.com 1College of Computer Science and Technology, Henan Polytechnic University, Jiaozuo, China Full list of author information is available at the end of the article Abstract In this paper, we consider a kind of p-Laplacian neutral Rayleigh equation with singularity of attractive type, (φp(u(t) – cu(t – δ))′)′ + f(t,u′(t)) + g(t,u(t)) = e(t). By applications of an extension of Mawhin’s continuation theorem, suﬃcient conditions for the existence of periodic solution are established. Keywords: Neutral operator; p-Laplacian; Periodic solution; Rayleigh equation; Singularity of attractive type Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 https://doi.org/10.1186/s13660-018-1654-6 Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 https://doi.org/10.1186/s13660-018-1654-6 1 Introduction As is well known, the Rayleigh equation can be derived from many ﬁelds, such as physics, mechanics and engineering technique ﬁelds, and an important question is whether this equation can support periodic solutions. In 1977, Gaines and Mawhin [1] introduced some continuation theorems and applied this theorem to discussing the existence of solutions for the Rayleigh equation [1, p. 99] u′′ + f u′ + g(t,u) = 0. Gaines and Mawhin’s work has attracted the attention of many scholars in the ﬁeld of the Rayleigh equations. More recently, the existence of periodic solutions for Rayleigh equa- tion was extensively studied (see [2–11] and the references therein). Some classical tools have been used to study Rayleigh equation in the literature, including the method of upper and lower solutions [6], the time map continuation theorem [7, 9], ﬁxed point theory [4], the Manásevich–Mawhin continuation theorem [10, 11], and coincidence degree theory [2, 3, 5, 8]. Recently there have been published some results on singular Rayleigh equations [12– 16]. In 2015, Wang and Ma [15] investigated the following singular Rayleigh equation: u′′ + f t,u′ + g(u) = p(t), Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 2 of 11 where g had a repulsive singularity at the origin, i.e., where g had a repulsive singularity at the origin, i.e., (1.1) lim u→0+ g(u) = –∞. (1.1) lim u→0+ g(u) = –∞. By applications of the limit properties of the time map, the authors obtained the result of the existence of periodic solution for this equation. Afterwards, by using topological degree theory, Chen and Lu [12] discussed that the existence of periodic solution for the following singular Rayleigh equations: By applications of the limit properties of the time map, the authors obtained the result of the existence of periodic solution for this equation. Afterwards, by using topological degree theory, Chen and Lu [12] discussed that the existence of periodic solution for the following singular Rayleigh equations: u′′ + f t,u′ + ϕ(t)u(t) – 1 ur(t) = h(t). (1.2) (1.2) The authors found new methods for estimating a lower priori bounds of periodic solutions to equation (1.2). Recently, Xin and Cheng [16] investigated a kind of neutral Rayleigh equation with singularity of repulsive type, u(t) – cu(t – δ) ′′ + f t,u′(t) + g t,u(t) = e(t), (1.3) (1.3) where g(t,u) = g1(t,u) + g0(u) and g0 had a strong singularity at u = 0, i.e., lim u→0+ u 1 g0(s)ds = +∞. (1.4) lim u→0+ u 1 g0(s)ds = +∞. (1.4) By applications of coincidence degree theory, the authors found the existence of positive periodic solution for equation (1.3). All the aforementioned results are related to Rayleigh equation or neutral Rayleigh equa- tion with singularity of repulsive type. Naturally, a new question arises: how p-Laplacian neutral Rayleigh equation works on singularity of attractive type? Besides practical inter- ests, the topic has obvious intrinsic theoretical signiﬁcance. 2 Preliminary lemmas For convenience, deﬁne C1 ω = u ∈C1(R,R) : u(t + ω) = u(t) , C1 ω = u ∈C1(R,R) : u(t + ω) = u(t) , which is a Banach space endowed with the norm ∥· ∥deﬁne by ∥u∥= max{∥u∥∞,∥u′∥∞}, for all x, and ∥u∥∞= max t∈[0,ω] u(t) , u′ ∞= max t∈[0,ω] u′(t) . ∥u∥∞= max t∈[0,ω] u(t) , u′ ∞= max t∈[0,ω] u′(t) . Lemma 2.1 (see [18]) If \|c\| ̸= 1, then the operator (Au)(t) := u(t)–cu(t–δ) has a continuous inverse A–1 on the space Cω, and satisfying Lemma 2.1 (see [18]) If \|c\| ̸= 1, then the operator (Au)(t) := u(t)–cu(t–δ) has a continuous inverse A–1 on the space Cω, and satisfying A–1f (t) = ⎧ ⎨ ⎩ f (t) + ∞ j=1 cjf (t – jδ), for \|c\| < 1,∀f ∈Cω, – f (t+δ) c – ∞ j=1 1 cj+1 f (t + (j + 1)δ), for \|c\| > 1,∀f ∈Cω. Lemma 2.2 If \|c\| ̸= 1, then operator A–1 satisfying Lemma 2.2 If \|c\| ̸= 1, then operator A–1 satisfying ω 0 A–1f (t) p dt ≤ 1 \|1 – \|c\|\|p ω 0 f (t) p dt, ∀f ∈Cω,here 1 ≤p < ∞. Proof We ﬁrst consider \|c\| < 1. From Lemma 2.1, we have Proof We ﬁrst consider \|c\| < 1. From Lemma 2.1, we have ω 0 A–1f (t) p dt = ω 0 ∞ j=0 cjf (t – jδ) p dt ≤ ω 0 ∞ j=0 cjf (t – jδ) p dt ≤ 1 (1 – \|c\|)p ω 0 f (t) p dt. Similarly, for \|c\| > 1, we can get Similarly, for \|c\| > 1, we can get ω 0 A–1f (t) p dt ≤ 1 (\|c\| – 1)p ω 0 f (t) p dt. Therefore, we have Therefore, we have T 0 A–1f (t) p dt ≤ 1 \|1 – \|c\|\|p T 0 f (t) p dt. □ T 0 A–1f (t) p dt ≤ 1 \|1 – \|c\|\|p T 0 f (t) p dt. To answer this question, in this paper, we consider a kind of p-Laplacian neutral Rayleigh equation with singularity of attractive type, φp u(t) – cu(t – δ) ′′ + f t,u′(t) + g t,u(t) = e(t), (1.5) (1.5) where p > 1, ϕp(u) = \|u\|p–2u for u ̸= 0 and ϕp(0) = 0; \|c\| ̸= 1 and δ is a constant with 0 ≤ δ < ω; e : R →R is continuous periodic functions with e(t + ω) – e(t) ≡0 and T 0 e(t)dt = 0; f is for continuous functions deﬁned on R2 and periodic in t with f (t,·) = f (t + ω,·) and f (t,0) = 0, g(t,u) = g0(u) + g1(t,u), here g1 : R × (0,+∞) →R is an L2-Carathéodory function, g1(t,·) = g1(t + ω,·); g0 ∈C((0,∞);R) has an attractive singularity at the origin, i.e., lim u→0+ u 1 g0(s)ds = –∞. (1.6) lim u→0+ u 1 g0(s)ds = –∞. (1.6) Obviously, the attractive condition (1.6) is in contradiction with the repulsive singularity of (1.1) and (1.4). Therefore, the above methods of [12, 15, 16] are no long applicable to the proof of existence of a periodic solution for (1.5) with singularity of attractive type. So we need to ﬁnd a new method to get over it. Obviously, the attractive condition (1.6) is in contradiction with the repulsive singularity of (1.1) and (1.4). Therefore, the above methods of [12, 15, 16] are no long applicable to the proof of existence of a periodic solution for (1.5) with singularity of attractive type. So we need to ﬁnd a new method to get over it. Page 3 of 11 Page 3 of 11 Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 In this paper, by applications of an extension of Mawhin’s continuation theorem in [17] and some analysis techniques, we see the existence of a positive periodic solution for (1.5). Our results improve and extend the results in [12, 15, 16]. Lemma 2.4 Assume that condition \|c\| ̸= 1, is an open bounded set in C1 ω. If: (i) for each λ ∈(0,1) the equation Lemma 2.4 Assume that condition \|c\| ̸= 1, is an open bounded set in C1 ω. If: (i) for each λ ∈(0,1) the equation Lemma 2.4 Assume that condition \|c\| ̸= 1, is an open bounded set in C1 ω. If: (i) for each λ ∈(0,1) the equation φp(Au)′(t) ′ + λf t,u′(t) + λg t,u(t) = λe(t) (2.1) (2.1) has no solution on ∂; has no solution on ∂; (ii) the equation has no solution on ∂; (ii) the equation (ii) the equation F(a) := 1 ω ω 0 g(t,a)dt = 0 has no solution on ∂ ∩R; (iii) the Brouwer degree deg{F, ∩R,0} ̸= 0, then Eq. (2.1) has at least one periodic solution on ¯. 2 Preliminary lemmas □ Lemma 2.3 (see [19]) If u ∈C1 ω(R,R), and there exists a point t∗∈[0,ω] such that \|u(t∗)\| < d, then ∥u∥∞≤d + 1 2 ω 0 u(t) ′ dt Page 4 of 11 Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 and ω 0 u(t) p dt 1 p ≤ ω πp ω 0 u′(t) 2 dt 1 p + dω 1 p , where 1 ≤p < ∞, πp = 2 (p–1)/p 0 ds (1– sp p–1 )1/p = 2π(p–1)1/p psin(π/p) . and and ω 0 u(t) p dt 1 p ≤ ω πp ω 0 u′(t) 2 dt 1 p + dω 1 p , where 1 ≤p < ∞, πp = 2 (p–1)/p 0 ds (1– sp p–1 )1/p = 2π(p–1)1/p psin(π/p) . The following lemma involves the consequences of Theorem 3.1 of [17]. The following lemma involves the consequences of Theorem 3.1 of [17]. 3 Main results: positive periodic solution for (1.5) 3 Main results: positive periodic solution for (1.5) In this section, we will consider the existence of a positive periodic solution for (1.5) with singularity. In this section, we will consider the existence of a positive periodic solution for (1.5) with singularity. Theorem 3.1 Assume that the following conditions hold: Theorem 3.1 Assume that the following conditions hold: (H1) there exists a positive constant K such that \|f (t,v)\| ≤K, for (t,v) ∈R × R; (H2) there exist positive constants D1 and D2 with D1 > D2 > 0 such that g(t,u) < –K for (t,u) ∈R × (D1,+∞) and g(t,u) > K for (t,u) ∈R × (0,D2); (H3) there exist positive constants a, b such that (H3) there exist positive constants a, b such that (H3) there exist positive constants a, b such that –g(t,u) ≤aup–1 + b, for all u > 0. –g(t,u) ≤aup–1 + b, for all u > 0. –g(t,u) ≤aup–1 + b, for all u > 0. Then (1.5) has at least one positive solution with period ω if ω(1+\|c\|) 1p a 1p \|1–\|c\|\| < 2 p–1 p . Proof Firstly, we will claim that the set of all possible ω-periodic solu bounded. Let u(t) ∈C1 ω be an arbitrary solution of (2.1) with period ω. We claim that there exists a point t0 ∈[0,ω] such that (3.1) 0 < u(t0) ≤D1. Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 5 of 11 Integrating both sides of (2.1) over [0,ω], we have Integrating both sides of (2.1) over [0,ω], we have Integrating both sides of (2.1) over [0,ω], we have Integrating both sides of (2.1) over [0,ω], we have ω 0 f t,u′(t) + g t,u(t) dt = 0. ω 0 f t,u′(t) + g t,u(t) dt = 0. (3.2) (3.2) Therefore, from (H1), we have –Kω ≤ ω 0 g t,u(t) dt ≤Kω. From (H2), we know that there exist two points t0, τ ∈(0,T), such that From (H2), we know that there exist two points t0, τ ∈(0,T), such that u(t0) ≤D1, and u(τ) > D2. (3.3) u(t0) ≤D1, and u(τ) > D2. (3.3) Since u(t) > 0, t ∈[0,ω], we get 0 < u(t0) ≤D1. Equation (3.1) is proved. Then, from Lemma 2.3, we have Since u(t) > 0, t ∈[0,ω], we get 0 < u(t0) ≤D1. Equation (3.1) is proved. Then, from Lemma 2.3, we have ∥u∥∞≤D1 + 1 2 ω 0 u′(s) ds. Theorem 3.1 Assume that the following conditions hold: Substituting (3.4) and (3.7) into (3.6), we have ω 0 (Au)′(t) p dt ≤ 1 + \|c\| ∥u∥∞ 2aω∥u∥p–1 ∞+ 2bω + 2Kω + ∥e∥∞ω ω 0 (Au)′(t) p dt ≤ 1 + \|c\| ∥u∥∞ 2aω∥u∥p–1 ∞+ 2bω + 2Kω + ∥e∥∞ω = 2 1 + \|c\| aω∥u∥p ∞+ 1 + \|c\| N1∥u∥∞ ≤2 1 + \|c\| aω D1 + 1 2 ω 0 u′(t) dt p + 1 + \|c\| N1 D1 + 1 2 ω 0 u′(t) dt = (1 + \|c\|)aω 2p–1 1 + 2D1 ω 0 \|u′(t)\|dt p ω 0 u′(t) dt p + 1 2 1 + \|c\| N1 ω 0 u′(t) dt + 1 + \|c\| N1D1, where N1 := 2bω + 2Kω + ∥e∥∞ω. For a given constant ζ > 0, which is only dependent on k > 0, we have (1 + u)k ≤1 + (1 + k)u, for u ∈[0,ζ]. (1 + u)k ≤1 + (1 + k)u, for u ∈[0,ζ]. Therefore, we have Therefore, we have ω 0 (Au)′(t) p dt ≤(1 + \|c\|)aω 2p–1 1 + 2D1p ω 0 \|u′(t)\|dt ω 0 u′(t) dt p + 1 2 1 + \|c\| N1 ω 0 u′(t) dt + 1 + \|c\| N1D1 = (1 + \|c\|)aω 2p–1 ω 0 u′(t) dt p + (1 + \|c\|)aωD1p 2p–2 ω 0 u′(t) dt p–1 + 1 2 1 + \|c\| N1 ω 0 u′(t) dt + 1 + \|c\| N1D1. (3.8) (3.8) By application of Lemma 2.1, we have By application of Lemma 2.1, we have By application of Lemma 2.1, we have ω 0 u′(t) dt = ω 0 A–1Au′ (t) dt ≤ ω 0 \|(Au)′(t)\|dt \|1 – \|c\|\| ≤ω 1 q ( ω 0 \|(Au)′(t)\|p dt) 1 p \|1 – \|c\|\| , (3.9) (3.9) since (Au′)(t) = (Au)′(t) and 1 p + 1 q = 1. Apply the inequality since (Au′)(t) = (Au)′(t) and 1 p + 1 q = 1. Apply the inequality (a + b)k ≤ak + bk, for a,b > 0,0 < k < 1. Theorem 3.1 Assume that the following conditions hold: (3.4) (3.4) Multiplying both sides of (2.1) by (Au)(t) and integrating over [0,ω], we get Multiplying both sides of (2.1) by (Au)(t) and integrating over [0,ω], we get ω 0 φp(Au)′(t) ′(Au)(t)dt + λ ω 0 f t,u′(t) (Au)(t)dt + λ ω 0 g t,u(t) (Au)(t)dt = λ ω 0 e(t)(Au)(t)dt, ω 0 φp(Au)′(t) ′(Au)(t)dt + λ ω 0 f t,u′(t) (Au)(t)dt + λ ω 0 g t,u(t) (Au)(t)dt = λ ω 0 e(t)(Au)(t)dt, i.e. ω 0 (Au)′(t) p dt = λ ω 0 f t,u′(t) (Au)(t)dt + λ ω 0 g t,u(t) (Au)(t)dt ω 0 (Au)′(t) p dt = λ ω 0 f t,u′(t) (Au)(t)dt + λ ω 0 g t,u(t) (Au)(t)dt – λ ω 0 e(t)(Au)(t)dt. (3.5 – λ ω 0 e(t)(Au)(t)dt. (3.5) (3.5) From (H1), we have From (H1), we have From (H1), we have om (H1), we have ω 0 (Au)′(t) p dt ω 0 (Au)′(t) p dt ≤ 1 + \|c\| ω 0 f t,u′(t) u(t) dt + ω 0 g t,u(t) u(t) dt + ω 0 e(t) u(t) dt ≤ 1 + \|c\| ∥u∥∞ ω 0 f t,u′(t) dt + ω 0 g t,u(t) dt + ω 0 e(t) dt ≤ 1 + \|c\| ∥u∥∞ Kω + ∥e∥∞ω + ω 0 g t,u(t) dt . (3.6) (3.6) We get from (H1), (H3) and (3.2) We get from (H1), (H3) and (3.2) We get from (H1), (H3) and (3.2) We get from (H1), (H3) and (3.2) ω 0 g t,u(t) dt = g(t,u(t))≥0 g+ t,u(t) dt – g(t,u(t))≤0 g– t,u(t) dt = –2 g(t,u(t))≤0 g– t,u(t) dt + ω 0 f t,u′(t) dt Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 6 of 11 ≤2a ω 0 u(t) p–1 dt + 2bω + Kω ≤2aω∥u∥p–1 ∞+ 2bω + Kω, (3.7) (3.7) ≤2aω∥u∥p–1 ∞+ 2bω + Kω, (3.7) where g– := min{g(t,u),0}. Theorem 3.1 Assume that the following conditions hold: (a + b)k ≤ak + bk, for a,b > 0,0 < k < 1. (a + b)k ≤ak + bk, for a,b > 0,0 < k < 1. Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 7 of 11 Substituting (3.8) into (3.9), we have Substituting (3.8) into (3.9), we have Substituting (3.8) into (3.9), we have ω 0 u′(t) dt ≤ ω 1 q ( (1+\|c\|)aω 2p–1 ) 1 p ω 0 \|u′(t)\|dt + ω 1 q ( (1+\|c\|)aωD1p 2p–2 ) 1 p ( ω 0 \|u′(t)\|dt) p–1 p \|1 – \|c\|\| + ω 1 q ( 1 2(1 + \|c\|)N1 ω 0 \|u′(t)\|dt) 1 p + ω 1 q ((1 + \|c\|)N1D1) 1 p \|1 – \|c\|\| . Since ω(1+\|c\|) 1p a 1p \|1–\|c\|\| < 2 p–1 p , it is easy to see that there exists a positive constant M′ 1 such that ω 0 u′(t) dt ≤M′ 1. ω 0 u′(t) dt ≤M′ 1. (3.10) (3.10) From (3.4) and (3.10), we have From (3.4) and (3.10), we have ∥u∥∞≤D1 + 1 2 ω 0 u′(t) dt ≤D1 + 1 2M′ 1 := M1. (3.11) (3.11) As (Au)(0) = (Au)(ω), there exists t1 ∈[0,ω] such that (Au)′(t1) = 0, while φp(0) = 0, we have φp (Au)′(t) = t t1 φp (Au)′(s) ′ ds ≤λ ω 0 f t,u′(t) dt + λ ω 0 g t,u(t) dt + λ ω 0 e(t) dt, (3.12) (3.12) where t ∈[t1,t1 + ω]. In view of (H1), (3.7) and (3.12), we have where t ∈[t1,t1 + ω]. In view of (H1), (3.7) and (3.12), we have φp(Au)′ ∞= max t∈[0,ω] φp (Au)′(t) ∞ = max t∈[t1,t1+ω] t t1 φp (Au)′(s) ′ ds ≤λ ω 0 f t,u′(t) dt + ω 0 g t,u(t) dt + ω 0 e(t) dt ≤λ Kω + 2aω∥u∥p–1 ∞+ 2bω + Kω + ∥e∥∞ω ≤λ 2aωMp–1 1 + 2Kω + 2bω + ∥e∥∞ω := λM′ 2. Theorem 3.1 Assume that the following conditions hold: (3.13) ≤λ ω 0 f t,u′(t) dt + ω 0 g t,u(t) dt + ω 0 e(t) dt ≤λ Kω + 2aω∥u∥p–1 ∞+ 2bω + Kω + ∥e∥∞ω ≤λ 2aωMp–1 1 + 2Kω + 2bω + ∥e∥∞ω := λM′ 2. (3.13) (3.13) We claim that there exists a positive constant M2 > M′ 2 + 1 such that, for all t ∈R, We claim that there exists a positive constant M2 > M′ 2 + 1 such that, for all t ∈R, u′ ∞≤M2. (3.14) u′ ∞≤M2. (3.14) In fact, if u′ is not bounded, there exists a positive constant M′′ 2 such that ∥u′∥∞> M′′ 2 for some u′ ∈R. Therefore, we have φp(Au)′ ∞= φp Au′ ∞= Au′ p–1 ∞ = 1 + \|c\| p–1 u′ p–1 ∞≥ 1 + \|c\| p–1M′′p–1 2 := M∗ 2. Then it is a contradiction. So (3.14) holds. Then it is a contradiction. So (3.14) holds. Then it is a contradiction. So (3.14) holds. Then it is a contradiction. So (3.14) holds. Then it is a contradiction. So (3.14) holds. Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 8 of 11 On the other hand, it follows by (2.1) that On the other hand, it follows by (2.1) that φp(Au)′(t) ′ + λf t,u′(t) + λ g0 u(t) + g1 t,u(t) = λe(t). (3.15) φp(Au)′(t) ′ + λf t,u′(t) + λ g0 u(t) + g1 t,u(t) = λe(t). (3.15) (3.15) Multiplying both sides of (3.15) by u′(t) we get Multiplying both sides of (3.15) by u′(t) we get φp(Au)′(t) ′u′(t) + λf t,u′(t) u′(t) + λ g0 u(t) + g1 t,u(t) u′(t) = λe(t)u′(t). (3.16) φp(Au)′(t) ′u′(t) + λf t,u′(t) u′(t) + λ g0 u(t) + g1 t,u(t) u′(t) = λe(t)u′(t). Theorem 3.1 Assume that the following conditions hold: (3.16) (3.16) Let τ ∈[0,ω] be as in (3.3), for any τ ≤t ≤ω, we integrate (3.16) on [τ,t] and get λ u(t) u(τ) g0(v)dv = λ t τ g0 u(s) u′(s)ds = – t τ φp(Au)′(s) ′u′(s)ds – λ t τ f s,u′(s) u′(s)ds – λ t τ g1 s,u(s) u′(s)ds + λ t τ e(s)u′(s)ds. (3.17) (3.17) By (3.7), (3.11) and (3.14), we have By (3.7), (3.11) and (3.14), we have t τ φp(Au)′(s) ′u′(s)ds ≤ T 0 φp(Au)′(s) ′u′(s) ds ≤λ u′ ∞ ω 0 f t,u′(t) dt + ω 0 g t,u(t) dt + ω 0 e(t) dt ≤λM2 Kω + 2aω\|u∥p–1 ∞+ 2bω + Kω + ∥e∥∞ω ≤λM2 2Kω + 2aωMp–1 1 + 2bω + ∥e∥∞ω . Moreover, from (H1) and (3.14) Moreover, from (H1) and (3.14) t τ f s,u′(s) u′(s)ds ≤ T 0 f s,u′(s) u′(s) ds ≤KM2ω, t τ g1 s,u(s) u′(s)ds ≤ T 0 g1 s,u(s) u′(s) ds ≤M2\|gM1\|√ω, t τ f s,u′(s) u′(s)ds ≤ T 0 f s,u′(s) u′(s) ds ≤KM2ω, t τ g1 s,u(s) u′(s)ds ≤ T 0 g1 s,u(s) u′(s) ds ≤M2\|gM1\|√ω, where gM1 = max0≤u≤M1 \|g1(t,u)\| ∈L2(0,ω). where gM1 = max0≤u≤M1 \|g1(t,u)\| ∈L2(0,ω). t τ e(s)u′(s)ds ≤ ω 0 e(s) u′(s) ds ≤∥e∥∞ωM2. With these inequalities we can derive from (3.17) that With these inequalities we can derive from (3.17) that u(t) u(τ) g0(v)dv ≤M2 3Kω + 2aωMp–1 1 + 2bω + 2∥e∥∞ω + \|gM1\|√ω . Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 9 of 11 In view of (1.6), we know there exists M3 > 0 such that In view of (1.6), we know there exists M3 > 0 such that u(t) ≥M3, ∀t ∈[τ,ω]. (3.18) u(t) ≥M3, ∀t ∈[τ,ω]. (3.18) The case t ∈[0,τ] can be treated similarly. The case t ∈[0,τ] can be treated similarly. The case t ∈[0,τ] can be treated similarly. The case t ∈[0,τ] can be treated similarly. Theorem 3.1 Assume that the following conditions hold: Having in mind (3.11), (3.14) and (3.18), we deﬁne = u ∈X : E1 < u(t) < E2 and u′(t) < E3 ∀t ∈R , where 0 < E1 < min{D2,M3}, E2 > max{M1,D1} and E3 > M2. We know that (2.1) has no solution on ∂ as λ ∈(0,1) and when u(t) ∈∂ ∩R, u(t) = E2 or u(t) = E1, from (3.4), we know that E2 + 1 > D1; therefore, from (H2) we see that where 0 < E1 < min{D2,M3}, E2 > max{M1,D1} and E3 > M2. We know that (2.1) has no solution on ∂ as λ ∈(0,1) and when u(t) ∈∂ ∩R, u(t) = E2 or u(t) = E1, from (3.4), we know that E2 + 1 > D1; therefore, from (H2) we see that 1 ω ω 0 g(t,E2)dt < 0 and 1 ω ω 0 g(t,E1)dt > 0. So condition (ii) is also satisﬁed. Set H(u,μ) = μu + (1 – μ) 1 ω ω 0 g(t,u)dt, where x ∈∂ ∩R, μ ∈[0,1], we have where x ∈∂ ∩R, μ ∈[0,1], we have uH(u,μ) = μu2 + (1 – μ) u ω ω 0 g(t,u)dt ̸= 0, uH(u,μ) = μu2 + (1 – μ) u ω ω 0 g(t,u)dt ̸= 0, and thus H(u,μ) is a homotopic transformation and deg{F, ∩R,0} = deg 1 ω ω 0 g(t,u)dt, ∩R,0 = deg{u, ∩R,0} ̸= 0. deg{F, ∩R,0} = deg 1 ω ω 0 g(t,u)dt, ∩R,0 = deg{u, ∩R,0} ̸= 0. deg{F, ∩R,0} = deg 1 ω ω 0 g(t,u)dt, ∩R,0 So condition (iii) is satisﬁed. In view of Lemma 2.1, there exists a solution with period ω. □ 4 Example Author details 1 1College of Computer Science and Technology, Henan Polytechnic University, Jiaozuo, China. 2School of Mathematics and Information Science, Henan Polytechnic University, Jiaozuo, China. 3Department of Mathematics, Sichuan University, Chengdu, China. Competing interests Competing interests The authors declare that they have no competing interests. 5 Conclusions In this article we introduce the existence of a periodic solution for a p-Laplacian neu- tral Rayleigh equation with singularity of attractive type. Due to the attractive condition being in contradiction with the repulsive condition, the methods of [12, 15, 16] are no long applicable to the proof of a periodic solution for equation (1.5) with singularity of at- tractive singularity. In this paper, we give attractive conditions (1.6) and (H3), and we see the existence of a periodic solution for (1.5) by applications of the extension of Mawhin’s continuation theorem [17]. Moreover, in view of the mathematical points, the results sat- isfying the conditions of an attractive singularity are valuable to understand the periodic solution for Rayleigh equations. 4 Example p Example 4.1 Consider the following p-Laplacian neutral Rayleigh equation with singular- ity: p Example 4.1 Consider the following p-Laplacian neutral Rayleigh equation with singular- ity: φp u(t) – 1 4u(t – δ) ′′ – cos2(2t)sinu′(t) – 1 3π4 (sin4t + 2)u3(t) + 1 uμ = sin2(2t), (4.1) φp u(t) – 1 4u(t – δ) ′′ – cos2(2t)sinu′(t) – 1 3π4 (sin4t + 2)u3(t) + 1 uμ = sin2(2t), (4.1) (4.1) where μ ≥1 and p = 4, δ is a constant and 0 ≤δ < ω. where μ ≥1 and p = 4, δ is a constant and 0 ≤δ < ω. Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 Page 10 of 11 Page 10 of 11 It is clear that ω = π 2 , c = 1 4, e(t) = sin2(2t), f (t,v) = –cos2(2t)sinv, g(t,u) = – 1 3π3 (sin4t + 2)u4(t) + 1 uμ(t). Choose K = 1, D1 = 2, D2 = 1, a = 1 π4 , it is obvious that (H1), (H2) and (H3) hold. Next, we consider ω(1 + \|c\|) 1 p a 1 p 2 p–1 p \|1 – \|c\|\| = π 2 (1 + 1 4) 1 4 ( 1 π4 ) 1 4 2 3 4 (1 – 1 4) ≈1.057 1.783 < 1. ω(1 + \|c\|) 1 p a 1 p 2 p–1 p \|1 – \|c\|\| = π 2 (1 + 1 4) 1 4 ( 1 π4 ) 1 4 2 3 4 (1 – 1 4) ≈1.057 1.783 < 1. Therefore, by Theorem 3.1, (4.1) has at least one nonconstant π 2 -periodic solution. Authors’ contributions YX, HML and ZBC worked together on the derivation of the mathematical results. All authors read and approved the ﬁnal manuscript. Acknowledgements YX HML d ZBC c o edge e ts YX, HML and ZBC would like to thank the referee for invaluable comments and insightful suggestions. This work was supported by National Natural Science Foundation of China (No. 11501170), Education Department of Henan Province project (No. 16B110006) and Henan Polytechnic University Outstanding Youth Fund (J2016-03). Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. Received: 4 January 2018 Accepted: 7 March 2018 Xin et al. Journal of Inequalities and Applications ( 2018) 2018:58 References 1. Gaines, R.E., Mawhin, J.L.: Coincidence Degree, and Nonlinear Diﬀerential Equations. Lecture Notes in Mathematics, vol. 568. Springer, Berlin (1977) 5. Du, B., Lu, S.P.: On the existence of periodic solutions to a p-Laplacian Rayleigh equation. Indian J. Pure Appl. Math. 40, 253–266 (2009) Page 11 of 11 6. Habets, P., Torres, P.: P: Some multiplicity results for periodic solutions of a Rayleigh diﬀerential equation. Dyn. Contin. Di t I l S t S A M th A l 8 335 351 (2001) 6. 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https://openalex.org/W4221004527	https://biomedical-engineering-online.biomedcentral.com/track/pdf/10.1186/s12938-022-00988-7	English	null	Effect of two types of shoulder prosthesis on the muscle forces using a generic multibody model for different arm motions	BioMedical engineering online	2,022	cc-by	9,996	© 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 mate- rial. 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/publi cdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Weisse et al. BioMedical Engineering OnLine (2022) 21:17 https://doi.org/10.1186/s12938-022-00988-7 Weisse et al. BioMedical Engineering OnLine (2022) 21:17 https://doi.org/10.1186/s12938-022-00988-7 BioMedical Engineering OnLine BioMedical Engineering OnLine Weisse et al. BioMedical Engineering OnLine https://doi.org/10.1186/s12938-022-00988-7 Open Access Bernhard Weisse* , Susan Lama, Gabor Piskoty, Christian Affolter and Ameet K. Aiyangar Bernhard Weisse* , Susan Lama, Gabor Piskoty, Christian Affolter and Ameet K. Aiyangar Correspondence: bernhard.weisse@empa.ch Laboratory for Mechanical Systems Engineering, Empa, Swiss Federal Laboratories for Materials Science and Technology, CH‑8600 Dübendorf, Switzerland Abstract Background: This study aims to analyze the effects of a novel dual-bearing shoulder prosthesis and a conventional reverse shoulder prosthesis on the deltoid and rota- tor cuff muscle forces for four different arm motions. The dual-bearing prosthesis is a glenoid-sparing joint replacement with a moving center of rotation. It has been developed to treat rotator cuff arthropathy, providing an increased post-operative functionality. Methods: A three-dimensional musculoskeletal OpenSim® model of an upper body, incorporating a natural gleno-humeral joint and a scapula-thoracic joint developed by Blana et al. (J Biomech 41: 1714-1721, 2008), was used as a reference for the natural shoulder. It was modified by integrating first a novel dual-bearing prosthesis, and sec- ond, a reverse shoulder prosthesis into the shoulder joint complex. Four different arm motions, namely abduction, scaption, internal and external rotation, were simulated using an inverse kinematics approach. For each of the three models, shoulder muscle forces and joint reaction forces were calculated with a 2 kg weight in the hand. Results: In general, the maximal shoulder muscle force and joint reaction force values were in a similar range for both prosthesis models during all four motions. The maxi- mal deltoid muscle forces in the model with the dual-bearing prosthesis were 18% lower for abduction and 3% higher for scaption compared to the natural shoulder. The maximal rotator cuff muscle forces in the model with the dual-bearing prosthesis were 36% lower for abduction and 1% higher for scaption compared to the natural shoul- der. Although the maximal deltoid muscle forces in the model with the dual-bearing prosthesis in internal and external rotation were 52% and 64% higher, respectively, compared to the natural shoulder, the maximal rotator cuff muscle forces were 27% lower in both motions. Conclusion: The study shows that the dual-bearing shoulder prosthesis is a feasi- ble option for patients with rotator cuff tear and has a strong potential to be used as secondary as well as primary joint replacement. The study also demonstrates that computer simulations can help to guide the continued optimization of this particular design concept for successful clinical outcomes. Correspondence: bernhard.weisse@empa.ch Laboratory for Mechanical Systems Engineering, Empa, Swiss Federal Laboratories for Materials Science and Technology, CH‑8600 Dübendorf, Switzerland Background Total shoulder arthroplasty is broadly classified into two categories: anatomical total shoulder arthroplasty and reverse total shoulder arthroplasty. Anatomical total shoul- der arthroplasty retains the orientation of the natural joint and involves replacing the gleno-humeral joint with an anatomical shoulder prosthesis (ASP), wherein the natural humeral head is replaced by a ceramic or highly polished metal ball connected to the humerus by a stem. The ball head articulates with a concave polyethylene (PE) compo- nent, which is fixed onto the glenoid surface. Damage to the polyethylene glenoid com- ponent of the ASP and loosening of its fixation are the most common modes of failure [1–3]. With the overall goal of reducing the high failure rates of the "conventional" ASP, P. Grammont developed in 1985 the reverse shoulder prosthesis, wherein the ball head is fixed in the glenoid cavity instead of the humerus, while the PE cup is implanted into the humerus [4–6]. Both, the anatomical and reverse prosthetic designs are based on the assumption that the gleno-humeral joint is well represented by a purely 3 degree-of-free- dom rotational joint with a fixed center of rotation. Nevertheless, during reverse arthro- plasty surgery, the medialized center of rotation (COR) of the artificial gleno-humeral joint is altered by setting it in a more medial location compared to the natural shoulder joint. This modification is intended to increase the contribution of the deltoid muscles to large shoulder movements such as abduction and scaption to the reaction moment at the shoulder without substantially increasing muscle effort. This is accomplished by increas- ing the deltoid’s lever arm by up to 42%, thus reducing deltoid muscle forces without significantly compromising the torque generated by these muscles to drive functional movements such as scaption [7, 8]. This solution is particularly beneficial when massive tears or other forms of damage to the rotator cuff muscles (e.g., supraspinatus) signifi- cantly reduce the otherwise substantial contribution of this muscle group to large shoul- der movements and overall shoulder stability. The worldwide acceptance of RSP attests to its efficacy [9], and indications for reverse shoulder arthroplasty have been expanded to all forms of gleno-humeral joint disease with rheumatoid arthritis and serious rotator cuff tears, presently accounting for more than 40% of the shoulder arthroplasty market [10, 11]. The growing popularity of reverse shoulder arthroplasty notwithstanding [12–14], this surgical intervention is also associated with some significant disadvantages. © 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 mate- rial. 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/publi cdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 19 Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Keywords: Reverse shoulder prosthesis, Dual-bearing glenoid-sparing shoulder prosthesis, Musculoskeletal model, OpenSim, shoulder biomechanics, Muscle activation Background The compli- cation rate for primary arthroplasty is around 24%, which increases to 40% with revision surgeries [15, 16]. Of these, glenoid component loosening is the weak link in shoulder replacement, accounting for nearly one-third of all TSA complications [17]. Outright implant failure rates at 5- and 10-years are 10% and 22%, respectively [18], which is high compared to knee and hip replacements [19–21]. Second, patients affected by these complications must undergo revision surgeries. However, for those with a compromised bone stock (from osteolysis or bone loss due to additional preparation requirements fol- lowing removal of primary prosthesis fixation), no reliable solution is currently available Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 3 of 19 on the market and arthrodesis, although sub-optimal, is the final solution. A third limi- tation pertains to the design based on a fixed COR and no translational degrees of free- dom common to both anatomical and reverse shoulder prostheses. Recent dynamic radiographic imaging studies seem to indicate otherwise [22–24]. More pertinently, while a fixed COR may simplify design and manufacturing of the prosthesis and pro- vide some advantages for arm elevation motion, it requires a precise implantation by the surgeon and may compromise certain muscle lever-arms for other shoulder movements crucial for independent daily living. In an attempt to address these limitations, a new shoulder prosthesis with a novel dual-bearing design, the so-called dual-bearing shoulder prosthesis (DBSP), has recently been developed and patented by the Swiss company 41hemiverse AG [25, 26]. One major advantage is that the prosthesis can be implanted with a less invasive surgical proce- dure compared to conventional shoulder prostheses. In addition, the proximal com- ponent does not need a fixation to the glenoid, which represents a benefit for patients with compromised bone stock, especially at revision surgery. Furthermore, the DBSP with two, eccentrically located rotational centers partially simulates a moving COR, thus theoretically providing an improved replication of natural shoulder gleno-humeral joint kinematics. Our overall goal is to assess the biomechanical performance of this new and promising prosthesis design and, eventually, to assess its performance in vivo and in comparison with existing solutions. An ideal scientific study would be to implant the DBSP into patients and compare per- formance and outcome with conventional designs. Joint reactions forcesi Considering first, the two large ROM movements, the novel DBSP model generated the lowest joint reaction forces of all the three models during abduction (Fig. 1A). JRF were 10% and almost 20% lower than the NS and RSP models, respectively. For scaption (Fig. 1B), the DBSP JRF were slightly higher (~ 6%) than the NS model, but were lower than the forces generated in the RSP model, which were almost 15% greater than for the natural shoulder (NS). On the other hand, the DBSP model generated the largest JRF for internal and external rotation (Fig. 1C and D). Nevertheless, they were comparable to the RSP model (~ 1% and 5% difference, respectively). Consequently, the differences between DBSP and NS models—12% for internal- and 20% for external rotation—were very similar to the corresponding differences between RSP and NS models (11.5% and 17%, respectively). Results Peak reaction forces at the joint, as well as peak forces generated in the individual shoulder muscles during four different movements were assessed in each of the three musculoskeletal models: NS, DBSP, and RSP model (Table 1). Of these, two are large range-of-motion (ROM) movements—abduction 0°–120°; and scaption 0°–120°—with internal rotation 0°–40°; and external rotation 40°–0° being the other two movements studied. Additionally, Table 1 also includes the peak muscle activation, as output by the OpenSim® simulation suite. Background However, from an ethical viewpoint, it is necessary to first make preliminary assessments to verify the claims with respect to superior (or at the very least equivalent) biomechanical performance compared to the existing solutions. Furthermore, measuring in vivo joint or muscle loads to assess biomechanical performance is not readily feasible, and computational musculoskeletal modeling and simulation is a valuable technique to analyze the influence of a prosthesis on the muscle forces in silico. Hence, in a first step, our study focuses on comparing the computed joint reaction forces as well as the demand on the muscles when using the novel implant with the conventional counterparts, since this is often an accepted design criterion as well as a criterion for determining placement of the implant by the surgeon during surgery. Several studies have shown the power of 3D dynamic rigid multibody simulation tools for determining movements and internal loads within the musculoskeletal system. Opensim® is one of the software suited for simulating and predicting muscle forces and also contains a repository of models developed by research groups worldwide. The soft- ware was developed by the National Centers for Biomedical Computing at Stanford Uni- versity and is an established open source software [27]. This tool was chosen because many studies demonstrated its usability for shoulder movement analysis [10, 13, 28–33] and it also offers validated basic models which can be adapted for comparative investi- gations. The main objective of the current study was to assess the influence of the new DBSP design on the muscle forces and joint reaction forces for four different motions. Additionally, the muscles forces and joint reaction forces estimated for the novel DBSP Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 4 of 19 prosthesis in question were compared with models representing the more conventional, but widely accepted RSP, as well as the natural shoulder (NS). Muscle forces for the different motions The shoulder muscles investigated fall into two groups: deltoid muscle group consisting of the anterior, middle and posterior deltoids, and the rotator cuff group, which com- prised the supraspinatus, infraspinatus teres minor and subscapularis. Individual muscle peak forces are delineated in Table 1. The total deltoid muscle group peak forces in the DBSP model are similar to those generated in the RSP model for abduction (~ 4% difference; Fig. 2A) and scaption (~ 1.7% difference; Fig. 2B)—the two large ROM motions, but 18% and 22% less, respectively, than those generated in the NS model. Differences in rotator cuff muscle forces between the DBSP and NS models were small during simulated abduction (6%; Fig. 3A) and scap- tion (3%; Fig. 3B). However, rotator cuff muscles elicited a significantly lower effort in the DBSP model compared to the RSP model in both abduction (45%; Fig. 3A) and scap- tion (22%; Fig. 3B). For internal and external rotation, the deltoid muscles generated substantially larger forces in the prosthesis models—both DBSP and RSP—compared to the NS model. However, the opposite occurred within the rotator cuff muscles. The biggest reductions compared to the NS model were seen in the subscapularis muscle force for both, DBSP and RSP models—82% and 87%, respectively, for internal rotation, and 85% and 87%, respectively, for external rotation. In external rotation, the second largest reduction Page 5 of 19 Weisse et al. ABD abduction, SCP scaption (SCP), IR internal rotation, ER external rotation Muscle forces for the different motions BioMedical Engineering OnLine (2022) 21:17 Page 6 of 19 Fig. 1 Peak total joint reaction forces for four simulated motions for the natural shoulder, the dual-bearing shoulder and the reverse shoulder prosthesis model for abduction (A), scaption (B), internal rotation (C), and external rotation (D) Fig. 1 Peak total joint reaction forces for four simulated motions for the natural shoulder, the dual-bearing shoulder and the reverse shoulder prosthesis model for abduction (A), scaption (B), internal rotation (C), and external rotation (D) (37%) after subscapularis muscle was seen in the teres minor for the DBSP model, com- pared to a relatively modest 12% reduction in the RSP model. Muscle activation Muscle activation is the ratio of actual muscle force to the maximum isometric force that a muscle can generate. Its value lies between 0 and 1 and it implies the amount of relative effort of the muscle element. For each muscle group, the sum of peak forces produced by each muscle element in that group during a particular motion was divided by the sum of maximal isometric forces of those muscle elements to calculate the peak muscle activa- tion in order to derive the average effort of the muscle system for comparison purpose. f Table 1 clearly shows that the muscle activation for all the models during four different motions is below 0.5, implying a realistic effort simulated by the models. It can be seen that the middle deltoid activation for the DBSP model is higher than for the RSP model. However, the activations for the posterior deltoid and rotator cuff muscles are lower for the DBSP model compared to the RSP model. Muscle forces for the different motions BioMedical Engineering OnLine (2022) 21:17 Table 1 Maximal muscle forces and peak joint reaction forces (JRF) in different ranges of motion for the natural shoulder model, the reverse shoulder prosthesis model and the dual-bearing shoulder prosthesis model prosthesis model ABD abduction, SCP scaption (SCP), IR internal rotation, ER external rotation Natural shoulder (NS) Reverse shoulder prosthesis (RSP) Dual-bearing shoulder prosthesis (DBSP) Force (N) Activation Force (N) % Change from NS Activation Force (N) % Change from NS Activation ABD Anterior deltoid 136.3 0.27 121.7 − 11 0.24 116.5 − 15 0.23 Middle deltoid 292.4 0.25 299.4 + 2 0.25 396.8 + 36 0.33 Posterior deltoid 330 0.20 156.5 − 53 0.10 107.9 − 67 0.07 Supraspinatus 56.1 0.09 – – – – – – Infraspinatus 95.4 0.07 152.7 + 60 0.11 130.2 + 36 0.09 Teres minor 36.6 0.07 118 + 222 0.24 68.9 + 88 0.14 Subscapularis 213.6 0.15 205 − 4 0.14 58.6 − 73 0.04 JRF 1054.0 1142.2 + 8 957.5 − 9 SCP Anterior deltoid 126.7 0.25 143.3 13 0.28 146.4 16 0.29 Middle deltoid 227.1 0.19 265.5 17 0.22 370.4 63 0.31 Posterior deltoid 281.8 0.17 195.2 − 31 0.12 137.2 − 51 0.08 Supraspinatus 53.4 0.09 – – – – – – Infraspinatus 220.9 0.15 255.9 16 0.18 259.5 17 0.18 Teres minor 38.6 0.08 139.5 261 0.28 80.7 109 0.16 Subscapularis 40.2 0.03 19.8 − 51 0.01 14.7 − 63 0.01 JRF 1031.6 1176.8 14 1095 6 IR Anterior deltoid 110.2 0.22 113.9 3 0.23 130.4 18 0.26 Middle deltoid 187.4 0.16 277.2 48 0.23 307.4 64 0.26 Posterior deltoid 19.2 0.01 30.9 61 0.02 43.1 124 0.03 Supraspinatus 50.2 0.08 – – – – – – Infraspinatus 374.8 0.26 338 − 10 0.24 392.3 5 0.27 Teres minor 180.3 0.36 162.5 − 10 0.33 117.6 − 35 0.24 Subscapularis 110.7 0.08 19.9 − 82 0.01 14.5 − 87 0.01 JRF 827.6 935.9 13 946.3 14 ER Anterior deltoid 122.7 0.24 112.4 − 8 0.22 133.7 9 0.26 Middle deltoid 157.8 0.13 292.4 85 0.25 309.9 96 0.26 Posterior deltoid 17.9 0.01 39 118 0.02 44.4 148 0.03 Supraspinatus 49.6 0.08 – – – – – – Infraspinatus 369.3 0.26 329.3 − 11 0.23 392.4 6 0.27 Teres minor 171.3 0.34 151.6 − 12 0.31 108.4 − 37 0.22 Subscapularis 111.6 0.08 16.2 − 85 0.01 14.6 − 87 0.01 JRF 779.5 935 20 982.5 26 Weisse et al. Discussion Our overall goal was to assess the biomechanical performance of the dual-bearing shoul- der prosthesis, which purportedly claims to address limitations in the currently avail- able reverse shoulder prosthesis designs. In this initial study, we adopted an in silico approach, wherein we deployed a validated computational musculoskeletal model of Weisse et al. BioMedical Engineering OnLine (2022) 21:17 (2022) 21:17 Page 7 of 19 Fig. 2 Deltoid muscle force vs abduction angle (A) and deltoid muscle force vs scaption angle (B) for different shoulder models Fig. 2 Deltoid muscle force vs abduction angle (A) and deltoid muscle force vs scaption angle (B) for different shoulder models the shoulder joint complex to estimate the gleno-humeral joint reaction forces, and the shoulder muscle forces and activations (muscle effort). We then compared these outputs between models adapted for the natural shoulder, the "conventional" reverse shoulder prosthesis and the "novel" dual-bearing shoulder prosthesis for four common shoulder movements: abduction, scaption, internal and external rotation. This approach enabled a verification of the claims with respect to superior, or, at the very least, equivalent bio- mechanical performance of the dual-bearing prosthesis compared to the "conventional" reverse shoulder prosthesis. In this regard, the biomechanical performance of the DBSP can be considered to benchmark against RSP, which is the currently available design. The results confirm the findings from other studies, that the RSP generates lower del- toid muscle forces by increasing the moment arm during abduction compared to the NS [4, 7, 8]. Further validation was also carried out by comparing results for abduction to the results of Bergmann et al. [34] and Costantini et al. [35], see details in the section "Method". Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 8 of 19 Fig. 3 Rotator cuff muscle force vs abduction angle (A) and rotator cuff muscle force vs scaption angle (B) for different shoulder models Fig. 3 Rotator cuff muscle force vs abduction angle (A) and rotator cuff muscle force vs scaption angle (B) for different shoulder models For the DBSP, the peak deltoid muscle forces were lower in abduction compared to the NS model (Fig. 2), in scaption the peak deltoid muscle force was higher dur- ing lower scaption angles (0–70°), but lower during higher scaption angles (70°–120°) compared to the NS and the RSP model (Fig. 4). Discussion Overall, the difference in peak values of deltoid muscle forces during abduction and scaption between the reverse and dual- bearing designs was not substantial. This indicates that the dual-bearing prosthesis is a viable option biomechanically for shoulder replacement surgery in future. Not only did the DBSP result in lower JRF than the RSP during abduction and scap- tion—the two large ROM movements, but these value were either equivalent or lower than those generated in the natural shoulder. Although the forces were much larger than for the natural shoulder during internal and external rotation, they were quite Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 9 of 19 Fig. 4 OpenSim® shoulder model incorporating the shoulder joint complex without muscles: head (1), thorax including spine (2), right clavicle (3), right scapula (4), right humerus (5), right ulna (6), right radius (7), and right hand (8) (A); OpenSim® shoulder model with all involved muscles (red), markers on bony landmarks (pink) and wrapping surface in humerus for the muscles (blue) at 30° scaption (B) Fig. 4 OpenSim® shoulder model incorporating the shoulder joint complex without muscles: head (1), thorax including spine (2), right clavicle (3), right scapula (4), right humerus (5), right ulna (6), right radius (7), and right hand (8) (A); OpenSim® shoulder model with all involved muscles (red), markers on bony landmarks (pink) and wrapping surface in humerus for the muscles (blue) at 30° scaption (B) similar to the joint forces developed in the RSP model. Assessing the magnitude of joint reaction forces is useful to understand the risk of implant failure due to disloca- tion or outright damage on the one hand, but also to assess longer term risks of wear- related complications. In this regard, given that the DBSP model generated lower or equivalent JRF to the RSP model indicate that the DBSP performed quite favorably in comparison to the RSP. The basic musculoskeletal shoulder model used in this research is a verified one originally built in SIMM [28], incorporating all involved joints from the shoulder: the gleno-humeral joint, the sterno-clavicular joint, the acromio-clavicular joint, and the scapula-thoracic joint [33]. With respect to muscle forces, peak DBSP forces in the deltoid muscle group were similar to those generated in the RSP for all the four movements investigated, again implying an equivalent biomechanical performance in this regard. Discussion The most notable outcome in this study related to the efforts (force and activation) elicited in the rotator cuff muscles for the four movements studied. The DBSP model generated significantly lower forces compared to the model representing the con- ventional reverse shoulder prosthesis. Furthermore, the subscapularis, which plays a substantial role during internal rotation, also developed significantly lower forces during internal rotation in the DBSP model compared to the NS model, but a much more modest, yet notable reduction compared to the RSP model. Similarly, the teres minor, which has an important role in external rotation, also developed significantly lower forces with the DBSP model compared with both, the natural shoulder and the Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 10 of 19 RSP models. With regard to abduction and scaption, it is generally accepted that the deltoid muscles will generate less force in the prostheses with respect to the natural shoulder. While this certainly held true, the novel DBSP model also revealed results similar to the RSP. This indicates that, under the made assumptions, the new design is better, or, at the very least not inferior to the conventionally available RSP design when assessing demands placed on the shoulder muscles. Overall, these results indicate that the novel DBSP design can be a feasible alterna- tive for patients suffering from severe rotator cuff arthropathy, following which rota- tor cuff muscle activity is severely compromised. f The values of the muscle activation reported in Table 1 imply that all of the mus- cle groups in the three models have generated efforts below 50% of their maximum capacity. Although the middle deltoid forces and the activations for the DBSP model are higher than for the RSP model, the forces and activations for rotator cuff muscles are lower for the DBSP model compared to the RSP model. This implies that the mid- dle deltoid in the DBSP model bears more load during arm motion to reduce forces in the rotator cuff muscles. This finding further supports the earlier statement that DBSP can be used for the treatment of patients with severe rotator cuff tear, since any weakness in the rotator cuff muscle group is compensated by the higher activity and greater force-generation in the middle deltoid muscle. In reality, the glenoid ring is just supported to the glenoid. Discussion However, the acromion, the coracoid process, and the surrounding muscles limit the motion of the ring within the shoulder joint complex. One shortcoming of this investigation is that the computer simulation predicts the behavior just after implantation and does not consider the effect of healing and muscle relaxation with the time. Furthermore, this study did not take into account patient-specific anatomic differences that may also affect muscle forces and JRF [10]. This limitation is not crucial, as the study was more focused on comparing the bio- mechanical behavior of the novel prosthesis with the RSP and the NS, therefore addi- tional anatomic variances would have gone beyond the scope of this study, and would have reduced the significance of the afore-mentioned results. i Although preliminary tests on cadavers with inserted dual-bearing prostheses revealed good functionality of the DBSP without dislocation, further tests will need to be conducted to quantify and confirm the stability of the novel design during different movements, which could not be addressed with the computer simulations. Despite the limitations of a computational study, as mentioned above, it should be noted that the results of this initial step in the evaluation of the DBSP are promis- ing. The results of this study can provide the basis for future investigations, includ- ing in vivo studies wherein the kinematics of the shoulder joint containing the novel prosthesis can be recorded using techniques such as motion capture or videometry. This dataset can then be further compared to the performance of the natural shoulder and reverse shoulder prostheses to help gain a better understanding of the behavior of this novel prosthesis in vivo. With the help of future validation studies and a proper understanding of how to incor- porate computer simulation testing in biomechanics, new prostheses and innovative design variants in TSA can be explored and optimized before their use in clinical trials. Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 11 of 19 Conclusion This research presents a biomechanical comparison of generic musculoskeletal shoulder models, including a NS model and two models including a shoulder prosthesis. Further- more, the study shows that the novel dual-bearing shoulder prosthesis can be a promis- ing option for patients with rotator cuff arthropathy because of its lower rotator cuff muscle forces during all four arm motions compared to the reverse shoulder prosthesis. Peak values of deltoid muscle force for reverse and dual-bearing designs were in a simi- lar range during abduction and scaption. Low peak muscle activation values in all mod- els for anterior, middle and posterior deltoids during the four motions reveals that the muscles were not investing high effort when holding 2 kg weight in the hand. Ultimately, this study demonstrates that computer simulation can be a useful tool in the develop- ment phase to assess the effects of prosthesis design on the biomechanics of the human musculoskeletal system. Model of the natural shoulder (NS) An existing OpenSim® 3D musculoskeletal model of an upper body incorporating the shoulder joint complex with all involved muscles was used as the NS model in this study (Fig. 4A and B). The model was originally built in SIMM (Software for Interac- tive Musculoskeletal Modeling, Motion Analysis Corporation, Rohnert Park, Califor- nia, [27]) by Blana et al. [28], using anatomical data from cadaver studies performed by Klein-Breteler et al. [36]. The well-validated generic model represents the right shoulder of an embalmed 57-year-old muscular man with an estimated height of 168 cm [36]. The model is composed of the following 8 rigid parts: head, thorax including spine, right clavicle, right scapula, right humerus, right ulna, right radius, and right hand (Fig. 4A). It incorporates a gleno-humeral ball–socket joint and a novel scapulo-thoracic joint plugin developed by Seth et al. [33]. The scapulo-thoracic joint plugin provides an improved representation of scapular and clavicular kinematics relative to the thorax during arm motion. The ligaments are not included in the model, as they do not significantly con- tribute to the joint reaction forces and the corresponding muscle forces [27, 28]. In the whole model, there are 29 muscle groups comprising a total of 138 muscle elements (Fig. 4B). The muscle groups acting over the gleno-humeral joint are shown in Fig. 5. The remaining muscles are listed in Table 2 as given by Blana et al. [28]. Each muscle is assumed as a tensile force-generating element. Muscle forces are calculated based on the Hill muscle model [37]. Major shoulder muscle groups consist of the deltoid group and the rotator cuff group. The deltoid group comprises three subgroups: anterior deltoid (or so-called deltoid clavicle, 4 muscle elements), middle deltoid (4 muscle elements), and posterior deltoid (7 muscle elements). The rotator cuff group comprises four subgroups: subscapularis (11 muscle elements), infraspinatus (6 muscle elements), teres minor (3 muscle elements) and supraspinatus (4 muscle elements). While the supraspinatus is included in the NS model, it is rendered inactive in the models with prosthesis, since this muscle is usually cut by the surgeon in order to enable placement of the implant during arthroplasty surgery. Tendons are implicitly included in the model as they are defined within the muscle model [27]. Page 12 of 19 Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Fig. 5 The deltoid and rotator cuff muscle groups acting over the gleno-humeral joint Fig. Model of the natural shoulder (NS) 5 The deltoid and rotator cuff muscle groups acting over the gleno-humeral joint Fig. 5 The deltoid and rotator cuff muscle groups acting over the gleno-humeral joint To develop the DBSP and RSP models, the NS model was then adapted, wherein the "natural" gleno-humeral joint was replaced by the virtual DBSP or RSP prosthetic joint respectively, as described in the next two sections. As in the original model, no fric- tion between any gliding surfaces was considered in all the three models, reducing the computational time significantly and increasing the numerical stability (convergence of results). This assumption relies on the low coefficient of friction within the natural joint and prostheses (below 0.05 between the UHMWPE and CoCr). An analytical calculation showed that the contribution of friction to the joint reaction moment is below 4% for the DBSP and RSP, see Appendix A. Hence, the contribution of friction is negligible, espe- cially for the present comparison purpose. Model including the dual‑bearing shoulder prosthesis (DBSP)h The dual-bearing shoulder prosthesis concept and design is based on two patents [25, 26]. The prosthesis is assembled by four components and is located between the Page 13 of 19 Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Table 2 Muscles (other than deltoid and rotator cuff muscle group) and the number of elements that represent each one that are included in the models of the DBSP, RSP, and NS Muscle Number of elements Trapezius, scapular part (mid and lower) 11 Trapezius, clavicular part (upper) 2 Levator scapulae 2 Pectoralis minor 4 Rhomboid 5 Serratus anterior 12 Coracobrachialis 3 Teres major 4 Biceps, long head 1 Biceps, short head 2 Triceps, long head 4 Triceps, medial head 5 Triceps, lateral head 5 Latissimus dorsi 6 Pectoralis major, thoracic part 6 Pectoralis major, clavicular part 2 Brachialis 7 Brachioradialis 3 Pronator teres 2 Supinator 5 Pronator quadratus 3 Anconeus 5 Table 2 Muscles (other than deltoid and rotator cuff muscle group) and the number of elements that represent each one that are included in the models of the DBSP, RSP, and NS Muscle Number of elements Trapezius, scapular part (mid and lower) 11 Trapezius, clavicular part (upper) 2 Levator scapulae 2 Pectoralis minor 4 Rhomboid 5 Serratus anterior 12 Coracobrachialis 3 Teres major 4 Biceps, long head 1 Biceps, short head 2 Triceps, long head 4 Triceps, medial head 5 Triceps, lateral head 5 Latissimus dorsi 6 Pectoralis major, thoracic part 6 Pectoralis major, clavicular part 2 Brachialis 7 Brachioradialis 3 Pronator teres 2 Supinator 5 Pronator quadratus 3 Anconeus 5 Table 2 Muscles (other than deltoid and rotator cuff muscle group) and the number of elements that represent each one that are included in the models of the DBSP, RSP, and NS Muscle Anconeus Fig. 6 Principal parts of the dual-bearing shoulder prosthesis (DBSP) assembly: glenoid ring (A), PE-bearing (B); ball head (C); and offset adapter (D) (left), DBSP shown in an explosion graphic (right) Fig. 6 Principal parts of the dual-bearing shoulder prosthesis (DBSP) assembly: glenoid ring (A), PE-bearing (B); ball head (C); and offset adapter (D) (left), DBSP shown in an explosion graphic (right) Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 14 of 19 humerus and the glenoid. For easier reference, the parts are designated with letters A–D, see Fig. 6. humerus and the glenoid. For easier reference, the parts are designated with letters A–D, see Fig. 6. Before being implanted, the ball head (C) is mounted in the PE-bearing (B). Then, the B–C assembly is inserted in the glenoid ring (A). A snap-in system avoids the unlocking between the B and A parts. The offset adapter (D) is then connected to the part C by press-fitting and secured with a screw. Finally, the A–B–C–D assembly is fixed to the humerus stem by press-fitting and secured with a screw and is supported proximally to the following bodies: medially by the glenoid (lateral region of the scapula, which nor- mally supports the humerus head), superiorly by the acromion and anteriorly by the coracoid. Although A and C parts are made of cobalt–chromium (CoCr) alloy, part B of polyethylene, and part D and the humerus stem of titanium (Ti) alloy, all components are assumed as rigid bodies in the model. Part B has a hinge joint in respect to part A which means that part B rotates about the central axis of part A. Part C is connected to part B with restriction due to the engagement of the ball head protrusion in the groove of part B. Furthermore, part C can also rotate about the central axis of its protrusion. Thus, the connection between part C and part B represents a gimbal joint. The scap- ula–A, C–D and D–humerus stem connections are assumed as welded together (fixed joint) in the model. The fixed joint between scapula and part A allows the analysis of the joint reaction forces (JRF) between scapula and glenoid ring in OpenSim®. In reality, the DBSP does not require glenoid component fixation, it is placed without anchorage. The stabilization is ensured by the acromion, the coracoid process, and the muscles. Muscle The prosthesis system possesses two CORs, one of the joints between the PE-bearing (B) and the glenoid ring (A) and the other between the ball head (C) and the PE-bearing (B). Both CORs are offset from each other. The connections between the parts of the prosthesis and between the prosthesis and the skeleton in the dual-bearing model are summarized in Table 3. The components of the implant are produced in different sizes in order to allow a best fit with the patient’s anatomy. For this comparative study, the prosthesis with a 55 mm outer diameter of the glenoid ring was chosen with a medium-sized (M) offset adapter. An ellipsoid wrapping surface with principle axes diameters of 75 mm, 75 mm and 28 mm was used in the model to avoid a penetration of the muscles into the DBSP. The wrapping surface provides a virtual deflection support for the middle line of the muscle. In order to deflect the deltoid muscle elements realistically, the diameter of the implant wrap surface was increased by half of the thickness of deltoid muscle elements Table 3 Joint type and connections in the dual-bearing shoulder prosthesis (DBSP) model DOF degrees of freedom, PE polyethylene Connecting parts Type of joint Degree of freedom (DOF) Parent Child Scapula Glenoid disk Fixed joint 0 Glenoid disk PE-bearing (inlay) Hinge joint 1 rotational PE-bearing (inlay) Ball head with a protrusion Gimbal joint 2 rotational Ball head with a protrusion Offset adapter Fixed joint 0 Offset adapter Humerus Fixed joint 0 Table 3 Joint type and connections in the dual-bearing shoulder prosthesis (DBSP) model DOF degrees of freedom, PE polyethylene Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 15 of 19 (estimated to 20 mm). The glenoid ring was fixed to the glenoid fossa and tilted 10° downwards to the glenoid plane (Fig. 7A), similar to the RSP. Model including the reverse shoulder prosthesis (RSP) Basically, the model is built up from a ball-and-socket joint. The RSP was kept in the recommended position for surgery [38–40] in the model as shown in Fig. 7B. A humeral cup was positioned at a suitable angle to the humeral shaft axis in such a way that the prosthesis and other segments do not interfere each other. The COR of the glenosphere was placed in the anteroposterior midline of the glenoid face, 18 mm above the inferior edge of the glenoid rim. As the real glenosphere of hemispherical shape had a diam- eter of 36 mm, a spherical wrapping surface with a diameter of 56 mm was used for this implant in the model, again considering the deltoid muscle thickness for realistic deflec- tion of the muscle, similar to the model including the DBSP. Simulated motions For the three models, four shoulder motions were simulated: abduction 0°–120°, scaption 0°–120°, internal rotation 0°–40° and external rotation 0°–40°. The inverse kinematics (IK) approach was used to derive rotational joint kinematics from ana- tomical surface marker-based motion capture data of a single subject, named subject S4, from the shoulder movement database available on SimTK.org [41]. The motions of the markers applied on the subject were measured at 100 Hz. The used mark- ers from the motion capture were duplicated as landmarks in the OpenSim® (V 4.0) model. The motion capture data were then used to drive the model in order to repli- cate the four shoulder motions. The static optimization (SO) method was applied to obtain muscle forces for individual muscle elements for the given shoulder motion. In the SO method, the muscle forces are resolved by minimizing the sum of squared Fig. 7 Dual-bearing shoulder prosthesis integrated in the model (A), reverse shoulder prosthesis integrated in the model (B) Fig. 7 Dual-bearing shoulder prosthesis integrated in the model (A), reverse shoulder prosthesis integrated in the model (B) Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 16 of 19 muscle activations, which are obtained based on muscle–tendon length–velocity dynamics at the particular time point during the simulation movement [27]. The IK motion results were noisy with sudden jumps, which resulted in unrealistic accelera- tions and constraint violation. Therefore, the SO solver failed to solve the equation for the muscle forces. IK results could have been filtered before using the SO solver to overcome the afore-mentioned problem. However, data filtering could have also altered the marker dataset and the filtered marker positions may differ from the real marker positions. Therefore, in order to achieve convergence of the results, the time scale of the original dataset was stretched by a factor of 1000. In that way, the sud- den acceleration jumps/noise present in the data were reduced by the same factor, without the need to filter the original data. Hence, each motion used to calculate muscle forces and JRFs in all the models resembles a quasi-static motion. No inertial forces were considered. All three models used the same basic set of muscles. The main physical muscle parameters, like the maximum isometric force, optimal fiber length, tendon slack length and the location of the insertion and origin points of the muscles, were taken unchanged from the NS model. Simulated motions Actuators were added to the thorax in all the models to better follow the driven motion. Elbow and wrist were kept fixed during the sim- ulations. The supraspinatus muscle was deactivated during simulation on RSP and DBSP models, considering that supraspinatus is usually impaired during surgery. Besides the self-weight of the body parts, a weight of 2 kg rigidly attached to the hand, simulating a held object, was defined for the simulation of the different arm motions in each of the three models. The starting position of IR and ER is the palm of the hand looking to posterior. Validation of the natural shoulder (NS) model In order to validate our model, the natural shoulder (NS) model simulation results for abduction motion were compared to a unique experimental study by Bergmann et al. Second, JRF results for abduction and scaption motion from our reverse shoul- der prosthesis model simulation were also compared to results reported by Constan- tini et al. [35] for the Newcastle reverse shoulder model. The shoulder joint reaction forces were calculated for the NS model with 2 kg load in the hand and compared with the in vivo gleno-humeral joint loads measured on patients using an instru- mented shoulder prosthesis [34, 42]. The maximal JRF at 90° abduction for the NS model amounts 1054 N, which is 9.9% higher than reported by Bergmann et al. [34] for slow motions. Notice, that the Bergmann’s study shows a strong variability from subject to subject. The JRF of our NS model is in the mid-range (i.e., 143%) of the results of the study varying between 69%BW and 188%BW. Besides this, joint reaction forces during abduction and scaption for the Newcastle reverse shoulder model used by Costantini et al. [18] and the reverse shoulder model used in this study were also found in a similar range for different lateral positions of the implant. Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Page 17 of 19 Appendix A The considered material combination and surface finish used for the DBSP ensure a low friction of about μ = 0.05 (UHMWPE against CoCr alloy). The maximal JRF in the COR of the prosthesis in abduction amounts to approximately 1000 N, thus the con- tribution of friction to the moment acting around the COR of the prosthesis is: Mf = μ * JRF * r, with μ friction UHMWPE–CoCr alloy (0.05); JRF: joint reaction force (~ 1000 N, see Table 1); r: moment arm for the friction force which is equal to the radius of the pros- thesis (DBSP: 12.5 mm, RSP: 18 mm); Mf DBSP = 0.05 * 1000 * 12.5 = 625 Nmm or 0.625 Nm; Mf RSP = 0.05 * 1000 * 18 = 900 Nmm or 0.900 Nm. The moment arising from the weight of the arm and the 2 kg weight in the hand is (Fig. 8): Mext = Mgl + mgL = 4.275 * 9.81 * 0.3 + 2 * 9.81 * 0.6 = 24.35 Nm, with M: mass of arm (4.275 kg, see [42]); m: mass in the hand (2 kg); l: moment arm of the arm weight (~ 0.3 m); L: moment arm of the hand (~ 0.6 m); g: gravity (9.81 m/s2); Mext is independent of the prosthesis type. The contribution of the friction Cf in the total moment acting around the COR of the corresponding prosthesis is: Cf DBSP = Mf DBSP / Mext = 0.625 Nm / 24.35 Nm = 2.6%, Cf DBSP = Mf DBSP / Mext = 0.625 Nm / 24.35 Nm = 2.6%, Cf RSP = Mf RSP / Mext = 0.900 Nm / 24.35 Nm = 3.7%. Cf RSP = Mf RSP / Mext = 0.900 Nm / 24.35 Nm = 3.7%. The contribution of friction to the joint reaction moment is < 4% for both prosthe- ses, the DBSP and RSP, thus its contribution can be neglected. Fig. 8 OpenSim® shoulder model used for the estimation of the contribution of the friction on the total moment around the gleno-humeral joint and the prosthesis COR, respectively. The axis around which the moment is calculated for abduction is shown with dash-dotted line, the arm is in horizontal position (90°). Fig. 8 OpenSim® shoulder model used for the estimation of the contribution of the friction on the total moment around the gleno-humeral joint and the prosthesis COR, respectively. Acknowledgements g This study was carried out in collaboration with 41hemiverse AG. We gratefully acknowledge the support in modelling of Malavika Harikrishnan and Gabriel Barreto de Oliveira, both engineering students and trainees at Empa in 2016 and 2017, respectively. Consent for publication p The authors certify that this paper is original and unpublished and is not being considered for publication elsewhere and if accepted will not be published elsewhere in the same form, in English or in any other language. Availability of data and materials Availability of data and materials Not applicable/the generated models of the current study are available in the OpenSim repository. Appendix A The axis around which the moment is calculated for abduction is shown with dash-dotted line, the arm is in horizontal position (90°). Page 18 of 19 Weisse et al. BioMedical Engineering OnLine (2022) 21:17 Abbreviations ASP: Anatomical shoulder prosthesis; BW: Body weight; COR: Center of rotation; DBSP: Dual-bearing shoulder prosthesis; DOF: Degree of freedom; IK: Inverse kinematics; JRF: Joint reaction force; NS: Natural shoulder; PE: Polyethylene; ROM: Range of motion; RSP: Reverse shoulder prosthesis; RTSA: Reverse total shoulder arthroplasty; SO: Static optimization; SIMM: Software for Interactive Musculoskeletal Modeling; TSA: Total shoulder arthroplasty. Competing interests Competing interests We certify that there were no financial and personal relationships with hemiverse41 or organizations that could inap- propriately influence our work. Received: 29 April 2021 Accepted: 7 March 2022 Received: 29 April 2021 Accepted: 7 March 2022 Declarations Ethics approval and consent to participate Not applicable. 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https://openalex.org/W2414478892	https://air.unimi.it/bitstream/2434/457526/2/PhysRevLett.117.182002.pdf	English	null	Measurement of the Inelastic Proton-Proton Cross Section at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>13</mml:mn><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mi>TeV</mml:mi></mml:math> with the ATLAS Detector at the LHC	Physical review letters	2,016	cc-by	23,307	week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC M. Aaboud et al.* (ATLAS Collaboration) (Received 9 June 2016; published 26 October 2016) This Letter presents a measurement of the inelastic proton-proton cross section using 60 μb−1 of pp collisions at a center-of-mass energy ﬃﬃﬃs p of 13 TeV with the ATLAS detector at the LHC. Inelastic interactions are selected using rings of plastic scintillators in the forward region (2.07<jηj<3.86) of the detector. A cross section of 68.1 1.4 mb is measured in the fiducial region ξ¼M2 X=s>10−6, where MX is the larger invariant mass of the two hadronic systems separated by the largest rapidity gap in the event. In this ξ range the scintillators are highly efficient. For diffractive events this corresponds to cases where at least one proton dissociates to a system with MX >13GeV. The measured cross section is compared with a range of theoretical predictions. When extrapolated to the full phase space, a cross section of 78.1 2.9 mb is measured, consistent with the inelastic cross section increasing with center-of-mass energy. DOI: 10.1103/PhysRevLett.117.182002 The rise of the total proton-proton (pp) cross section with center-of-mass energy ﬃﬃﬃs p , predicted by Heisenberg [1] and observed at the CERN Intersecting Storage Rings [2], probes the nonperturbative regime of quantum chromodynamics (QCD). Arguments based on unitarity, analyticity, and factorization imply an upper bound on the high-energy behavior of total hadronic cross sections that prevents them from rising more rapidly than ln2ðsÞ [3–5]. diffractive dissociation processes in which neither proton dissociates into a system, X, of mass MX > 13 GeV, or equivalently, ξ ¼ M2 X=s > 10−6. The cross-section meas- urement is reported in this fiducial region, ξ > 10−6, and after extrapolation to the total inelastic cross section using models of inelastic interactions. The ATLAS detector is a cylindrical particle detector composed of several subdetector layers [17]. The inner tracking detector (ID) is immersed in a 2 T magnetic field provided by a superconducting solenoid. Around the tracker is a system of electromagnetic and hadronic calorimeters, which use liquid argon and lead, copper, or tungsten absorber for the electromagnetic and forward (jηj > 1.7) [18] hadronic components of the detector, and scintillator-tile active material and steel absorber for the central (jηj < 1.7) hadronic component. Many experiments have measured σinel and found an increase with ﬃﬃﬃs p [6]. Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distri- bution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Full author list given at the end of the article. Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distri- bution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Full author list given at the end of the article. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC To facilitate back- ground studies, data were also collected with the same selection when no proton bunch (“empty”) or a single proton bunch from only one of the two beams (“single 182002-1 © 2016 CERN, for the ATLAS Collaboration 0031-9007=16=117(18)=182002(19) week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) beam”) was passing through the center of ATLAS. All of these triggers require at least one MBTS hit above thresh- old. Two additional triggers were used to collect data to determine the MBTS trigger efficiency, requiring either hits in a forward (5.6 < jηj < 5.9) Cherenkov detector (LUCID) or a far forward (jηj > 8.4) tungsten-scintillator calorimeter detector (LHCf [19]) located at z ¼ 17 m and 140 m, respectively. The LHCf detector is an indepen- dent detector, but for the runs considered in this analysis, its trigger signals were incorporated into the ATLAS readout. The fiducial region of the measurement is determined using MC simulation. In each generated event, the largest rapidity gap between any two final-state hadrons is used to define the boundary between two collections of hadrons. These collections define the dissociation systems in an event-generator-independent manner. The invariant mass of each collection is calculated, and the larger of the two masses, denoted MX, is used to define ξ ¼ M2 X=s. The variable ξ is constrained to be >6 × 10−9 by the elastic limit of m2p=s where mp is the proton mass. This measurement is restricted to ξ > 10−6, the region in which the event selection efficiency exceeds 50%. Monte Carlo (MC) simulation samples were produced to correct the fiducial measurement and to compare to the data. The detector response is modeled using a simulation based on GEANT4 [20–22]. The data and MC simulated events are passed through the same reconstruction and analysis software. y Two samples of data events passing the MBTS trigger requirements are selected: an inclusive sample and a single- sided sample. The inclusive selection requires at least two MBTS counters with a charge above 0.15 pC (nMBTS ≥2). This threshold is chosen to be well above the electronic noise level of the counters. Requiring two hits rather than one substantially reduces background due to collision- induced radiation and activation. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC To constrain the diffrac- tive component of the cross section and reduce the uncertainty in extrapolation to σinel, an additional single- sided selection is defined, requiring hits in at least two counters on one side of the detector and no hits on the other. In the data, 4 159 074 events pass the inclusive selection and 442 192 events pass the single-sided selection. The primary MC samples are based on the PYTHIA8 generator [23,24] either with the A2 [25] set of tuned underlying-event parameters and the MSTW 2008 LO PDF set [26] or with the Monash [27] set of tuned parameters and the NNPDF 2.3 LO PDF set [28]. The samples are divided into four components: single-dissociation (SD, pp →pX), double-dissociation (DD, pp →XY), central- dissociation (CD, pp →pXp), all involving colorless exchange, and nondiffractive dissociation (ND) wherein color flow is present between the two colliding protons. For all dissociation event types, the Monash tune is used. The fiducial cross section is determined by PYTHIA8 uses a pomeron-based diffraction model [29] to describe colorless exchange with a default pomeron flux model by Schuler and Sjöstrand (SS) [30,31]. Alternative MC samples are generated with the pomeron flux model of Donnachie and Landshoff (DL) [32] and with the minimum-bias Rockefeller (MBR) model [33]. In the DL model, the pomeron Regge trajectory is given by αðtÞ ¼ 1 þ ε þ α0t, where ε and α0 are free parameters. In most samples used for this analysis, the value of α0 is 0.25, the PYTHIA8 default. The ε parameter is varied from 0.06 to 0.10 (the PYTHIA8 default is 0.085). An additional sample produced with α0 ¼ 0.35 is found to be statistically con- sistent with the α0 ¼ 0.25 default samples in each aspect of thisanalysis. Therangesofε andα0 consideredaremotivated by previous total, inelastic, elastic, and diffractive cross- section measurements, including measurements of low- mass diffraction by the ATLAS and CMS collaborations [34,35]. For the DL and SS models the CD component is neglected. The MBR model is tuned to data as described in Ref. [33] and includes a small CD component. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC The TOTEM and ATLAS collabo- rations determined σinel at ﬃﬃﬃs p ¼ 7 and 8 TeV using the optical theorem and a measurement of the elastic cross section with Roman pot detectors [7–11]. Using a variety of alternative techniques, the ATLAS, CMS, ALICE, and LHCb experiments have made measurements of σinel at ﬃﬃﬃs p ¼ 7 TeV [12–15] and ﬃﬃﬃs p ¼ 2.76 TeV (ALICE) [14]. The Pierre Auger Collaboration measured the inelastic p-air cross section at ﬃﬃﬃs p ¼ 57 TeV and extracted σinel using the Glauber model [16]. At z ¼ 3.6 m, thin plastic scintillation counters, the minimum-bias trigger scintillators (MBTS), are installed on the front face of each endcap calorimeter. These detectors cover the region 2.07 < jηj < 3.86. They are similar to those described in Ref. [17] but were rebuilt during 2014, when the coverage was slightly extended from 2.08 < jηj < 3.75 after the ﬃﬃﬃs p ¼ 7 TeV run. The MBTS are divided into inner (4 counters in 149 < r < 445 mm) and outer (8 counters in 444.5 < r < 895 mm) octagonal rings. This Letter presents a measurement of the inelastic cross section σinel using pp collisions at ﬃﬃﬃs p ¼ 13 TeV with the ATLAS detector at the Large Hadron Collider (LHC). It is performed using two sets of scintillation counters in a data set corresponding to an integrated luminosity of 60.1 1.1 μb−1 collected in June 2015. In inelastic interactions, one or both protons dissociate as a result of colored (nondiffractive) or colorless (diffractive) exchange. The counters are insensitive to elastic pp scattering and The ATLAS experiment uses a multistage trigger to select events at about 1 kHz for offline analysis. Three trigger configurations were used to collect data for this analysis. The primary triggers use the MBTS detector and constant-fraction discriminators to select events when two proton bunches collide in the detector. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC I E W L E T T E R S week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) f 0.1 0.15 0.2 0.25 0.3 0.35 0.4 SS R 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Data 2015 Pythia8 SS =0.085 ε Pythia8 DL, =0.060 ε Pythia8 DL, =0.10 ε Pythia8 DL, Pythia8 MBR EPOS LHC QGSJET-II ATLAS -1 b μ =13 TeV, L=60.1 s agrees with that in the colliding bunches to within 10%. The radiation and activation-induced backgrounds are implicitly part of this background estimate. Double- counting of these components is removed using estimates from empty events. The total background contributions to the inclusive and single-sided data samples are determined to be 1.2% and 5.8%, respectively. The classification of single-sided events as double-sided due to noise or other backgrounds is estimated to be below 0.1%. A systematic uncertainty of 50% is assigned to the background based on studies of the background composition and the relative contributions of the background components. This uncer- tainty is treated as fully correlated between the single-sided and inclusive selections. FIG. 1. The ratio of the number of single-sided to inclusive events (RSS) as a function of the fraction of the cross section that is diffractive according to each model (fD). The default value of fD in each model is shown with a marker. The trigger efficiency for events passing the inclusive selection, ϵtrig, is measured with respect to events selected with the LUCID detector after subtracting the background. A trigger efficiency of 99.7% (97.4%) is measured for the inclusive (single-sided) event sample. In both cases the statistical uncertainty is below 0.1%. The efficiency is also measured with events selected by the LHCf detector and agrees within 0.3% with the LUCID determination. This difference is taken as a systematic uncertainty. with the DL models, particularly in the low-nMBTS range. The MBR-based distribution provides a slightly worse description of the data. The PYTHIA8 sample using the SS model does not describe data well in the low- multiplicity region. EPOS LHC and QGSJET-II also do not describe the data well, particularly in the single-sided hit multiplicity distribution. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC Therefore, the PYTHIA8 DL model with ε ¼ 0.085 is chosen as the nominal MC model for the ϵsel and fξ<10−6 corrections, and only the DL and MBR models are considered for systematic uncertainties related to the MC corrections. The ratio of the number of events passing the single- sided event selection to the number passing the inclusive selection (RSS) is used to adjust, for each model, the fractional contribution of the single- and double-diffractive dissociative cross section (σSD þ σDD) to the inelastic cross section, fD ¼ ðσSD þ σDDÞ=σinel [12]. The measured value is RSS ¼ 10.4% with a total uncertainty of 0.4%. The dominant systematic uncertainty arises from the back- ground subtraction in the single-sided sample. For each MC model, fD is varied until it matches the observed RSS value in data. The data uncertainty is used to set the error in the constrained fD for each model. An additional uncer- tainty in the ratio of single- to double-diffractive events is determined by taking the diffractive events to be entirely SD or to be evenly divided between SD and DD. The event selection efficiency, ϵsel, depends upon the MBTS counter sensitivity. This sensitivity is tested using isolated charged particles, reconstructed as ID tracks in the region 2.07 < jηj < 2.5 where the coverages of the MBTS and ID overlap. Over the full coverage of the MBTS counters, the calorimeter is used to measure the counter efficiency with respect to particles that deposit sufficient energy in the calorimeter to seed a topological energy cluster [39]. Differences between the efficiencies in data and MC simulation are accounted for by adjusting the MBTS charge threshold in MC simulation until the simulated efficiencies match those observed in the data. The residual uncertainty in the counter efficiency after these corrections is 0.5% for the outer and 1.0% for the inner counters. Additionally, an uncertainty arising from the knowledge of the material in front of the MBTS detector is estimated using MC samples with an increased amount of material in front of the MBTS. Based on the MC samples, the uncertainty in the efficiency measurement due to modeling of hadronization and the underlying event is estimated to be negligible. Using this method, the fitted fD in the PYTHIA8 samples is between 25% and 31%, depending on the model (the default value is 28%). Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC σfid inelðξ > 10−6Þ ¼ N −NBG ϵtrig × L × 1 −fξ<10−6 ϵsel ; ð1Þ ð1Þ where N is the number of observed events passing the inclusive selection, NBG is the number of background events, ϵtrig and ϵsel are factors accounting for the trigger and event selection efficiencies, 1 −fξ<10−6 accounts for the migration of events with ξ < 10−6 into the fiducial region, and L is the integrated luminosity of the sample. g g y p Sources of background include interactions between the beam and residual gas in the beam pipe; interactions between the beam and collimators upstream of the detector, which can send charged particles through the detector parallel to the beam; collision-induced radiation; and activation backgrounds. Backgrounds from cosmic rays and instrumental noise are negligible. The mean number of pp collisions in the same LHC bunch crossing was 2.3 × 10−3 for the recorded data set. Thus, the contribution from multiple collisions is also negligible. The beam- related background components are extracted from sin- gle-beam events and dominate the total background. They are normalized by scaling the number of selected single- beam events by a factor of 37=4 × 2, accounting for the 37 colliding pairs of bunches and 4 bunches producing the single-beam data in this run. The factor of 2 accounts for the presence of two colliding bunches. The number of protons per bunch producing these single-beam events The EPOS LHC and QGSJET-II event generators are also used to simulate pp collisions. EPOS LHC [36] uses a “cut pomeron” model for diffraction and differs significantly from PYTHIA8 in its modeling of hadronization and the underlying event. QGSJET-II [37,38] uses Reggeon field theory to describe pomeron-pomeron interactions. Both EPOS LHC and QGSJET-II have been developed primarily to model cosmic-ray showering in the atmosphere. 182002-2 Df 0.1 0.15 0.2 0.25 0.3 0.35 0.4 SS R 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Data 2015 Pythia8 SS =0.085 ε Pythia8 DL, =0.060 ε Pythia8 DL, =0.10 ε Pythia8 DL, Pythia8 MBR EPOS LHC QGSJET-II ATLAS -1 b μ =13 TeV, L=60.1 s FIG. 1. The ratio of the number of single-sided to inclusive events (RSS) as a function of the fraction of the cross section that is diffractive according to each model (fD). The default value of fD in each model is shown with a marker. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC The models shown here use the fD value determined from the RSS measurement. MBTS n d events n d events n 1 2 − 10 1 − 10 1 Data Pythia8 SS = 0.06 ε Pythia8 DL, = 0.085 ε Pythia8 DL, = 0.10 ε Pythia8 DL, MBR EPOS LHC QGSJET-II ATLAS -1 b μ 13 TeV, 60.1 Inclusive selection MBTS n 2 4 6 8 10 12 14 16 18 20 22 24 MC/data 0.5 1 1.5 TABLE I. Inputs to the calculation of the measured cross section and their systematic uncertainties. TABLE I. Inputs to the calculation of the measured cross section and their systematic uncertainties. Factor Value Relative uncertainty Number of events passing the inclusive selection (N) 4 159 074 Number of background events (NBG) 51 187 50% Integrated luminosity [μb−1] (L) 60.1 1.9% Trigger efficiency (ϵtrig) 99.7% 0.3% MC correction factor (CMC) 99.3% 0.5% ATLAS -1 b μ 13 TeV, 60.1 Inclusive selection -1 b μ 13 TeV, 60.1 Inclusive selection variation in CMC found by comparing the PYTHIA8 DL and MBR models. Of these sources of uncertainty, the last is most important at 0.5%. The value of CMC is ð99.3 0.5Þ%. The uncertainty also implicitly contains an uncertainty due to the CD contribution, since this is included in only some of the models. MBTS n d events n d events n 1 1 − 10 Data Pythia8 SS = 0.06 ε Pythia8 DL, = 0.085 ε Pythia8 DL, = 0.10 ε Pythia8 DL, MBR EPOS LHC QGSJET-II ATLAS -1 b μ 13 TeV, 60.1 Single-sided selection MBTS n 2 4 6 8 10 12 MC/data 0.5 1 1.5 The uncertainty in the integrated luminosity is 1.9%. It is derived, following a methodology similar to that detailed in Refs. [40,41], from a calibration of the luminosity scale using x-y beam-separation scans performed in August 2015. This calibration uncertainty is slightly smaller than what has been reported in Ref. [42] because the low- luminosity data set used in this Letter is not affected by the uncertainties related to high-luminosity runs. The components of the fiducial cross-section calculation [Eq. (1)] are shown in Table I with their systematic uncertainties. The statistical uncertainties are negligible. The measured fiducial cross section is determined to be σfid inel ¼ 68.1 0.6ðexpÞ 1.3ðlumÞ mb; FIG. 2. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC For the QGSJET-II (EPOS LHC) model the fitted fD is 35% (37%), differing significantly from the default value of 21% (28%). The observed RSS and the MC predictions of its dependence on fD are shown in Fig. 1. The fitted fD is used when determining the acceptance corrections ϵsel and fξ<10−6 for each model. ξ In Fig. 2 the nMBTS distributions in data are compared to the ones from MC simulated samples utilizing the fitted fD values for both the inclusive and single-sided selections. The estimated background is subtracted from the measured distribution, and the trigger efficiency measured in data is applied to the simulation. The data distributions and MC simulation are peaked at high multiplicity values. In the single-sided case, nMBTS ¼ 12 corresponds to hits in all counters on one side of the detector. The data agree best After adjusting the counter charge threshold, ϵsel is determined from the nominal PYTHIA8 DL MC simula- tions, using the fitted fD corresponding to this model, to be 99.34% with a statistical uncertainty of 0.03%. The uncertainty in the MBTS counter efficiencies results in 182002-3 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) MBTS n d events n d events n 1 2 − 10 1 − 10 1 Data Pythia8 SS = 0.06 ε Pythia8 DL, = 0.085 ε Pythia8 DL, = 0.10 ε Pythia8 DL, MBR EPOS LHC QGSJET-II ATLAS -1 b μ 13 TeV, 60.1 Inclusive selection MBTS n 2 4 6 8 10 12 14 16 18 20 22 24 MC/data 0.5 1 1.5 MBTS n d events n d events n 1 1 − 10 Data Pythia8 SS = 0.06 ε Pythia8 DL, = 0.085 ε Pythia8 DL, = 0.10 ε Pythia8 DL, MBR EPOS LHC QGSJET-II ATLAS -1 b μ 13 TeV, 60.1 Single-sided selection MBTS n 2 4 6 8 10 12 MC/data 0.5 1 1.5 FIG. 2. The background-subtracted distribution of the number of MBTS counters (nMBTS) above threshold in data and MC simulation for (top) the inclusive selection and (bottom) the single-sided selection. The ratio of the MC models to the data is also shown. The experimental uncertainty is shown as a shaded band around the data points. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC The systematic uncertainties include the counter efficiency variations, the impact of the material uncertainty, the uncertainty in the fitted value of fD, and the ð2Þ The term σ7 TeVðξ < 5 × 10−6Þ ¼ σ7 TeV inel −σ7 TeVðξ > 5 × 10−6Þ ¼ 9.9 2.4 mb is the difference between σinel 182002-4 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) FIG. 3. The inelastic proton-proton cross section versus ﬃﬃﬃs p . Measurements from other hadron collider experiments [6,7,9,14,15] and the Pierre Auger experiment [16] are also shown. Some LHC data points have been slightly shifted in the horizontal position for display purposes. The data are compared to the PYTHIA8, EPOS LHC and QGSJET-II MC generator predictions. The uncertainty in the ATLAS ALFA measurement is smaller than the marker size. cross section increasing with center-of-mass energy, as observed at lower energies. cross section increasing with center-of-mass energy, as observed at lower energies. We thank CERN for the very successful operation of the LHC,aswellasthesupportstafffromourinstitutionswithout whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/ NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, FP7, Horizon 2020 and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, Région Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. The crucial computing support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA), the Tier-2 facilities worldwide and large non-WLCG resource providers. Major contributors of computing resources are listed in Ref. [48]. Swiss National Science Foundation FIG. 3. The inelastic proton-proton cross section versus ﬃﬃﬃs p . Measurements from other hadron collider experiments [6,7,9,14,15] and the Pierre Auger experiment [16] are also shown. Some LHC data points have been slightly shifted in the horizontal position for display purposes. The data are compared to the PYTHIA8, EPOS LHC and QGSJET-II MC generator predictions. The uncertainty in the ATLAS ALFA measurement is smaller than the marker size. measured at 7 TeVusing the ALFA detector [8], σ7 TeV inel , and σinel measured at 7 TeV for ξ > 5 × 10−6 using the MBTS [12] (The 7 TeV result is corrected upward by 1.9% following an improved luminosity calibration [40]). The uncertainties of the two measurements are uncorrelated. The PYTHIA8 DL and PYTHIA8 MBR MC samples are used to assess the systematic uncertainty in the MC-derived ratio of cross sections in Eq. (2), which is determined to be 1.015 0.081. (The value of the ratio arises from an approximately 20% increased cross section from increasing ﬃﬃﬃs p which is largely compensated by a 15% decrease due to the change in the ξ distribution.) These models also agree with the measurement of σ7 TeVðξ < 5 × 10−6Þ to within 2σ. The measured value for σinel is σinel ¼ 78.1 0.6ðexpÞ 1.3ðlumÞ 2.6ðextrapÞ mb: This and other inelastic cross-section measurements are compared to several Monte Carlo models in Fig. 3. Additional predictions range between 76.6 and 81.6 mb [43–47]. [1] W. Heisenberg, Production of mesons as a shock wave problem, Z. Phys. 133, 65 (1952). [2] U. Amaldi, R. Biancastelli, C. Bosio, G. Matthiae, J. V. Allaby, W. Bartel, G. Cocconi, A. N. Diddens, R. W. Dobinson, and A. M. Wetherell, The energy dependence of the proton-proton total cross-section for centre-of-mass Measurement of the Inelastic Proton-Proton Cross Section at ﬃﬃs p = 13 TeV with the ATLAS Detector at the LHC The background-subtracted distribution of the number of MBTS counters (nMBTS) above threshold in data and MC simulation for (top) the inclusive selection and (bottom) the single-sided selection. The ratio of the MC models to the data is also shown. The experimental uncertainty is shown as a shaded band around the data points. The models shown here use the fD value determined from the RSS measurement. where the first uncertainty refers to all experimental uncertainties apart from the luminosity and the second refers to the luminosity only. The PYTHIA8 DL model predicts values of 71.0 mb, 69.1 mb, and 68.1 mb for ε ¼ 0.06, 0.085, and 0.10, respectively, all of which are compatible with the meas- urement. The PYTHIA8 MBR model predicts 70.1 mb, also in agreement with the measurement. The EPOS LHC (71.2 mb) and QGSJET-II (72.7 mb) predictions exceed the data by 2–3σ. The PYTHIA8 SS model predicts 74.4 mb, and thus exceeds the measured value by ∼4σ. only a 0.1% uncertainty in the overall event selection efficiency, because many counters are hit in typical events. In addition, an uncertainty of 0.2% in ϵsel arises from the knowledge of the material in front of the MBTS. 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Beck,18,h K. Becker,120 M. Becker,84 M. Beckingham,169 C. Becot,110 A J Beddall 20e A Beddall 20b V A Bednyakov 66 M Bedognetti 107 C P Bee 148 L J Beemster 107 T A Beermann 32 emin,161 P. Bechtle,23 H. P. Beck,18,h K. Becker,120 M. Becker,84 M. Beckingham,169 C. Becot P. H. Beauchemin,161 P. Bechtle,23 H. P. Beck,18,h K. Becker,120 M. Becker,84 M. Beckingham,169 C. Becot,110 20e 20b 66 107 148 107 32 A. Beddall,20b V. A. Bednyakov,66 M. Bedognetti,107 C. P. Bee,148 L. J. Beemster,107 T. A. Bee M. Begel,27 J. K. Behr,44 C. Belanger-Champagne,88 A. S. Bell,79 G. Bella,153 L. Bellagamba,22a A. Bellerive,31 ,87 K. Belotskiy,98 O. Beltramello,32 N. L. P H Y S I C A L R E V I E W L E T T E R S Alexa,28b G. Alexander,153 T. Alexopoulos,10 M. Alhroob,113 B. Ali,128 M. Aliev,74a,74b G. Alimonti,92a J. Alison,33 S. P. Alkire,37 B. M. M. Allbrooke,149 B. W. Allen,116 P. P. Allport,19 A. Aloisio,104a,104b 38 72 138 86 32 166 I. N. Aleksandrov, C. Alexa, G. Alexander, T. Alexopoulos, M. Alhroob, B. Ali, M. Aliev, G. Alimonti,92a J. Alison,33 S. P. Alkire,37 B. M. M. Allbrooke,149 B. W. Allen,116 P. P. Allport,19 A. Aloisio,104a,104b A. Alonso,38 F. Alonso,72 C. Alpigiani,138 M. Alstaty,86 B. Alvarez Gonzalez,32 D. Álvarez Piqueras,166 M G Alviggi 104a,104b B T Amadio 16 K Amako 67 Y Amaral Coutinho 26a C Amelung 25 D Amidei 90 S. P. Amor Dos Santos,126a,126c A. Amorim,126a,126b S. Amoroso,32 G. Amundsen,25 C. Anastopoulos,139 L. S. Ancu,51 N A d i 19 T A d 11 C F A d 59b G A d 32 J K A d 75 K J A d 33 A A d 92a 92b V A d i 59a S. Angelidakis,9 I. Angelozzi,107 P. Anger,46 A. Angerami,37 F. Anghinolfi,32 A. V. Anisenkov,109,d N. Anjos,13 A. Annovi,124a,124b C. Antel,59a M. Antonelli,49 A. Antonov,98,a F. Anulli,132a M. Aoki,67 L. Aperio Bella,19 G. Arabidze,91 A. Annovi, C. Antel, M. Antonelli, A. Antonov, F. Anulli, M. Aoki, L. Aperio Bella, G. Arabidze, Y. Arai,67 J. P. Araque,126a A. T. H. Arce,47 F. A. Arduh,72 J-F. Arguin,95 S. Argyropoulos,64 M. Arik,20a A. J. Armbruster,143 Y. Arai,67 J. P. Araque,126a A. T. H. Arce,47 F. A. Arduh,72 J-F. Arguin,95 S. Argyropoulos,64 M. Arik,20a A. J. Armbruster,143 L. J. Armitage,77 O. Arnaez,32 H. Arnold,50 M. Arratia,30 O. Arslan,23 A. Artamonov,97 G. Artoni,120 S. Artz,84 S. Asai,155 N. Asbah,44 A. Ashkenazi,153 B. Åsman,146a,146b L. Asquith,149 K. Assamagan,27 R. Astalos,144a M. Atkinson,165 N. B. Atlay,141 K. Augsten,128 G. Avolio,32 B. Axen,16 M. K. Ayoub,117 G. Azuelos,95,e M. A. Baak,32 A. E. Baas,59a M. J. Baca,19 H. Bachacou,136 K. Bachas,74a,74b M. Backes,148 M. Backhaus,32 P. Bagiacchi,132a,132b P. Bagnaia,132a,132b M. J. Baca,19 H. Bachacou,136 K. Bachas,74a,74b M. Backes,148 M. Backhaus,32 P. Bagiacchi,132a,132b P. Bagnaia,132a,132b Y. Bai,35a J. T. Baines,131 O. K. Baker,175 E. M. Baldin,109,d P. Balek,171 T. Balestri,148 F. Balli,136 W. K. Balunas,122 E. Banas,41 Sw. Banerjee,172,f A. A. E. Bannoura,174 L. Barak,32 E. L. Barberio,89 D. Barberis,52a,52b M. Barbero,86 Y. Bai, J. T. Baines, O. K. Baker, E. M. Baldin, , P. Balek, T. Balestri, F. Balli, W. K. Balunas, E. Banas,41 Sw. Banerjee,172,f A. A. E. Bannoura,174 L. P H Y S I C A L R E V I E W L E T T E R S D’amen,22a,22b S. D’Auria,55 M. D’Onofrio,75 M. J. Da Cunha Sargedas De Sousa,126a,126b C. Da Via,85 W. Dabrowski,40a T. Dado,144a T. Dai,90 O. Dale,15 F. Dallaire,95 C. Dallapiccola,87 M. Dam,38 J. R. Dandoy,33 N. P. Dang,50 A. C. Daniells,19 N. S. Dann,85 M. Danninger,167 M. Dano Hoffmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W. Davey,23 C. David,168 T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8 ann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W. Davey,23 C. D 29 153 9 89 139 8 . , V. , G. , S. , J. , . g p , W. v y, C. v , T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8 104a 113 22a 22b 81 106 107 83 T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8 R de Asmundis 104a A De Benedetti 113 S De Castro 22a,22b S De Cecco 81 N De Groot 106 P de Jong 107 H De la Torre 83 29 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. D y 04a A. De Benedetti,113 S. De Castro,22a,22b S. De Cecco,81 N. De Groot,106 P. de Jong,107 H. De la Asmundis,104a A. De Benedetti,113 S. De Castro,22a,22b S. De Cecco,81 N. De Groot,106 P. de Jong g F. De Lorenzi,65 A. De Maria,56 D. De Pedis,132a A. De Salvo,132a U. De Sanctis,149 A. De Santo,149 J. B. De Vivie De Regie, W. J. Dearnaley, R. Debbe, C. Debenedetti, D. V. Dedovich, N. Dehghanian, I. Deigaard,107 M. Del Gaudio,39a,39b J. Del Peso,83 T. Del Prete,124a,124b D. Delgove,117 F. Deliot,136 C. M. Delitzsch,51 A. Dell’Acqua,32 L. Dell’Asta,24 M. Dell’Orso,124a,124b M. Della Pietra,104a,l D. della Volpe,51 M. Delmastro,5 P. A. Delsart,57 g y g I. Deigaard,107 M. Del Gaudio,39a,39b J. Del Peso,83 T. Del Prete,124a,124b D. Delgove,117 F. Deliot,136 C. M. Delitzsch,51 32 24 124a 124b 104a l 51 5 57 g y I. Deigaard,107 M. Del Gaudio,39a,39b J. Del Peso,83 T. Del Prete,124a,124b D. Del A. Dell’Acqua,32 L. Dell’Asta,24 M. Dell’Orso,124a,124b M. Della Pietra,104a,l D. de 182002-8 P H Y S I C A L R E V I E W L E T T E R S Cherkaoui El Moursli,135e V. Chernyatin,27,a E. Cheu,7 L. Chevalier,136 V. Chiarella,49 G. Chiarelli,124a,124b G. Chiodini,74a A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100 79 32 127 88 41 115 D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 S. Chekanov,6 S. V. Chekulaev,159a G. A. Chelkov,66,k M. A. Chelstowska,90 C. Chen,65 H. Chen,27 K. Chen,148 S. Chen,35c S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130 R. Cherkaoui El Moursli,135e V. Chernyatin,27,a E. Cheu,7 L. Chevalier,136 V. Chiarella,49 G. Chiarelli,124a,124b G. Chiodini,74a A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100 V. Christodoulou,79 D. Chromek-Burckhart,32 J. Chudoba,127 A. J. Chuinard,88 J. J. Chwastowski,41 L. Chytka,115 G. Ciapetti,132a,132b A. K. Ciftci,4a D. Cinca,45 V. Cindro,76 I. A. Cioara,23 C. Ciocca,22a,22b A. Ciocio,16 F. Cirotto,104a,104b Z. H. Citron,171 M. Citterio,92a M. Ciubancan,28b A. Clark,51 B. L. Clark,58 M. R. Clark,37 P. J. Clark,48 R. N. Clarke,16 C. Clement,146a,146b Y. Coadou,86 M. Cobal,163a,163c A. Coccaro,51 J. Cochran,65 L. Colasurdo,106 B. Cole,37 A. P. Colijn,107 J. Collot, T. Colombo, G. Compostella, P. Conde Muiño, E. Coniavitis, S. H. Connell, I. A. Connelly, V. Consorti,50 S. Constantinescu,28b G. Conti,32 F. Conventi,104a,l M. Cooke,16 B. D. Cooper,79 A. M. Cooper-Sarkar,120 K. J. R. Cormier,158 T. Cornelissen,174 M. Corradi,132a,132b F. Corriveau,88,m A. Corso-Radu,162 A. Cortes-Gonzalez,32 V. Consorti,50 S. Constantinescu,28b G. Conti,32 F. Conventi,104a,l M. Cooke,16 B. D. Cooper,79 A. M. Cooper-Sarkar,120 K. J. R. Cormier,158 T. Cornelissen,174 M. Corradi,132a,132b F. Corriveau,88,m A. Corso-Radu,162 A. Cortes-Gonzalez,32 G. Cortiana,101 G. Costa,92a M. J. Costa,166 D. Costanzo,139 G. Cottin,30 G. Cowan,78 B. E. Cox,85 K. Cranmer,110 K. J. R. Cormier, T. Cornelissen, M. Corradi, F. Corriveau, A. Corso Radu, A. Cortes Gonzalez, G. Cortiana,101 G. Costa,92a M. J. Costa,166 D. Costanzo,139 G. Cottin,30 G. Cowan,78 B. E. Cox,85 K. Cranmer,110 S J C l 55 G C 31 S C é é R di 57 F C i li 81 W A C ibb 146a,146b M C i i O t 120 S. J. Crawley,55 G. Cree,31 S. Crépé-Renaudin,57 F. Crescioli,81 W. A. Cribbs,146a,146b M. Crispin Ortuzar,120 M. Cristinziani,23 V. Croft,106 G. Crosetti,39a,39b A. Cueto,83 T. Cuhadar Donszelmann,139 J. Cummings,175 M. Curatolo,49 g J. Cúth,84 H. Czirr,141 P. Czodrowski,3 G. P H Y S I C A L R E V I E W L E T T E R S Cattai,32 J Caudron 23 V Cavaliere 165 E Cavallaro 13 D Cavalli 92a M Cavalli Sforza 13 V Cavasinni 124a,124b F Ceradini 134a,134b R. Castelijn,107 A. Castelli,107 V. Castillo Gimenez,166 N. F. Castro,126a,j A. Catinaccio,32 J. R. Catmore,119 A. Cattai,32 23 165 13 92a 13 124a 124b 134a 134b L. Cerda Alberich,166 B. C. Cerio,47 A. S. Cerqueira,26b A. Cerri,149 L. Cerrito,133a,133b F. Cerutti,16 M. Cerv,32 A. Cervelli,18 S. A. Cetin,20d A. Chafaq,135a D. Chakraborty,108 S. K. Chan,58 Y. L. Chan,61a P. Chang,165 J. D. Chapman,30 L. Cerda Alberich,166 B. C. Cerio,47 A. S. Cerqueira,26b A. Cerri,149 L. Cerrito,133a,133b F. Cerutti,16 M. Cerv,32 A. Cervelli,18 S. A. Cetin,20d A. Chafaq,135a D. Chakraborty,108 S. K. Chan,58 Y. L. Chan,61a P. Chang,165 J. D. Chapman,30 , q, y, , , g, p , D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 S. Chekanov,6 S. V. Chekulaev,159a G. A. Chelkov,66,k M. A. Chelstowska,90 C. Chen,65 H. Chen,27 K. Chen,148 S. Chen,35c S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130 R. Cherkaoui El Moursli,135e V. Chernyatin,27,a E. Cheu,7 L. Chevalier,136 V. Chiarella,49 G. Chiarelli,124a,124b G. Chiodini,74a A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100 V. Christodoulou,79 D. Chromek-Burckhart,32 J. Chudoba,127 A. J. Chuinard,88 J. J. Chwastowski,41 L. Chytka,115 G. Ciapetti,132a,132b A. K. Ciftci,4a D. Cinca,45 V. Cindro,76 I. A. Cioara,23 C. Ciocca,22a,22b A. Ciocio,16 F. Cirotto,104a,104b 1 1 92 28b 51 58 3 48 16 D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 S Chekanov 6 S V Chekulaev 159a G A Chelkov 66,k M A Chelstowska 90 C Chen 65 H Chen 27 K Chen 148 S Chen 35c D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 S. Chekanov,6 S. V. Chekulaev,159a G. A. Chelkov,66,k M. A. Chelstowska,90 C. Chen,65 H. Chen,27 K. Chen,148 S. Chen,35c S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130 R. P H Y S I C A L R E V I E W L E T T E R S Belyaev,98 O. Benary,153 D. Benchekroun,135a M. B M. Bellomo,87 K. Belotskiy,98 O. Beltramello,32 N. L. Belyaev,98 O. Benary,153 D. Benchekroun,135a M. Bender,100 K. Bendtz,146a,146b N. Benekos,10 Y. Benhammou,153 E. Benhar Noccioli,175 J. Benitez,64 D. P. Benjamin,47 J. R. Bensinger,25 107 120 49 107 164 5 16 K. Bendtz,146a,146b N. Benekos,10 Y. Benhammou,153 E. Benhar Noccioli,175 J. Benitez,64 D. P. Benjamin,47 J. R. Bensinger,25 nekos,10 Y. Benhammou,153 E. Benhar Noccioli,175 J. Benitez,64 D. P. Benjamin,47 J. R. Bensing 120 49 107 164 5 S. Bentvelsen,107 L. Beresford,120 M. Beretta,49 D. Berge,107 E. Bergeaas Kuutmann,16 57 87 110 23 78 129 en,107 L. Beresford,120 M. Beretta,49 D. Berge,107 E. Bergeaas Kuutmann,164 N. Berger,5 J. Be 87 110 23 78 129 84 S. Berlendis,57 N. R. Bernard,87 C. Bernius,110 F. U. Bernlochner,23 T. Berry,78 P. Berta,129 C. Bertella,84 G. Bertoli,146a,146b 124a 124b 73 44 113 38 146a 146b N. R. Bernard,87 C. Bernius,110 F. U. Bernlochner,23 T. Berry,78 P. Berta,129 C. Bertella,84 G. Be S. Berlendis,57 N. R. Bernard,87 C. Bernius,110 F. U. Bernlochner,23 T. Berry,78 P. Berta,129 C. Bertella,84 G. Bertoli,146a,146b F. Bertolucci,124a,124b I. A. Bertram,73 C. Bertsche,44 D. Bertsche,113 G. J. Besjes,38 O. Bessidskaia Bylund,146a,146b I. A. Bertram,73 C. Bertsche,44 D. Bertsche,113 G. J. Besjes,38 O. Bessidskaia Bylund,146a 136 50 57 101 77 125 M. Bessner,44 N. Besson,136 C. Betancourt,50 A. Bethani,57 S. Bethke,101 A. J. Bevan,77 R. M. Bianchi,125 L. Bianchini,25 32 100 17 85 124 124b 134 51 g T. R. V. Billoud,95 H. Bilokon,49 M. Bindi,56 S. Binet,117 A. Bingul,20b C. Bini,132a,132b S. Biondi,22a,22b T. Bisanz,56 D. M. Bjergaard,47 C. W. Black,150 J. E. Black,143 K. M. Black,24 D. Blackburn,138 R. E. Blair,6 J.-B. Blanchard,136 T. Blazek,144a I. Bloch,44 C. Blocker,25 W. Blum,84,a U. Blumenschein,56 S. Blunier,34a G. J. Bobbink,107 V. S. Bobrovnikov,109,d S. S. Bocchetta,82 A. Bocci,47 C. Bock,100 M. Boehler,50 D. Boerner,174 J. A. Bogaerts,32 D. M. Bjergaard,47 C. W. Black,150 J. E. Black,143 K. M. Black,24 D. Blackburn,138 R. E. Blair,6 J.-B. Blanchard,136 T. Blazek,144a I. Bloch,44 C. Blocker,25 W. Blum,84,a U. Blumenschein,56 S. Blunier,34a G. J. Bobbink,107 109 d 82 47 100 50 174 32 182002-7 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) D. Bogavac,14 A. G. Bogdanchikov,109 C. Bohm,146a V. Boisvert,78 P. Bokan,14 T. P H Y S I C A L R E V I E W L E T T E R S Bold,40a A. S. Boldyrev,163a,163c M. Bomben,81 M. Bona,77 M. Boonekamp,136 A. Borisov,130 G. Borissov,73 J. Bortfeldt,32 D. Bortoletto,120 V. Bortolotto,61a,61b,61c K. Bos,107 D. Boscherini,22a M. Bosman,13 J. D. Bossio Sola,29 J. Boudreau,125 J. Bouffard,2 G. Bruni,22a L. S. Bruni,107 BH Brunt,30 M. Bruschi,22a N. Bruscino,23 P. Bryant,33 L. Bryngemark,82 T. Buanes,15 Q. Buat,142 P. Buchholz,141 A. G. Buckley,55 I. A. Budagov,66 F. Buehrer,50 M. K. Bugge,119 O. Bulekov,98 D. Bullock,8 H. Burckhart,32 S. Burdin,75 C. D. Burgard,50 B. Burghgrave,108 K. Burka,41 S. Burke,131 I. Burmeister,45 J. T. P. Burr,120 E. Busato,36 D. Büscher,50 V. Büscher,84 P. Bussey,55 J. M. Butler,24 C. M. Buttar,55 J. M. B 27 55 109 d 166 128 W. Buttinger,27 A. Buzatu,55 A. R. Buzykaev,109,d S. Cabrera Urbán,166 D. Caforio,128 V. M. Cairo,39a,39b O. Cakir,4a N. Calace,51 P. Calafiura,16 A. Calandri,86 G. Calderini,81 P. Calfayan,100 G. Callea,39a,39b L. P. Caloba,26a y S. Calvente Lopez,83 D. Calvet,36 S. Calvet,36 T. P. Calvet,86 R. Camacho Toro,33 S. Camarda,32 P. Camarri,133a,133b S. Calvente Lopez,83 D. Calvet,36 S. Calvet,36 T. P. Calvet,86 R. Camacho Toro,33 S. Camarda,32 P. Camarri,133a,133b D. Cameron,119 R. Caminal Armadans,165 C. Camincher,57 S. Campana,32 M. Campanelli,79 A. Camplani,92a,92b S. Calvente Lopez,83 D. Calvet,36 S. Calvet,36 T. P. Calvet,86 R. Camacho Toro,33 S. Camarda,32 P. Camarri,133a,133b D. Cameron,119 R. Caminal Armadans,165 C. Camincher,57 S. Campana,32 M. Campanelli,79 A. Camplani,92a,92b A. Campoverde,141 V. Canale,104a,104b A. Canepa,159a M. Cano Bret,35e J. Cantero,114 R. Cantrill,126a T. Cao,42 p , , , , , , , D. Cameron,119 R. Caminal Armadans,165 C. Camincher,57 S. Campana,32 M. Campanelli,79 A. Camplani,92a,92b A. Campoverde,141 V. Canale,104a,104b A. Canepa,159a M. Cano Bret,35e J. Cantero,114 R. Cantrill,126a T. Cao,42 32 28b 28b 39 39b 84 37 133 D. Cameron, R. Caminal Armadans, C. Camincher, S. Campana, M. Campanelli, A. Camplani, A. Campoverde,141 V. Canale,104a,104b A. Canepa,159a M. Cano Bret,35e J. Cantero,114 R. Cantrill,126a T. Cao,42 b b b A. Campoverde,141 V. Canale,104a,104b A. Canepa,159a M. Cano Bret,35e J. Cantero,114 R. Cantrill,126a T. Cao,42 M. D. M. Capeans Garrido,32 I. Caprini,28b M. Caprini,28b M. Capua,39a,39b R. Caputo,84 R. M. Carbone,37 R. Cardarelli,133a F. Cardillo,50 I. Carli,129 T. Carli,32 G. Carlino,104a L. Carminati,92a,92b S. Caron,106 E. Carquin,34b G. D. Carrillo-Montoya,32 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,162 E. Castaneda-Miranda,145a R. Castelijn,107 A. Castelli,107 V. Castillo Gimenez,166 N. F. Castro,126a,j A. Catinaccio,32 J. R. Catmore,119 A. 182002-8 Gray,32 E G i i 134a Z D G d 80,r C G f 23 K G 79 I M G 44 P G i 143 K G t 5 J G iffith 8 Gilles,174 D. M. Gingrich,3,e N. Giokaris,9 M. P. Giordani,163a,163c F. M. Giorgi,22a F. M. Giorg romini,58 D. Giugni,92a F. Giuli,120 C. Giuliani,101 M. Giulini,59b B. K. Gjelsten,119 S. Gkaitatz I. Gkialas,154 E. L. Gkougkousis,117 L. K. Gladilin,99 C. Glasman,83 J. Glatzer,50 P. C. F. Glaysher,48 A. Glazov,44 M. Goblirsch-Kolb,25 J. Godlewski,41 S. Goldfarb,89 T. Golling,51 D. Golubkov,130 A. Gomes,126a,126b,126d R. Gonçalo,126a Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. Gonzá J. Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. González de la Hoz,166 13 51 32 97 27 105 32 G. Gonzalez Parra,13 S. Gonzalez-Sevilla,51 L. Goossens,32 P. A. Gorbounov,97 H. A. Gordon,27 I. Gorelov,105 B. Gorini,32 E. Gorini,74a,74b A. Gorišek,76 E. Gornicki,41 A. T. Goshaw,47 C. Gössling,45 M. I. Gostkin,66 C. R. Goudet,117 A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowska-Bold,40a P. O. J. G D. Goujdami,135c A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowska-Bold,40a P. O. J. Gradin,57 P. Grafström,22a,22b J. Gramling,51 E. Gramstad,119 S. Grancagnolo,17 V. Gratchev,123 P. M. Gravila,28e H. M. Gray,32 E. Graziani,134a Z. D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K. Grevtsov,5 J. Griffiths,8 A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross,171 J. Grosse-Knetter,56 G. C. Grossi,80 Z. J. Grout,79 L. Guan,90 W. Guan,172 J. Guenther,63 F. Guescini,51 D. Guest,162 , g, , g , , , y, E. Graziani,134a Z. D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K. Grevtsov,5 J. Griffiths,8 A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross,171 J. Grosse-Knetter,56 G. C. Grossi,80 Z. J. Grout,79 L. Guan,90 W. Guan,172 J. Guenther,63 F. Guescini,51 D. Guest,162 , , , , , , , , O. Gueta,153 E. Guido,52a,52b T. Guillemin,5 S. Guindon,2 U. Gul,55 C. Gumpert,32 J. Guo,35e Y. Guo,35b,p R. Gupta,42 120 132 132b 113 9 46 136 120 O. Gueta,153 E. Guido,52a,52b T. Guillemin,5 S. Guindon,2 U. Gul,55 C. Gumpert,32 J. Guo,35e Y. Guo,35b,p R. Gupta,42 S. Gupta,120 G. Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. Gutschow,46 C. Guyot,136 C. Gwenlan,120 182002-9 182002-8 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) j j j C. Doglioni,82 J. Dolejsi,129 Z. Dolezal,129 M. Donadelli,26d S. Donati,124a,124b P. Dondero,121a,1 g j p A. Doria,104a M. T. Dova,72 A. T. Doyle,55 E. Drechsler,56 M. Dris,10 Y. Du,35d J. Duarte-Campderros,153 E. Duchovni,171 G. Duckeck,100 O. A. Ducu,95,n D. Duda,107 A. Dudarev,32 A. Chr. Dudder,84 E. M. Duffield,16 L. Duflot,117 M. Dührssen,32 M. Dumancic,171 M. Dunford,59a H. Duran Yildiz,4a M. Düren,54 A. Durglishvili,53b D. Duschinger,46 B. Dutta,44 g g M. Dyndal,44 C. Eckardt,44 K. M. Ecker,101 R. C. Edgar,90 N. C. Edwards,48 T. Eifert,32 G. Eigen,15 K. Einsweiler,16 g g M. Dyndal,44 C. Eckardt,44 K. M. Ecker,101 R. C. Edgar,90 N. C. Edwards,48 T. Eifert,32 G. Eigen,15 K. Einsweiler,16 T Ekelof 164 M El Kacimi 135c V Ellajosyula 86 M Ellert 164 S Elles 5 F Ellinghaus 174 A A Elliot 168 N Ellis 32 T. Ekelof,164 M. El Kacimi,135c V. Ellajosyula,86 M. Ellert,164 S. Elles,5 F. Ellinghaus,174 A , , j y , , , g , , , J. Elmsheuser,27 M. Elsing,32 D. Emeliyanov,131 Y. Enari,155 O. C. Endner,84 J. S. Ennis,169 J. Erdmann,45 A. Ereditato,18 174 2 27 165 84 117 45 125 49 J. Elmsheuser,27 M. Elsing,32 D. Emeliyanov,131 Y. Enari,155 O. C. Endner,84 J. S. Ennis,169 J. Erdmann,45 A. Ereditato,18 G. Ernis,174 J. Ernst,2 M. Ernst,27 S. Errede,165 E. Ertel,84 M. Escalier,117 H. Esch,45 C. Escobar,125 B. Esposito,49 136 153 62 123 22 22b 22 22b 33 130 G. Ernis,174 J. Ernst,2 M. Ernst,27 S. Errede,165 E. Ertel,84 M. Escalier,117 H. Esch,45 C. E A. I. Etienvre,136 E. Etzion,153 H. Evans,62 A. Ezhilov,123 F. Fabbri,22a,22b L. Fabbri,22a,22b G. Facini,33 R. M. Fakhrutdinov,130 S. Falciano,132a R. J. Falla,79 J. Faltova,32 Y. Fang,35a M. Fanti,92a,92b A. Farbin,8 A. Farilla,134a C. Farina,125 121a 121b 13 16 169 32 135e 32 g E. M. Farina,121a,121b T. Farooque,13 S. Farrell,16 S. M. Farrington,169 P. Farthouat,32 F. Fassi,135e P. Fassnacht,32 9 78 52 52b 120 117 123 167 D. Fassouliotis,9 M. Faucci Giannelli,78 A. Favareto,52a,52b W. J. Fawcett,120 L. Fayard,117 O. L S. Feigl,119 L. Feligioni,86 C. Feng,35d E. J. Feng,32 H. Feng,90 A. B. Fenyuk,130 L. Feremenga,8 P. Fernandez Martinez,166 55 5 S. Fernandez Perez,13 J. Ferrando,55 A. Ferrari,164 P. Ferrari,107 R. D. A. DeMarco,158 S. Demers,175 M. Demichev,66 A. Demilly,81 S. P. Denisov,130 D. Denysiuk,136 D. Derendarz,41 J. E. Derkaoui,135d F. Derue,81 P. Dervan,75 K. Desch,23 C. Deterre,44 K. Dette,45 P. O. Deviveiros,32 A. Dewhurst,131 S. Dhaliwal,25 A. Di Ciaccio,133a,133b L. Di Ciaccio,5 W. K. Di Clemente,122 C. Di Donato,132a,132b A. Di Girolamo,32 B. Di Girolamo,32 B. Di Micco,134a,134b R. Di Nardo,32 A. Di Simone,50 R. Di Sipio,158 D. Di Valentino,31 C. Diaconu,86 M. Diamond,158 F. A. Dias,48 M. A. Diaz,34a E. B. Diehl,90 J. Dietrich,17 S. Diglio,86 A. Dimitrievska,14 J. Dingfelder,23 P. Dita,28b S. Dita,28b F. Dittus,32 F. Djama,86 T. Djobava,53b J. I. Djuvsland,59a M. A. B. do Vale,26c D. Dobos,32 M. Dobre,28b C. Doglioni,82 J. Dolejsi,129 Z. Dolezal,129 M. Donadelli,26d S. Donati,124a,124b P. Dondero,121a,121b J. Donini,36 J. Dopke,131 A Doria 104a M T Dova 72 A T Doyle 55 E Drechsler 56 M Dris 10 Y Du 35d J Duarte Campderros 153 E Duchovni 171 182002-8 Ferrari,121a D. E. Ferreira de Lima,59b A. Ferrer,166 D. Ferrere,51 C. Ferretti,90 A. Ferretto Parodi,52a,52b F. Fiedler,84 A. Filipčič,76 M. Filipuzzi,44 F. Filthaut,106 M. Fincke-Keeler,168 K. D. Finelli,150 M. C. N. Fiolhais,126a,126c L. Fiorini,166 A. Firan,42 A. Fischer,2 C. Fischer,13 J. Fischer,174 W. C. Fisher,91 N. Flaschel,44 I. Fleck,141 P. Fleischmann,90 G. T. Fletcher,139 R. R. M. Fletcher,122 T. Flick,174 D. Fournier,117 H. Fox,73 S. Fracchia,13 P. Francavilla,81 M. Franchini,22a,22b D. Francis,32 L. Franconi,119 M. Franklin,58 M. Frate,162 M. Fraternali,121a,121b D. Freeborn,79 S. M. Fressard-Batraneanu,32 F. Friedrich,46 D. Froidevaux,32 J. A. Frost,120 C. Fukunaga,156 E. Fullana Torregrosa,84 T. Fusayasu,102 J. Fuster,166 C. Gabaldon,57 O. Gabizon,174 A. Gabrielli,22a,22b A. Gabrielli,16 G. P. Gach,40a S. Gadatsch,32 S. Gadomski,51 G. Gagliardi,52a,52b L. G. Gagnon,95 P. Gagnon,62 C. Galea,106 B. Galhardo,126a,126c E. J. Gallas,120 B. J. Gallop,131 P. Gallus,128 G. Galster,38 K. K. Gan,111 J. Gao,35b,86 Y. Gao,48 Y. S. Gao,143,g F. M. Garay Walls,48 C. García,166 J. E. García Navarro,166 M. Garcia-Sciveres,16 R. W. Gardner,33 N. Garelli,143 V. Garonne,119 A. Gascon Bravo,44 K. Gasnikova,44 C. Gatti,49 A. Gaudiello,52a,52b G. Gaudio,121a L. Gauthier,95 I. L. Gavrilenko,96 C. Gay,167 G. Gaycken,23 E. N. Gazis,10 Z. Gecse,167 C. N. P. Gee,131 Ch. Geich-Gimbel,23 M. Geisen,84 M. P. Geisler,59a C. Gemme,52a M. H. Genest,57 C. Geng,35b,p S. Gentile,132a,132b C. Gentsos,154 S. George,78 D. Gerbaudo,13 A. Gershon,153 S. Ghasemi,141 H. Ghazlane,135b M. Ghneimat,23 B. Giacobbe,22a S. Giagu,132a,132b P. Giannetti,124a,124b B. Gibbard,27 S. M. Gibson,78 M. Gignac,167 M. Gilchriese,16 T. P. S. Gillam,30 D. Gillberg,31 G. Gilles,174 D. M. Gingrich,3,e N. Giokaris,9 M. P. Giordani,163a,163c F. M. Giorgi,22a F. M. Giorgi,17 P. F. Giraud,136 P. Giromini,58 D. Giugni,92a F. Giuli,120 C. Giuliani,101 M. Giulini,59b B. K. Gjelsten,119 S. Gkaitatzis,154 I. Gkialas,154 E. L. Gkougkousis,117 L. K. Gladilin,99 C. Glasman,83 J. Glatzer,50 P. C. F. Glaysher,48 A. Glazov,44 M. Goblirsch-Kolb,25 J. Godlewski,41 S. Goldfarb,89 T. Golling,51 D. Golubkov,130 A. Gomes,126a,126b,126d R. Gonçalo,126a J. Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. González de la Hoz,166 G. Gonzalez Parra,13 S. Gonzalez-Sevilla,51 L. Goossens,32 P. A. Gorbounov,97 H. A. Gordon,27 I. Gorelov,105 B. Gorini,32 E. Gorini,74a,74b A. Gorišek,76 E. Gornicki,41 A. T. Goshaw,47 C. Gössling,45 M. I. Gostkin,66 C. R. Goudet,117 D. Goujdami,135c A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowska-Bold,40a P. O. J. Gradin,57 P. Grafström,22a,22b J. Gramling,51 E. Gramstad,119 S. Grancagnolo,17 V. Gratchev,123 P. M. Gravila,28e H. M. 182002-9 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) C. B. Gwilliam,75 A. Haas,110 C. Haber,16 H. K. Hadavand,8 N. Haddad,135e A. Hadef,86 S. Ha C. B. Gwilliam, A. Haas, C. Haber, H. K. Hadavand, N. Haddad, A. Hadef, S. Hageböck, Z. Hajduk, H. Hakobyan,176,a M. Haleem,44 J. Haley,114 G. Halladjian,91 G. D. Hallewell,86 K. Hamacher,174 P. Hamal,115 168 145 139 44 35b 67 155 137 g j H. Hakobyan,176,a M. Haleem,44 J. Haley,114 G. Halladjian,91 G. D. Hallewell,86 K. Hamacher,174 P. Hamal,115 168 145 139 44 35b 67 t 155 137 H. Hakobyan,176,a M. Haleem,44 J. Haley,114 G. Halladjian,91 G. D. Hallewell,86 K. Ham Hamano,168 A. Hamilton,145a G. N. Hamity,139 P. G. Hamnett,44 L. Han,35b K. Hanagaki,67,t K. H B. Haney,122 S. Hanisch,32 P. Hanke,59a R. Hanna,136 J. B. Hansen,38 J. D. Hansen,38 M. C. B. Haney,122 S. Hanisch,32 P. Hanke,59a R. Hanna,136 J. B. Hansen,38 J. D. Hansen,38 M. C. Hansen,23 P. H. Hansen,38 y K. Hara,160 A. S. Hard,172 T. Harenberg,174 F. Hariri,117 S. Harkusha,93 R. D. Harrington,48 P. F N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46 M. Havranek,127 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Hays,77 H. S. Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 T. Heim,16 16 100 110 54 127 32 146a 146b 32 H. S. Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 T. Heim,16 B. Heinemann,16 J. J. Heinrich,100 L. Heinrich,110 C. Heinz,54 J. Hejbal,127 L. Helary,32 S. Hellman,146a,146b C. Helsens,32 nderson,120 R. C. W. Henderson,73 Y. Heng,172 S. Henkelmann,167 A. M. Henriques Correia,32 g q G. H. Herbert,17 V. Herget,173 Y. Hernández Jiménez,166 G. Herten,50 R. Hertenberger,100 L. Hervas,32 G. G. Hesketh,79 10 42 166 168 30 44 19 N. P. Hessey,107 J. W. Hetherly,42 R. Hickling,77 E. Higón-Rodriguez,166 E. Hill,168 J. C. Hill,30 K N. P. Hessey, J. W. Hetherly, R. Hickling, E. Higón-Rodriguez, E. Hill, J. C. Hill, K. H. Hiller, S. J. Hillier, I. Hinchliffe,16 E. Hines,122 R. R. Hinman,16 M. Hirose,50 D. Hirschbuehl,174 J. Hobbs,148 N. Hod,159a M. C. Hodgkinson,139 y y g g g I. Hinchliffe,16 E. 182002-9 Hines,122 R. R. Hinman,16 M. Hirose,50 D. Hirschbuehl,174 J. Hobbs,148 N. Hod,159a M. C. Hodgkinson,139 139 32 105 100 23 16 45 125 P. Hodgson,139 A. Hoecker,32 M. R. Hoeferkamp,105 F. Hoenig,100 D. Hohn,23 T. R. Holmes,16 M. Homann,45 T. M. Hong,125 B. H. Hooberman,165 W. H. Hopkins,116 Y. Horii,103 A. J. Horton,142 J-Y. Hostachy,57 S. Hou,151 A. Hoummada,135a J. Howarth, M. Hrabovsky, I. Hristova, J. Hrivnac, T. Hryn ova, A. Hrynevich, C. Hsu, P. J. Hsu, S.-C. Hsu,138 D. Hu,37 Q. Hu,35b S. Hu,35e Y. Huang,44 Z. Hubacek,128 F. Hubaut,86 F. Huegging,23 T. B. Huffman,120 37 73 32 148 66 91 58 51 , y, , , y , y , , , S.-C. Hsu,138 D. Hu,37 Q. Hu,35b S. Hu,35e Y. Huang,44 Z. Hubacek,128 F. Hubaut,86 F. Huegging,23 T. B. Huffman,120 E. W. Hughes,37 G. Hughes,73 M. Huhtinen,32 P. Huo,148 N. Huseynov,66,c J. Huston,91 J. Huth,58 G. Iacobucci,51 E. W. Hughes,37 G. Hughes,73 M. Huhtinen,32 P. Huo,148 N. Huseynov,66,c J. Huston,91 J. g g y G. Iakovidis,27 I. Ibragimov,141 L. Iconomidou-Fayard,117 E. Ideal,175 Z. Idrissi,135e P. Iengo,32 O. Igonkina,107,v T. Iizawa,170 Y. Ikegami,67 M. Ikeno,67 Y. Ilchenko,11,w D. Iliadis,154 N. Ilic,143 T. Ince,101 G. Introzzi,121a,121b K. Iordanidou,37 V. Ippolito,58 N. Ishijima,118 M. Ishino,155 M. Ishitsuka,157 R. Ishmukhametov,111 C. Issever,120 S. Istin,20a F. Ito,160 J. M. Iturbe Ponce,85 R. Iuppa,133a,133b W. Iwanski,41 H. Iwasaki,67 J. M. Izen,43 V. Izzo,104a S. Jabbar,3 B. Jackson,122 P. Jackson,1 V. Jain,2 K. B. Jakobi,84 K. Jakobs,50 S. Jakobsen,32 T. Jakoubek,127 D. O. Jamin,114 D. K. Jana,80 y g y J. Jejelava,53a,x G.-Y. Jeng,150 D. Jennens,89 P. Jenni,50,y C. Jeske,169 S. Jézéquel,5 H. Ji,172 J. Jia,148 H. Jiang,65 Y. Jiang,35b S. Jiggins,79 J. Jimenez Pena,166 S. Jin,35a A. Jinaru,28b O. Jinnouchi,157 H. Jivan,145c P. Johansson,139 K. A. Johns,7 W. J. Johnson,138 K. Jon-And,146a,146b G. Jones,169 R. W. L. Jones,73 S. Jones,7 T. J. Jones,75 J. Jongmanns,59a S. Jiggins,79 J. Jimenez Pena,166 S. Jin,35a A. Jinaru,28b O. Jinnouchi,157 H. Jivan,145c P. Johansson,139 K. A. Johns,7 W. J. Johnson,138 K. Jon-And,146a,146b G. Jones,169 R. W. L. Jones,73 S. Jones,7 T. J. Jones,75 J. Jongmanns,59a P. M. Jorge,126a,126b J. Jovicevic,159a X. Ju,172 A. Juste Rozas,13,s M. K. Köhler,171 A. Kaczmarska,41 M. Kado,117 P. M. Jorge,126a,126b J. Jovicevic,159a X. Ju,172 A. Juste Rozas,13,s M. K. Köhler,171 A. Kaczmarska,41 M. Kado,117 H. Kagan,111 M. Kagan,143 S. J. Kahn,86 T. Kaji,170 E. Kajomovitz,47 C. W. Kalderon,120 A. Kaluza,84 S. 182002-9 Luehring,62 W L k 63 L L i i 132a O L db 146a,146b B L d J 147 P M L i 81 D L 27 R L k 127 E L tk 82 g y y y V. Lyubushkin,66 H. Ma,27 L. L. Ma,35d Y. Ma,35d G. Maccarrone,49 A. Macchiolo,101 C. M. Macdonald,139 B. Maček,76 V. Lyubushkin,66 H. Ma,27 L. L. Ma,35d Y. Ma,35d G. Maccarrone,49 A. Macchiolo,101 C. M. Macdonald,139 B. Maček,76 122 126b 86 36 164 46 44 68 S. Maeland,15 T. Maeno,27 A. Maevskiy,99 E. Magradze,56 J. Mahlstedt,107 C. Maiani,117 C. Maidantchik,26a A. A. Maier,101 T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41 V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166 G. Mancini,49 B. Mandelli,32 L. Mandelli,92a I. Mandić,76 J. Maneira,126a,126b L. Manhaes de Andrade Filho,26b T. Maier, A. Maio, S. Majewski, Y. Makida, N. Makovec, B. Malaescu, Pa. Malecki, V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166 G. Mancini,49 B. Mandelli,32 L. Mandelli,92a I. Mandić,76 J. Maneira,126a,126b L. Manhaes de Andrade Filho,26b J. Manjarres Ramos,159b A. Mann,100 A. Manousos,32 B. Mansoulie,136 J. D. Mansour,35a R. Mantifel,88 M. Mantoani,56 S. Manzoni,92a,92b L. Mapelli,32 G. Marceca,29 L. March,51 G. Marchiori,81 M. Marcisovsky,127 M. Marjanovic,14 D. E. Marley,90 F. Marroquim,26a S. P. Marsden,85 Z. Marshall,16 S. Marti-Garcia,166 B. Martin,91 T. A. Martin,169 y, q , , , , , , V. J. Martin,48 B. Martin dit Latour,15 M. Martinez,13,s V. I. Martinez Outschoorn,165 S. Martin-Haugh,131 V. S. Martoiu,28b A. C. Martyniuk,79 M. Marx,138 A. Marzin,32 L. Masetti,84 T. Mashimo,155 R. Mashinistov,96 J. Masik,85 A. L. Maslennikov,109,d I. Massa,22a,22b L. Massa,22a,22b P. Mastrandrea,5 A. Mastroberardino,39a,39b T. Masubuchi,155 P. Mättig,174 J. Mattmann,84 J. Maurer,28b S. J. Maxfield,75 D. A. Maximov,109,d R. Mazini,151 S. M. Mazza,92a,92b N. C. Mc Fadden,105 G. Mc Goldrick,158 S. P. Mc Kee,90 A. McCarn,90 R. L. McCarthy,148 T. G. McCarthy,101 79 89 79 56 131 168 y y M. Medinnis,44 S. Meehan,138 S. Mehlhase,100 A. Mehta,75 K. Meier,59a C. Meineck,100 B. Meirose,43 D. Melini,166 145 144 18 22 22b 101 161 1 P. Mermod,51 L. Merola,104a,104b C. Meroni,92a F. S. Merritt,33 A. Messina,132a,132b J. Metcalfe,6 A. S. Mete,162 C. Meyer,84 C. Meyer,122 J-P. Meyer,136 J. Meyer,107 H. Meyer Zu Theenhausen,59a F. Miano,149 R. P. Middleton,131 S. Miglioranzi,52a,52b L. Mijović,48 G. 182002-9 Mikenberg,171 M. Mikestikova,127 M. Mikuž,76 M. Milesi,89 A. Milic,63 D. W. Miller,33 C. Mills,48 j g A. Milov,171 D. A. Milstead,146a,146b A. A. Minaenko,130 Y. Minami,155 I. A. Minashvili,66 A. 66 172 13 122 170 100 A. Milov, D. A. Milstead, , A. A. Minaenko, Y. Minami, I. A. Minashvili, A. I. Mincer, B. Mindur, M. Mineev,66 Y. Ming,172 L. M. Mir,13 K. P. Mistry,122 T. Mitani,170 J. Mitrevski,100 V. A. Mitsou,166 A. Miucci,18 P. S. Miyagawa,139 J. U. Mjörnmark,82 T. Moa,146a,146b K. Mochizuki,95 S. Mohapatra,37 S. Molander,146a,146b R. Moles-Valls,23 R. Monden,69 M. C. Mondragon,91 K. Mönig,44 J. Monk,38 E. Monnier,86 A. Montalbano,148 32 72 92 92b 3 117 21 56 182002-11 182002-11 A. Kruse,172 M. C. Kruse,47 M. Kruskal,24 T. Kubota,89 H. Kucuk,79 S. Kuday,4b J. T. Kuechler,174 S. Kuehn,50 A. Kugel,59c F. Kuger,173 A. Kuhl,137 T. Kuhl,44 V. Kukhtin,66 R. Kukla,136 Y. Kulchitsky,93 S. Kuleshov,34b M. Kuna,132a,132b T. Kunigo,69 A. Kupco,127 H. Kurashige,68 Y. A. Kurochkin,93 V. Kus,127 E. S. Kuwertz,168 M. Kuze,157 J. Kvita,115 T. Kwan,168 D. Kyriazopoulos,139 A. La Rosa,101 J. L. La Rosa Navarro,26d L. La Rotonda,39a,39b C. Lacasta,166 F. Lacava,132a,132b 31 17 81 166 66 5 81 175 56 182002-9 Lehmann Miotto,32 X. Lei,7 W. A. Leight,31 A. Leisos,154,cc A. G. Leister,175 M. A. L. Leite,26d F. Ledroit-Guillon,57 C. A. Lee,27 S. C. Lee,151 L. Lee,1 B. Lefebvre,88 G. Lefebvre,81 M. Lefebvre,168 F. Legger,100 C. Leggett,16 A. Lehan,75 G. Lehmann Miotto,32 X. Lei,7 W. A. Leight,31 A. Leisos,154,cc A. G. Leister,175 M. A. L. Leite,26d , , , , , , , gg , C. Leggett,16 A. Lehan,75 G. Lehmann Miotto,32 X. Lei,7 W. A. Leight,31 A. Leisos,154,cc A. G. Leister,175 M. A. L. Leite,26d R. Leitner,129 D. Lellouch,171 B. Lemmer,56 K. J. C. Leney,79 T. Lenz,23 B. Lenzi,32 R. Leone,7 S. Leone,124a,124b C. Leonidopoulos,48 S. Leontsinis,10 G. Lerner,149 C. Leroy,95 A. A. J. Lesage,136 C. G. Lester,30 M. Levchenko,123 J Levêque 5 D Levin 90 L J Levinson 171 M Levy 19 D Lewis 77 A M Leyko 23 M Leyton 43 B Li 35b,p C Li 35b H Li 148 R. Leitner, D. Lellouch, B. Lemmer, K. J. C. Leney, T. Lenz, B. Lenzi, R. Leone, S. Leone, C. Leonidopoulos,48 S. Leontsinis,10 G. Lerner,149 C. Leroy,95 A. A. J. Lesage,136 C. G. Lester,30 M. Levchenko,123 J L ê 5 D L i 90 L J L i 171 M L 19 D L i 77 A M L k 23 M L 43 B Li 35b p C Li 35b H Li 148 H. L. Li,33 L. Li,47 L. Li,35e Q. Li,35a S. Li,47 X. Li,85 Y. Li,141 Z. Liang,35a B. Liberti,133a A. Liblong,158 P. Lichard,32 E. Lipeles,122 A. Lipniacka,15 M. Lisovyi,59b T. M. Liss,165 A. Lister,167 A. M. Litke,137 B. Liu,151,ee D. Liu,151 H. Liu,90 J. Llorente Merino,35a S. L. Lloyd,77 F. Lo Sterzo,151 E. Lobodzinska,44 P. Loch,7 W. S. Lockman,137 F. K. Loebinger,85 , , g , , , j , g, J. D. Long,165 R. E. Long,73 L. Longo,74a,74b K. A. Looper,111 L. Lopes,126a D. Lopez Mateos,58 B. Lopez Paredes,139 I. Lopez Paz,13 A. Lopez Solis,81 J. Lorenz,100 N. Lorenzo Martinez,62 M. Losada,21 P. J. Lösel,100 X. Lou,35a A. Lounis,117 6 73 61a 90 138 132a 132b 57 50 62 I. Lopez Paz, A. Lopez Solis, J. Lorenz, N. Lorenzo Martinez, M. Losada, P. J. Lösel, X. Lou, A. Lounis, J. Love,6 P. A. Love,73 H. Lu,61a N. Lu,90 H. J. Lubatti,138 C. Luci,132a,132b A. Lucotte,57 C. Luedtke,50 F. 182002-9 Kama,42 P. M. Jorge, J. Jovicevic, X. Ju, A. Juste Rozas, M. K. Köhler, A. Kaczmarska, M. Kado, H. Kagan,111 M. Kagan,143 S. J. Kahn,86 T. Kaji,170 E. Kajomovitz,47 C. W. Kalderon,120 A. Kaluza,84 S. Kama,42 A. Kamenshchikov,130 N. Kanaya,155 S. Kaneti,30 L. Kanjir,76 V. A. Kantserov,98 J. Kanzaki,67 B. Kaplan,110 L. S. Kaplan,172 A. Kapliy,33 D. Kar,145c K. Karakostas,10 A. Karamaoun,3 N. Karastathis,10 M. J. Kareem,56 E. Karentzos,10 L. Kashif,172 R. D. Kass,111 A. Kastanas,15 Y. Kataoka,155 C. Kato,155 A. Katre,51 J. Katzy,44 K. Kawagoe,71 T. Kawamoto,155 G. Kawamura,56 V. F. Kazanin,109,d R. Keeler,168 R. Kehoe,42 J. S. Keller,44 J. J. Kempster,78 K Kentaro,103 H. Keoshkerian,158 O. Kepka,127 B. P. Kerševan,76 S. Kersten,174 R. A. Keyes,88 M. Khader,165 F. Khalil-zada,12 A Khanov 114 A G Kharlamov 109,d T J Khoo 51 V Khovanskiy 97 E Khramov 66 J Khubua 53b,z S Kido 68 C R Kilby 78 T. Kishimoto,155 D. Kisielewska,40a F. Kiss,50 K. Kiuchi,160 O. Kivernyk,136 E. Kladiva,144b M. H. Klein,37 M. Klein,75 U. Klein,75 K. Kleinknecht,84 P. Klimek,108 A. Klimentov,27 R. Klingenberg,45 J. A. Klinger,139 T. Klioutchnikova,32 g g g E.-E. Kluge,59a P. Kluit,107 S. Kluth,101 J. Knapik,41 E. Kneringer,63 E. B. F. G. Knoops,86 A. Knue,55 A. Kobayashi,155 D. Kobayashi,157 T. Kobayashi,155 M. Kobel,46 M. Kocian,143 P. Kodys,129 N. M. Koehler,101 T. Koffas,31 E. Koffeman,107 T. Koi,143 H. Kolanoski,17 M. Kolb,59b I. Koletsou,5 A. A. Komar,96,a Y. Komori,155 T. Kondo,67 N. Kondrashova,44 182002-10 182002-10 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) y g F. Kuger,173 A. Kuhl,137 T. Kuhl,44 V. Kukhtin,66 R. Kukla,136 Y. Kulchitsky,93 S. Kuleshov,34b M. Kuna,132a,132b T. Kunigo,69 A. Kupco,127 H. Kurashige,68 Y. A. Kurochkin,93 V. Kus,127 E. S. Kuwertz,168 M. Kuze,157 J. Kvita,115 T. Kwan,168 D. Kyriazopoulos,139 A. La Rosa,101 J. L. La Rosa Navarro,26d L. La Rotonda,39a,39b C. Lacasta,166 F. Lacava,132a,132b Lammers,62 W. Lampl,7 E. Lançon,136 U. Landgraf,50 M. P. J. Landon,77 M. C. Lanfermann,51 V. S A. J. Lankford,162 F. Lanni,27 K. Lantzsch,23 A. Lanza,121a S. Laplace,81 C. Lapoire,32 J. F. L M. Lazzaroni,92a,92b B. Le,89 O. Le Dortz,81 E. Le Guirriec,86 E. P. Le Quilleuc,136 M. LeBla F. Ledroit-Guillon,57 C. A. Lee,27 S. C. Lee,151 L. Lee,1 B. Lefebvre,88 G. Lefebvre,81 M. Lefebvre,168 F. Legger,100 C. Leggett,16 A. Lehan,75 G. 182002-11 Pettersson,87 A Peyaud 136 R Pezoa 34b P W Phillips 131 G Piacquadio 143,ii E Pianori 169 A Picazio 87 E Piccaro 77 M Piccinini 22a,22b A. Peyaud,136 R. Pezoa,34b P. W. Phillips,131 G. Piacquadio,143,ii E. Pianori,169 A. Picazio,87 E. Piccaro,77 M. Piccinini,22a,22b M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,jj J. L. Pinfold,3 y , , p , q , , , , , M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,jj J. L. Pinfold,3 Pingel,38 S. Pires,81 H. Pirumov,44 M. Pitt,171 L. Plazak,144a M.-A. Pleier,27 V. Pleskot,84 E. Plo A. Pingel, S. Pires, H. Pirumov, M. Pitt, L. Plazak, M. A. Pleier, V. Pleskot, E. Plotnikova, P. Plucinski, D. Pluth,65 R. Poettgen,146a,146b L. Poggioli,117 D. Pohl,23 G. Polesello,121a A. Poley,44 A. Policicchio,39a,39b R. Polifka,158 A. Polini,22a C. S. Pollard,55 V. Polychronakos,27 K. Pommès,32 L. Pontecorvo,132a B. G. Pope,91 G. A. Popeneciu,28c g , , , , , , , , , D. Pluth,65 R. Poettgen,146a,146b L. Poggioli,117 D. Pohl,23 G. Polesello,121a A. Poley,44 A. Policicchio,39a,39b R. Polifka,158 22a 55 27 32 132a 91 28c A. Poppleton,32 S. Pospisil,128 K. Potamianos,16 I. N. Potrap,66 C. J. Potter,30 C. T. Potter,116 V. Pozdnyakov,66 M. E. Pozo Astigarraga,32 P. Pralavorio,86 A. Pranko,16 S. Prell,65 D. Price,85 L. E. Price,6 M. Primavera,74a S. Prince,88 K. Prokofiev,61c F. Prokoshin,34b S. Protopopescu,27 J. Proudfoot,6 M. Przybycien,40a D. Puddu,134a,134b M. Purohit,27,kk P. Puzo,117 J. Qian,90 G. Qin,55 Y. Qin,85 A. Quadt,56 W. B. Quayle,163a,163b M. Queitsch-Maitland,85 55 119 27 120 148 116 89 92a 92b , , Q , Q , Q , Q , Q y , Q , D. Quilty,55 S. Raddum,119 V. Radeka,27 V. Radescu,120 S. K. Radhakrishnan,148 P. Radloff,116 P. Rados,89 F. Ragusa,92a,92b G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. Ratti,92a,92b F. Rauscher,100 S. Rave,84 T. Ravenscroft,55 I. Ravinovich,171 M. Raymond,32 A. L. Read,119 N. P. Readioff,75 M. Reale,74a,74b D. M. Rebuzzi,121a,121b A. Redelbach,173 G. Redlinger,27 R. Reece,137 K. Reeves,43 L. Rehnisch,17 J. Reichert,122 H. Reisin,29 D. Quilty, S. Raddum, V. Radeka, V. Radescu, S. K. Radhakrishnan, P. Radloff, P. Rados, F. Ragusa, G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. Ratti,92a,92b F. 182002-11 Rauscher,100 S 84 f 55 i i h 171 d 32 A d 119 di ff 75 l 74a 74b G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. Ratti,92a,92b F. Rauscher,100 S. Rave,84 T. Ravenscroft,55 I. Ravinovich,171 M. Raymond,32 A. L. Read,119 N. P. Readioff,75 M. Reale,74a,74b y D. M. Rebuzzi,121a,121b A. Redelbach,173 G. Redlinger,27 R. Reece,137 K. Reeves,43 L. Rehnisch,17 J. Reichert,122 H. Reisin,29 C. Rembser,32 H. Ren,35a M. Rescigno,132a S. Resconi,92a O. L. Rezanova,109,d P. Reznicek,129 R. Rezvani,95 R. Richter,101 S. Richter,79 E. Richter-Was,40b O. Ricken,23 M. Ridel,81 P. Rieck,17 C. J. Riegel,174 J. Rieger,56 O. Rifki,113 , , g , , , , , , S. Richter,79 E. Richter-Was,40b O. Ricken,23 M. Ridel,81 P. Rieck,17 C. J. Riegel,174 J. Rieger,56 O. Rifki,113 j E. Rizvi,77 C. Rizzi,13 S. H. Robertson,88,m A. Robichaud-Veronneau,88 D. Robinson,30 J. E. M. Robinson,44 A. Robson,55 124a 124b 86 13 166 32 58 119 E. Rizvi,77 C. Rizzi,13 S. H. Robertson,88,m A. Robichaud-Veronneau,88 D. Robinson,30 J. E. M. Robinson,44 A. Robson,55 C. Roda,124a,124b Y. Rodina,86 A. Rodriguez Perez,13 D. Rodriguez Rodriguez,166 S. Roe,32 C. S. Rogan,58 O. Røhne,119 182002-12 182002-12 P. Morettini,52a D. Mori,142 T. Mori,155 M. Morii,58 M. Morinaga,155 V. Morisbak,119 S. Moritz,84 A. K. Morley,150 G. Mornacchi,32 J. D. Morris,77 S. S. Mortensen,38 L. Morvaj,148 M. Mosidze,53b J. Moss,143 K. Motohashi,157 R. Mount,143 E. Mountricha,27 S. V. Mouraviev,96,a E. J. W. Moyse,87 S. Muanza,86 R. D. Mudd,19 F. Mueller,101 J. Mueller,125 R. S. P. Mueller,100 T. Mueller,30 D. Muenstermann,73 P. Mullen,55 G. A. Mullier,18 F. J. Munoz Sanchez,85 J. A. Murillo Quijada,19 W. J. Murray,169,131 H. Musheghyan,56 M. Muškinja,76 A. G. Myagkov,130,ff M. Myska,128 B. P. Nachman,143 O. Nackenhorst,51 K. Nagai,120 R. Nagai,67,aa K. Nagano,67 Y. Nagasaka,60 K. Nagata,160 M. Nagel,50 E. Nagy,86 A. M. Nairz,32 Y. Nakahama,103 K. Nakamura,67 T. Nakamura,155 I. Nakano,112 H. Namasivayam,43 R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a I. Naryshkin,123 T. Naumann,44 G. Navarro,21 R. Nayyar,7 H. A. Neal,90 P. Yu. Nechaeva,96 T. J. Neep,85 A. Negri,121a,121b M. Negrini,22a S. Nektarijevic,106 C. Nellist,117 A. Nelson,162 S. Nemecek,127 P. Nemethy,110 A. A. Nepomuceno,26a M. Nessi,32,gg M. S. Neubauer,165 M. Neumann,174 R. M. Neves,110 P. Nevski,27 P. R. Newman,19 D. H. Nguyen,6 T. Nguyen Manh,95 R. B. Nickerson,120 R. Nicolaidou,136 J. Nielsen,137 A. Nikiforov,17 V. Nikolaenko,130,ff I. Nikolic-Audit,81 K. Nikolopoulos,19 J. K. Nilsen,119 P. Nilsson,27 Y. Ninomiya,155 A. Nisati,132a R. Nisius,101 T. Nobe,155 M. Nomachi,118 I. Nomidis,31 T. Nooney,77 S. Norberg,113 M. Nordberg,32 N. Norjoharuddeen,120 O. Novgorodova,46 S. Nowak,101 M. Nozaki,67 L. Nozka,115 K. Ntekas,10 E. Nurse,79 F. Nuti,89 F. O’grady,7 D. C. O’Neil,142 A. A. O’Rourke,44 V. O’Shea,55 F. G. Oakham,31,e H. Oberlack,101 T. Obermann,23 J. Ocariz,81 A. Ochi,68 I. Ochoa,37 J. P. Ochoa-Ricoux,34a S. Oda,71 S. Odaka,67 H. Ogren,62 A. Oh,85 S. H. Oh,47 C. C. Ohm,16 H. Ohman,164 H. Oide,32 H. Okawa,160 Y. Okumura,155 T. Okuyama,67 A. Olariu,28b L. F. Oleiro Seabra,126a S. A. Olivares Pino,48 D. Oliveira Damazio,27 A. Olszewski,41 J. Olszowska,41 A. Onofre,126a,126e K. Onogi,103 P. U. E. Onyisi,11,w M. J. Oreglia,33 Y. Oren,153 D. Orestano,134a,134b N. Orlando,61b R. S. Orr,158 B. Osculati,52a,52b R. Ospanov,85 G. Otero y Garzon,29 H. Otono,71 M. Ouchrif,135d F. Ould-Saada,119 A. Ouraou,136 K. P. Oussoren,107 Q. Ouyang,35a M. Owen,55 R. E. Owen,19 V. E. Ozcan,20a N. Ozturk,8 K. Pachal,142 A. Pacheco Pages,13 L. Pacheco Rodriguez,136 C. Padilla Aranda,13 M. Pagáčová,50 S. Pagan Griso,16 F. Paige,27 P. Pais,87 K. Pajchel,119 G. Palacino,159b S. Palazzo,39a,39b S. Palestini,32 M. Palka,40b D. Pallin,36 E. St. Panagiotopoulou,10 C. E. Pandini,81 J. G. Panduro Vazquez,78 P. Pani,146a,146b S. Panitkin,27 D. Pantea,28b L. Paolozzi,51 Th. D. Papadopoulou,10 K. Papageorgiou,154 A. Paramonov,6 D. Paredes Hernandez,175 A. J. Parker,73 M. A. Parker,30 K. A. Parker,139 F. Parodi,52a,52b J. A. Parsons,37 U. Parzefall,50 V. R. Pascuzzi,158 E. Pasqualucci,132a S. Passaggio,52a Fr. Pastore,78 G. Pásztor,31,hh S. Pataraia,174 J. R. Pater,85 T. Pauly,32 J. Pearce,168 B. Pearson,113 L. E. Pedersen,38 M. Pedersen,119 S. Pedraza Lopez,166 R. Pedro,126a,126b S. V. Peleganchuk,109,d O. Penc,127 C. Peng,35a H. Peng,35b J. Penwell,62 B. S. Peralva,26b M. M. Perego,136 D. V. Perepelitsa,27 E. Perez Codina,159a L. Perini,92a,92b H. Pernegger,32 S. Perrella,104a,104b R. Peschke,44 V. D. Peshekhonov,66 K. Peters,44 R. F. Y. Peters,85 B. A. Petersen,32 T. C. Petersen,38 5 5 b 182002-11 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) P. Morettini,52a D. Mori,142 T. Mori,155 M. Morii,58 M. Morinaga,155 V. Morisbak,119 S. Moritz,84 A. K. Morley,150 G. Mornacchi,32 J. D. Morris,77 S. S. Mortensen,38 L. Morvaj,148 M. Mosidze,53b J. Moss,143 K. Motohashi,157 R. Mount,143 J. A. Murillo Quijada,19 W. J. Murray,169,131 H. Musheghyan,56 M. Muškinja,76 A. G. Myagk gy . F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a I. Naryshkin,123 T. Naumann,44 G. H. A. Neal,90 P. Yu. Nechaeva,96 T. J. Neep,85 A. Negri,121a,121b M. Negrini,22a S. Nektarijevic,106 C. Nellist,117 A. Nelson,162 k,127 P. Nemethy,110 A. A. Nepomuceno,26a M. Nessi,32,gg M. S. Neubauer,165 M. Neumann,174 R P. Nevski,27 P. R. Newman,19 D. H. Nguyen,6 T. Nguyen Manh,95 R. B. Nickerson,120 R. Nicolaidou,136 J. Nielsen,137 17 130 ff 81 19 119 27 155 P. Nevski,27 P. R. Newman,19 D. H. Nguyen,6 T. Nguyen Manh,95 R. B. Nickerson,120 R. Nicol A. Nikiforov,17 V. Nikolaenko,130,ff I. Nikolic-Audit,81 K. Nikolopoulos,19 J. K. Nilsen,119 P. Nilsson,27 Y. Ninomiya,155 A. Nisati,132a R. Nisius,101 T. Nobe,155 M. Nomachi,118 I. Nomidis,31 T. Nooney,77 S. Norberg,113 M. Nordberg,32 y . Norjoharuddeen,120 O. Novgorodova,46 S. Nowak,101 M. Nozaki,67 L. Nozka,115 K. Ntekas,1 7 142 44 55 31 101 H. Ohman,164 H. Oide,32 H. Okawa,160 Y. Okumura,155 T. Okuyama,67 A. Olariu,28b L. F. Oleiro Seabra,126a S. A. Olivares Pino,48 D. Oliveira Damazio,27 A. Olszewski,41 J. Olszowska,41 A. Onofre,126a,126e K. Onogi,103 P. U. E. Onyisi,11,w M. J. Oreglia,33 Y. Oren,153 D. Orestano,134a,134b N. Orlando,61b R. S. Orr,158 B. Osculati,52a,52b 85 29 71 135d 119 136 107 g P. U. E. Onyisi,11,w M. J. Oreglia,33 Y. Oren,153 D. Orestano,134a,134b N. Orlando,61b R. S. Orr,158 B. Osculati,52a,52b P. U. E. Onyisi,11,w M. J. Oreglia,33 Y. Oren,153 D. Orestano,134a,134b N. Orlando,61b R. S. Orr, Ospanov,85 G. Otero y Garzon,29 H. Otono,71 M. Ouchrif,135d F. Ould-Saada,119 A. Ouraou,136 E. Petit,57 A. Petridis,1 C. Petridou,154 P. Petroff,117 E. Petrolo,132a M. Petrov,120 F. Petrucci,134a,134b N. E. Pettersson,87 A. Peyaud,136 R. Pezoa,34b P. W. Phillips,131 G. Piacquadio,143,ii E. Pianori,169 A. Picazio,87 E. Piccaro,77 M. Piccinini,22a,22b 120 29 33 85 85 163 163 jj 3 E. Petit,57 A. Petridis,1 C. Petridou,154 P. Petroff,117 E. Petrolo,132a M. Petrov,120 F. Petrucci,134a,134b N. E. 182002-12 Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D. Sidorov,114 A. Sid E. Shulga,98 M. A. Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D. Sidorov,114 A. Sidoti,22a,22b 46 14 126 126d 146 128 14 117 84 E. Shulga,98 M. A. Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D. Sidorov,114 A. Sidoti,22a,22b F. Siegert,46 Dj. Sijacki,14 J. Silva,126a,126d S. B. Silverstein,146a V. Simak,128 Lj. Simic,14 S. Simion,117 E. Simioni,84 Sijacki,14 J. Silva,126a,126d S. B. Silverstein,146a V. Simak,128 Lj. Simic,14 S. Simion,117 E. Sim B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Siragusa,173 S. Yu. Sivoklokov,99 J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161 R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98 L. N. Smirnova,99,nn O. Smirnova,82 M. N. K. Smith,37 R. W. Smith,37 M. Smizanska,73 K. Sm ,nn O. Smirnova,82 M. N. K. Smith,37 R. W. Smith,37 M. Smizanska,73 K. Smolek,128 A. A. Sn 168 m 46 153 151 76 32 L. N. Smirnova,99,nn O. Smirnova,82 M. N. K. Smith,37 R. W. Smith,37 M. Smizanska,73 K. Smolek,128 A. A. Snesarev,96 S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. Solar,128 L. N. Smirnova, O. Smirnova, M. N. K. Smith, R. W. Smith, M. Smizanska, K. Smolek, A. A. Snesarev, S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. Solar,128 98 166 130 66 130 123 50 , , , , , obie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Sol v,98 U. Soldevila,166 A. A. Solodkov,130 A. Soloshenko,66 O. V. Solovyanov,130 V. Solovyev,123 P E. Yu. Soldatov,98 U. Soldevila,166 A. A. Solodkov,130 A. Soloshenko,66 O. V. Solovyanov,130 V , , , , y , y , , H. Son,161 H. Y. Song,35b,oo A. Sood,16 A. Sopczak,128 V. Sopko,128 V. Sorin,13 D. Sosa,59b C. L. Sotiropoulou,124a,124b R. Soualah,163a,163c A. M. Soukharev,109,d D. South,44 B. C. Sowden,78 S. Spagnolo,74a,74b M. Spalla,124a,124b 6 berg,169 F. Spanò,78 D. Sperlich,17 F. Spettel,101 R. Spighi,22a G. Spigo,32 L. A. Spiller,89 M. S M. Spangenberg,169 F. Spanò,78 D. Sperlich,17 F. Spettel,101 R. 182002-12 Spighi,22a G. Spigo,32 L. A. Spiller,89 M. Spousta,129 R. D. St. Denis,55,a A. Stabile,92a R. Stamen,59a S. Stamm,17 E. Stanecka,41 R. W. Stanek,6 C. Stanescu,134a M. Stanescu-Bellu,44 M. M. Stanitzki,44 S. Stapnes,119 E. A. Starchenko,130 G. H. Stark,33 J. Stark,57 P. Staroba,127 P. Starovoitov,59a S. Stärz,32 R. Staszewski,41 P. Steinberg,27 B. Stelzer,142 H. J. Stelzer,32 O. Stelzer-Chilton,159a 54 55 23 88 88 28b 56 101 R. D. St. Denis,55,a A. Stabile,92a R. Stamen,59a S. Stamm,17 E. Stanecka,41 R. W. Stanek,6 C. Stanescu,134a M. Stanescu-Bellu,44 M. M. Stanitzki,44 S. Stapnes,119 E. A. Starchenko,130 G. H. Stark,33 J. Stark,57 P. Staroba,127 59 32 41 27 142 32 159 R. D. St. Denis,55,a A. Stabile,92a R. Stamen,59a S. Stamm,17 E. Stanecka,41 R. W. Stanek,6 C. Stanescu,134a 44 44 119 130 33 57 127 R. D. St. Denis,55,a A. Stabile,92a R. Stamen,59a S. Stamm,17 E. Stanecka,41 R. W. Stanek, ,a A. Stabile,92a R. Stamen,59a S. Stamm,17 E. Stanecka,41 R. W. Stanek,6 C. Stanescu,134a R. D. St. Denis, A. Stabile, R. Stamen, S. Stamm, E. Stanecka, R. W. Stanek, C. Stanescu, M. Stanescu-Bellu,44 M. M. Stanitzki,44 S. Stapnes,119 E. A. Starchenko,130 G. H. Stark,33 J. Stark,57 P. Staroba,127 P. Starovoitov,59a S. Stärz,32 R. Staszewski,41 P. Steinberg,27 B. Stelzer,142 H. J. Stelzer,32 O. Stelzer-Chilton,159a M. M. Stanitzki,44 S. Stapnes,119 E. A. Starchenko,130 G. H. Stark,33 J. Stark,57 P. Staroba,127 M. Stanescu-Bellu,44 M. M. Stanitzki,44 S. Stapnes,119 E. A. Starchenko,130 G. H. Stark,33 J. S M. Stanescu-Bellu, M. M. Stanitzki, S. Stapnes, E. A. Starchenko, G. H. Stark, J. Stark, P. Staroba, P. Starovoitov,59a S. Stärz,32 R. Staszewski,41 P. Steinberg,27 B. Stelzer,142 H. J. Stelzer,32 O. Stelzer-Chilton,159a p P. Starovoitov,59a S. Stärz,32 R. Staszewski,41 P. Steinberg,27 B. Stelzer,142 H. J. Stelzer,32 O. Stelzer-Chilton,159a tenzel,54 G. A. Stewart,55 J. A. Stillings,23 M. C. Stockton,88 M. Stoebe,88 G. Stoicea,28b P. Sto Stewart,55 J. A. Stillings,23 M. C. Stockton,88 M. Stoebe,88 G. Stoicea,28b P. Stolte,56 S. Stonje g j A. R. Stradling,8 A. Straessner,46 M. E. Stramaglia,18 J. Strandberg,147 S. Strandberg,146a,146b A. Strandlie,119 M. Strauss,113 A. R. Stradling,8 A. Straessner,46 M. E. Stramaglia,18 J. Strandberg,147 S. Strandberg,146a,146b A. P. Strizenec,144b R. Ströhmer,173 D. M. Strom,116 R. Stroynowski,42 A. Strubig,106 S. A. Stucci,2 D. Su,143 J. Su,125 S. Suchek,59a Y. Sugaya,118 M. Suk,128 V. V. Sulin,96 S. Sultansoy,4c T. Sumida,69 S. Sun,58 X. Sun,35a D. Su,143 J. Su,125 S. Suchek,59a Y. Sugaya,118 M. Suk,128 V. V. Sulin,96 S. Sultansoy,4c T. 182002-12 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) A. Romaniouk,98 M. Romano,22a,22b S. M. Romano Saez,36 E. Romero Adam,166 N. Rompotis,1 E. Ros,166 S. Rosati,132a K. Rosbach,50 P. Rose,137 O. Rosenthal,141 N.-A. Rosien,56 V. Rosse E. Ros,166 S. Rosati,132a K. Rosbach,50 P. Rose,137 O. Rosenthal,141 N.-A. Rosien,56 V. Rossetti,146a,146b E. Rossi,104a,104b L. P. Rossi,52a J. H. N. Rosten,30 R. Rosten,138 M. Rotaru,28b I. Roth,171 J. Rothberg,138 D. Ro A. Rozanov,86 Y. Rozen,152 X. Ruan,145c F. Rubbo,143 M. S. Rudolph,158 F. Rühr,50 A. Ruiz-Martinez,31 Z. Rurikova,50 N. A. Rusakovich,66 A. Ruschke,100 H. L. Russell,138 J. P. Rutherfoord,7 N. Ruthmann,32 Y. F. Ryabov,123 M. Rybar,165 G. Rybkin,117 S. Ryu,6 A. Ryzhov,130 G. F. Rzehorz,56 A. F. Saavedra,150 G. Sabato,107 S. Sacerdoti,29 H. F-W. Sadrozinski,137 R. Sadykov,66 F. Safai Tehrani,132a P. Saha,108 M. Sahinsoy,59a M. Saimpert,136 T. Saito,155 H. Sakamoto,155 Y. Sakurai,170 G. Salamanna,134a,134b A. Salamon,133a,133b J. E. Salazar Loyola,34b D. Salek,107 P. H. Sales De Bruin,138 D. Salihagic,101 A. Salnikov,143 J. Salt,166 D. Salvatore,39a,39b F. Salvatore,149 A. Salvucci,61a A S l b 32 D S l 50 D S idi 154 A S h 104a 104b J Sá h 166 V S h M ti 166 P. H. Sales De Bruin,138 D. Salihagic,101 A. Salnikov,143 J. Salt,166 D. Salvatore,39a,39b F. Salvatore,149 A. Salvucci,61a A. Salzburger,32 D. Sammel,50 D. Sampsonidis,154 A. Sanchez,104a,104b J. Sánchez,166 V. Sanchez Martinez,166 A. Salzburger,32 D. Sammel,50 D. Sampsonidis,154 A. Sanchez,104a,104b J. Sánchez,166 V. Sa H. Sandaker,119 R. L. Sandbach,77 H. G. Sander,84 M. Sandhoff,174 C. Sandoval,21 R. Sandstroem,101 D. P. C. Sankey,131 M. Sannino,52a,52b A. Sansoni,49 C. Santoni,36 R. Santonico,133a,133b H. Santos,126a I. Santoyo C y pp A. Sapronov,66 J. G. Saraiva,126a,126d B. Sarrazin,23 O. Sasaki,67 Y. Sasaki,155 K. Sato,160 G. Sauvage,5,a E. Sauvan,5 G S 78 P S d 158,e N S i 101 C S 131 L S 80,r J S 33 C Sb 22a A Sb i i 22a,22b T S l 79 A. Sapronov,66 J. G. Saraiva,126a,126d B. Sarrazin,23 O. Sasaki,67 Y. Sasaki,155 K. Sato,160 G. Sauvage,5,a E. Sauvan,5 G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79 G. Savage, P. Savard, , N. Savic, C. Sawyer, L. Sawyer, , J. Saxon, C. Sbarra, A. Sbrizzi, , T. 182002-12 Scanlon, D. A. Scannicchio,162 M. Scarcella,150 V. Scarfone,39a,39b J. Schaarschmidt,171 P. Schacht,101 B. M. Schachtner,100 g y y D. A. Scannicchio,162 M. Scarcella,150 V. Scarfone,39a,39b J. Schaarschmidt,171 P. Schacht,101 B. M. Schachtner,100 Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. Sch p D. Schaile,100 R. D. Schamberger,148 V. Scharf,59a V. A. Schegelsky,123 D. Scheirich,129 M. Schernau,162 C. Schiavi,52a,52b 5 b S. Schier,137 C. Schillo,50 M. Schioppa,39a,39b S. Schlenker,32 K. R. Schmidt-Sommerfeld,101 K. Schmieden,32 C. Schmitt,84 S. Schmitt,44 S. Schmitz,84 B. Schneider,159a U. Schnoor,50 L. Schoeffel,136 A. Schoening,59b B. D. Schoenrock,91 E. Schopf,23 M. Schott,84 J. Schovancova,8 S. Schramm,51 M. Schreyer,173 N. Schuh,84 A. Schulte,84 M. J. Schultens,23 H.-C. Schultz-Coulon,59a H. Schulz,17 M. Schumacher,50 B. A. Schumm,137 Ph. Schune,136 A. Schwartzman,143 T. A. Schwarz,90 H. Schweiger,85 Ph. Schwemling,136 R. Schwienhorst,91 J. Schwindling,136 T. Schwindt,23 G. Sciolla,25 F. Scuri,124a,124b F. Scutti,89 J. Searcy,90 P. Seema,23 S. C. Seidel,105 A. Seiden,137 F. Seifert,128 J. M. Seixas,26a T. A. Schwarz, H. Schweiger, Ph. Schwemling, R. Schwienhorst, J. Schwindling, T. Schwindt, G. Sciolla, F. Scuri,124a,124b F. Scutti,89 J. Searcy,90 P. Seema,23 S. C. Seidel,105 A. Seiden,137 F. Seifert,128 J. M. Seixas,26a p L. Serkin,163a,163b M. Sessa,134a,134b R. Seuster,168 H. Severini,113 T. Sfiligoj,76 F. Sforza,32 A. Sfyrla,51 E. Shabalina,56 N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b S M Shaw 85 A Shcherbakova 146a,146b C Y Shehu 149 P Sherwood 79 L Shi 151,ll S Shimizu 68 C O Shimmin 162 p L. Serkin,163a,163b M. Sessa,134a,134b R. Seuster,168 H. Severini,113 T. Sfiligoj,76 F. Sforza,32 A. Sfyrla,51 E. Shabalina,56 N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b S. M. Shaw,85 A. Shcherbakova,146a,146b C. Y. Shehu,149 P. Sherwood,79 L. Shi,151,ll S. Shimizu,68 C. O. Shimmin,162 M Shi ji 102 M Shi k 66 mm A Sh l 96 D Sh l h S di 95 M J Sh h 33 S Sh j ii 92a 92b S Sh h 111 S. M. Shaw,85 A. Shcherbakova,146a,146b C. Y. Shehu,149 P. Sherwood,79 L. Shi,151,ll S. Shimizu,68 C. O. Shimmin,162 M. Shimojima,102 M. Shiyakova,66,mm A. Shmeleva,96 D. Shoaleh Saadi,95 M. J. Shochet,33 S. S M. Shiyakova,66,mm A. Shmeleva,96 D. Shoaleh Saadi,95 M. J. Shochet,33 S. Shojaii,92a,92b S. Shr . 182002-12 Vigani,120 M. Villa,22a,22b M. Villaplana Perez,92a,92b E. Vilucc G. H. A. Viehhauser,120 S. Viel,16 L. Vigani,120 M. Villa,22a,22b M. Villaplana Perez,92a,92b E. Vilucchi,49 M. G. Vincter,31 66 22 22b 149 10 128 174 128 124 124b g p Vinogradov,66 C. Vittori,22a,22b I. Vivarelli,149 S. Vlachos,10 M. Vlasak,128 M. Vogel,174 P. Voka Vittori,22a,22b I. Vivarelli,149 S. Vlachos,10 M. Vlasak,128 M. Vogel,174 P. Vokac,128 G. Volpi,124a,1 M. Volpi,89 H. von der Schmitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75 N. Vranjes, M. Vranjes Milosavljevic, V. Vrba, M. Vreeswijk, R. Vuillermet, I. Vukotic, Z. Vykydal, P. Wagner,23 W. Wagner,174 H. Wahlberg,72 S. Wahrmund,46 J. Wakabayashi,103 J. Walder,73 R. Walker,100 W. Walkowiak,141 V. Wallangen,146a,146b C. Wang,35c C. Wang,35d,86 F. Wang,172 H. Wang,16 H. Wang,42 J. Wang,44 J. Wang,150 K. Wang,88 P. Wagner,23 W. Wagner,174 H. Wahlberg,72 S. Wahrmund,46 J. Wakabayashi,103 J. Walder,73 R. Walker,100 W. Walkowiak,141 V W ll 146a,146b C W 35c C W 35d,86 F W 172 H W 16 H W 42 J W 44 J W 150 K W 88 R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30 D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85 79 84 18 173 6 120 62 56 M. Waugh,79 S. Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. W B. M. Waugh,79 S. Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56 50 10 32 2 32 32 23 50 65 ,107 P. S. Wells,32 T. Wenaus,27 T. Wengler,32 S. Wenig,32 N. Wermes,23 M. Werner,50 M. D. Wer Weiser,50 H. Weits,107 P. S. Wells,32 T. Wenaus,27 T. Wengler,32 S. Wenig,32 N. Wermes,23 M. W P. Werner,32 M. Wessels,59a J. Wetter,161 K. Whalen,116 N. L. Whallon,138 A. M. Wharton,73 A. White,8 M. J. White,1 R. White,34b D. Whiteson,162 F. J. Wickens,131 W. Wiedenmann,172 M. Wielers,131 P. Wienemann,23 C. Wiglesworth,38 L. A. M. Wiik-Fuchs,23 A. Wildauer,101 F. Wilk,85 H. G. Wilkens,32 H. H. Williams,122 S. Williams,107 C. Willis,91 S. Willocq,87 J. A. Wilson,19 I. Wingerter-Seez,5 F. Winklmeier,116 O. J. Winston,149 B. T. Winter,23 M. Wittgen,143 100 107 41 126 126 131 41 32 J. Wittkowski,100 T. M. H. Wolf,107 M. W. Wolter,41 H. Wolters,126a,126c S. D. Worm,131 B. K. M. J. , , , , , , , M. J. Woudstra,85 K. W. Wozniak,41 M. Wu,57 M. Wu,33 S. L. Wu,172 X. Wu,51 Y. Wu,90 T. R. Wyatt,85 B. M. Wynne,48 S. Xella,38 D. Xu,35a L. Xu,27 B. Yabsley,150 S. Yacoob,145a D. Yamaguchi,157 Y. Yamaguchi,118 A. Yamamoto,67 S. Yamamoto,155 T. Yamanaka,155 K. Yamauchi,103 Y. Yamazaki,68 Z. Yan,24 H. Yang,35e H. Yang,172 Y. Yang,151 Z. Yang,15 W-M. Yao,16 Y. C. Yap,81 Y. Yasu,67 E. Yatsenko,5 K. H. Yau Wong,23 J. Ye,42 S. Ye,27 I. Yeletskikh,66 A. L. Yen,58 E. Yildirim,84 K. Yorita,170 R. Yoshida,6 K. Yoshihara,122 C. Young,143 C. J. S. Young,32 S. Youssef,24 D. R. Yu,16 J. Yu,8 J. M. Yu,90 J. Yu,65 L. Yuan,68 S. P. Y. Yuen,23 I. Yusuff,30,tt B. Zabinski,41 R. Zaidan,64 A. M. Zaitsev,130,ff N. Zakharchuk,44 J. Zalieckas,15 A. Zaman,148 S. Zambito,58 L. Zanello,132a,132b D. Zanzi,89 C. Zeitnitz,174 M. Zeman,128 A. Zemla,40a J. C. Zeng,165 Q. Zeng,143 K. Zengel,25 O. Zenin,130 T. Ženiš,144a D. Zerwas,117 D. Zhang,90 F. Zhang,172 G. Zhang,35b,oo H. Zhang,35c J. Zhang,6 L. Zhang,50 R. Zhang,23 R. Zhang,35b,uu X. Zhang,35d Z. Zhang,117 X. Zhao,42 Y. Zhao,35d Z. Zhao,35b A. Zhemchugov,66 J. Zhong,120 B. Zhou,90 C. Zhou,47 L. Zhou,37 L. Zhou,42 M. Zhou,148 N. Zhou,35f C. G. Zhu,35d J. Tanaka,155 M. Tanaka,157 R. Tanaka,117 S. Tanaka,67 B. B. Tannenwald,111 S. Tapia Araya,34b S. Tapprogge,84 S. Tarem,152 G. F. Tartarelli,92a P. Tas,129 M. Tasevsky,127 T. Tashiro,69 E. Tassi,39a,39b A. Tavares Delgado,126a,126b Y. Tayalati,135e A. C. Taylor,105 G. N. Taylor,89 P. T. E. Taylor,89 W. Taylor,159b F. A. Teischinger,32 P. Teixeira-Dias,78 K. K. Temming,50 D. Temple,142 H. Ten Kate,32 P. K. Teng,151 J. J. Teoh,118 F. Tepel,174 S. Terada,67 K. Terashi,155 J. Terron,83 S. Terzo,13 M. Testa,49 R. J. Teuscher,158,m T. Theveneaux-Pelzer,86 J. P. Thomas,19 J. Thomas-Wilsker,78 E. N. Thompson,37 P. D. Thompson,19 A. S. Thompson,55 L. A. Thomsen,175 E. Thomson,122 M. Thomson,30 M. J. Tibbetts,16 R. E. Ticse Torres,86 V. O. Tikhomirov,96,pp Yu. A. Tikhonov,109,d S. Timoshenko,98 P. Tipton,175 S. Tisserant,86 K. Todome,157 T. Todorov,5,a S. Todorova-Nova,129 J. Tojo,71 S. Tokár,144a K. Tokushuku,67 E. Tolley,58 L. Tomlinson,85 M. Tomoto,103 L. Tompkins,143,qq K. Toms,105 B. Tong,58 E. Torrence,116 H. Torres,142 E. Torró Pastor,138 J. Toth,86,rr 6 5 182002-12 Sumida,69 S. Sun,58 X. Sun,35a J. E. Sundermann,50 K. Suruliz,149 G. Susinno,39a,39b M. R. Sutton,149 S. Suzuki,67 M. Svatos,127 M. Swiatlowski,33 I. Sykora,144a T. Sykora,129 D. Ta,50 C. Taccini,134a,134b K. Tackmann,44 J. Taenzer,158 A. Taffard,162 R. Tafirout,159a N. Taiblum,153 H. Takai,27 R. Takashima,70 T. Takeshita,140 Y. Takubo,67 M. Talby,86 A. A. Talyshev,109,d K. G. Tan,89 , , , , , , , I. Sykora,144a T. Sykora,129 D. Ta,50 C. Taccini,134a,134b K. Tackmann,44 J. Taenzer,158 A. Taffard,162 R. Tafirout,159a 153 27 70 140 67 86 109 d 89 I. Sykora,144a T. Sykora,129 D. Ta,50 C. Taccini,134a,134b K. Tackmann,44 J. Taenzer,158 A. Taffard,162 R. Tafirout,159a N. Taiblum,153 H. Takai,27 R. Takashima,70 T. Takeshita,140 Y. Takubo,67 M. Talby,86 A. A. Talyshev,109,d K. G. Tan,89 182002-13 182002-13 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) D. Temple,142 H. Ten Kate,32 P. K. Teng,151 J. J. Teoh,118 F. Tepel,174 S. Terada,67 K. Terashi,155 J. Terron,83 S. Terzo,13 M. Testa,49 R. J. Teuscher,158,m T. Theveneaux-Pelzer,86 J. P. Thomas,19 J. Thomas-Wilsker,78 E. N. Thompson,37 P. D. Thompson,19 A. S. Thompson,55 L. A. Thomsen,175 E. Thomson,122 M. Thomson,30 M. J. Tibbetts,16 R. E. Ticse Torres,86 V. O. Tikhomirov,96,pp Yu. A. Tikhonov,109,d S. Timoshenko,98 P. Tipton,175 S. Tisserant,86 K. Todome,157 T. Todorov,5,a S. Todorova-Nova,129 J. Tojo,71 S. Tokár,144a K. Tokushuku,67 E. Tolley,58 L. Tomlinson,85 j M. Tomoto,103 L. Tompkins,143,qq K. Toms,105 B. Tong,58 E. Torrence,116 H. Torres,142 E. T M. Tomoto,103 L. Tompkins,143,qq K. Toms,105 B. Tong,58 E. Torrence,116 H. Torres,142 E. Torró Pastor,138 J. Toth,86,rr 86 139 173 27 159 81 13 F. Touchard,86 D. R. Tovey,139 T. Trefzger,173 A. Tricoli,27 I. M. Trigger,159a S. Trincaz-Duvoid,81 M. F. Tripiana,13 W. Trischuk,158 B. Trocmé,57 A. Trofymov,44 C. Troncon,92a M. Trottier-McDonald,16 M. Trovatelli,168 L. Truong,163a,163c y g gg p W. Trischuk,158 B. Trocmé,57 A. Trofymov,44 C. Troncon,92a M. Trottier-McDonald,16 M. Trovatelli,168 L. Truong,163a,163c M. Trzebinski,41 A. Trzupek,41 J. C-L. Tseng,120 P. V. Tsiareshka,93 G. Tsipolitis,10 N. Tsirintanis,9 S. Tsiskaridze,13 V. Tsiskaridze,50 E. G. Tskhadadze,53a K. M. Tsui,61a I. I. Tsukerman,97 V. Tsulaia,16 S. Tsuno,67 D. Tsybychev,148 Y. Tu,61b ki,41 A. Trzupek,41 J. C-L. Tseng,120 P. V. Tsiareshka,93 G. Tsipolitis,10 N. Tsirintanis,9 S. Tsis 50 E. G. Tskhadadze,53a K. M. Tsui,61a I. I. Tsukerman,97 V. Tsulaia,16 S. Tsuno,67 D. Tsybychev, y y V. 182002-12 Tudorache,28b A. N. Tuna,58 S. A. Tupputi,22a,22b S. Turchikhin,66 D. Turecek,128 D. Turge 55 6 6 A. Tudorache,28b V. Tudorache,28b A. N. Tuna,58 S. A. Tupputi,22a,22b S. Turchikhin,66 D. Tu R. Turra,92a,92b A. J. Turvey,42 P. M. Tuts,37 M. Tyndel,131 G. Ucchielli,22a,22b I. Ueda,155 M. Ughetto,146a,146b F. Ukegawa,160 G. Unel,162 F. C. Ungaro,89 Y. Unno,67 C. Unverdorben,100 J. Urban,144b P. Urquijo,89 P. Urrejola G. Unal,32 A. Undrus,27 G. Unel,162 F. C. Ungaro,89 Y. Unno,67 C. Unverdorben,100 J. Urban,144b P. Urquijo,89 P. Urrejola,84 G. Usai,8 A. Usanova,63 L. Vacavant,86 V. Vacek,128 B. Vachon,88 C. Valderanis,100 E. Valdes Santurio,146a,146b G. Unal, A. Undrus, G. Unel, F. C. Ungaro, Y. Unno, C. Unverdorben, J. Urban, G. Usai,8 A. Usanova,63 L. Vacavant,86 V. Vacek,128 B. Vachon,88 C. Valderanis,100 E. V g q j j G. Usai,8 A. Usanova,63 L. Vacavant,86 V. Vacek,128 B. Vachon,88 C. Valderanis,100 E. Valdes Santurio,146a,146b Valencic,107 S. Valentinetti,22a,22b A. Valero,166 L. Valery,13 S. Valkar,129 J. A. Valls Ferrer,166 W. inetti,22a,22b A. Valero,166 L. Valery,13 S. Valkar,129 J. A. Valls Ferrer,166 W. Van Den Wollenberg, P. C. Van Der Deijl,107 H. van der Graaf,107 N. van Eldik,152 P. van Gemmeren,6 J. Van Nieuwkoop,142 I. van Vulpen,107 P. C. Van Der Deijl,107 H. van der Graaf,107 N. van Eldik,152 P. van Gemmeren,6 J. Van N j . C. van Woerden,32 M. Vanadia,132a,132b W. Vandelli,32 R. Vanguri,122 A. Vaniachine,130 P. Vank 132 7 42 81 8 150 R. Vari,132a E. W. Varnes,7 T. Varol,42 D. Varouchas,81 A. Vartapetian,8 K. E. Varvell,150 J. G. Vasquez,175 F. Vazeille,36 T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a R. Vari,132a E. W. Varnes,7 T. Varol,42 D. Varouchas,81 A. Vartapetian,8 K. E. Varvell,150 J. G. Vasquez,175 F. Vazeille,36 T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107 107 46 142 82 165 139 139 T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107 q g A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107 M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke, ulen,107 A. Vest,46,ss M. C. Vetterli,142,e O. Viazlo,82 I. Vichou,165,a T. Vickey,139 O. E. Vickey B y A. Viehhauser,120 S. Viel,16 L. H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84 S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b M. zur Nedden,17 and L. Zwalinski32 H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84 S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b M. zur Nedden,17 and L. Zwalinski32 (ATLAS Collaboration) (ATLAS Collaboration) 1Department of Physics, University of Adelaide, Adelaide, Australia 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, AB, Canada 4aDepartment of Physics, Ankara University, Ankara, Turkey 4bIstanbul Aydin University, Istanbul, Turkey 4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France 6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA 7Department of Physics, University of Arizona, Tucson, Arizona, USA 8Department of Physics, The University of Texas at Arlington, Arlington, Texas, USA 9Physics Department, University of Athens, Athens, Greece 10Physics Department, National Technical University of Athens, Zografou, Greece 11Department of Physics, The University of Texas at Austin, Austin, Texas, USA 12Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain, Spain 14Institute of Physics, University of Belgrade, Belgrade, Serbia 15Department for Physics and Technology, University of Bergen, Bergen, Norway 16Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA 17Department of Physics, Humboldt University, Berlin, Germany 18Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 19School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 20aDepartment of Physics, Bogazici University, Istanbul, Turkey 20bDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey 20dIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey, Turkey 20eBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey, Turkey 21Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22aINFN Sezione di Bologna, Italy 22bDipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy 23Physikalisches Institut, University of Bonn, Bonn, Germany 24Department of Physics, Boston University, Boston, Massachusetts, USA 25Department of Physics, Brandeis University, Waltham, Massachusetts, USA 26aUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 26bElectrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Brazil 26cFederal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei, Brazil 26dInstituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 27Physics Department, Brookhaven National Laboratory, Upton, New York, USA 28aTransilvania University of Brasov, Brasov, Romania, Romania 28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 28cNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca, Romania 28dUniversity Politehnica Bucharest, Bucharest, Romania 28eWest University in Timisoara, Timisoara, Romania 29Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 30Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 31Department of Physics, Carleton University, Ottawa, ON, Canada 32CERN, Geneva, Switzerland 33Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA 34aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile 34bDepartamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 35aInstitute of High Energy Physics, Chinese Academy of Sciences, Beijing, China 35bDepartment of Modern Physics, University of Science and Technology of China, Anhui, China 35cDepartment of Physics, Nanjing University, Jiangsu, China 13Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain, Spain 14 14Institute of Physics, University of Belgrade, Belgrade, Serbia 15Department for Physics and Technology, University of Bergen, Bergen, Norway instein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 19School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 32CERN, Geneva, Switzerland 182002-15 182002-15 182002-12 Woudstra,85 K. W. Wozniak,41 M. Wu,57 M. Wu,33 S. L. Wu,172 X. Wu,51 Y. Wu,90 T. R. Wyatt,85 B. M. Wynne,48 S. Xella,38 D. Xu,35a L. Xu,27 B. Yabsley,150 S. Yacoob,145a D. Yamaguchi,157 Y. Yamaguchi,118 A. Yamamoto,67 S. Yamamoto,155 T. Yamanaka,155 K. Yamauchi,103 Y. Yamazaki,68 Z. Yan,24 H. Yang,35e H. Yang,172 Y. Yang,151 Z. Yang,15 W-M. Yao,16 Y. C. Yap,81 Y. Yasu,67 E. Yatsenko,5 K. H. Yau Wong,23 J. Ye,42 S. Ye,27 I. Yeletskikh,66 A. L. Yen,58 E. Yildirim,84 K. Yorita,170 R. Yoshida,6 K. Yoshihara,122 C. Young,143 C. J. S. Young,32 S. Youssef,24 D. R. Yu,16 J. Yu,8 J. M. Yu,90 J. Yu,65 L. Yuan,68 S. P. Y. Yuen,23 I. Yusuff,30,tt B. Zabinski,41 R. Zaidan,64 A. M. Zaitsev,130,ff N. Zakharchuk,44 J. Zalieckas,15 A. Zaman,148 S. Zambito,58 L. Zanello,132a,132b D. Zanzi,89 C. Zeitnitz,174 M. Zeman,128 A. Zemla,40a J. C. Zeng,165 Q. Zeng,143 K. Zengel,25 O. Zenin,130 T. Ženiš,144a D. Zerwas,117 D. Zhang,90 F. Zhang,172 G. Zhang,35b,oo H. Zhang,35c J. Zhang,6 L. Zhang,50 R. Zhang,23 R. Zhang,35b,uu X. Zhang,35d Z. Zhang,117 X. Zhao,42 Y. Zhao,35d Z. Zhao,35b A. Zhemchugov,66 J. Zhong,120 B. Zhou,90 C. Zhou,47 L. Zhou,37 L. Zhou,42 M. Zhou,148 N. Zhou,35f C. G. Zhu,35d 182002-14 182002-14 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84 S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b M. zur Nedden,17 and L. Zwalinski32 182002-15 week ending 28 OCTOBER 2016 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) 35dSchool of Physics, Shandong University, Shandong, China 35eDepartment of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai Jiao Tong University, Shanghai; (also affiliated with PKU-CHEP), China 35fPhysics Department, Tsinghua University, Beijing 100084, China 36Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 37Nevis Laboratory, Columbia University, Irvington, New York, USA 38Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 39aINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 39bDipartimento di Fisica, Università della Calabria, Rende, Italy 40aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 41Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 42Physics Department, Southern Methodist University, Dallas, Texas, USA 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA 44DESY, Hamburg and Zeuthen, Germany 45Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 47Department of Physics, Duke University, Durham, North Carolina, USA 48SUPA—School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49INFN Laboratori Nazionali di Frascati, Frascati, Italy 50Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 51Section de Physique, Université de Genève, Geneva, Switzerland 52aINFN Sezione di Genova, Italy 52bDipartimento di Fisica, Università di Genova, Genova, Italy 53aE. Andronikashvili Institute of Physics, Iv. Clermont-Ferrand, France 37Nevis Laboratory, Columbia University, Irvington, New York, USA 38 37Nevis Laboratory, Columbia University, Irvington, New York, USA NFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 42Physics Department, Southern Methodist University, Dallas, Texas, USA hysics Department, University of Texas at Dallas, Richardson, Texas, USA 44 hl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany t für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 47 47Department of Physics, Duke University, Durham, North Carolina, USA 49INFN Laboratori Nazionali di Frascati, Frascati, Italy ultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 51 51Section de Physique, Université de Genève, Geneva, Switzerland 52 4aINFN Sezione di Lecce, Italy 74bDipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 5 74bDipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 75Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 76Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slovenia 77School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 78Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 79Department of Physics and Astronomy, University College London, London, United Kingdom 80Louisiana Tech University, Ruston, Los Angeles, USA 81L b i d Ph i N lé i d H E i UPMC d U i i é P i Did d CNRS/IN2P3 P i F hysics and Astronomy, University College London, London, United Kingdom 80 80Louisiana Tech University, Ruston, Los Angeles, USA 81Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 182002-16 182002-16 52aINFN Sezione di Genova, Italy 52bDipartimento di Fisica, Università di Genova, Genova, Italy 57Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 58 cs, The University of Hong Kong, Hong Kong, China sity of Science and Technology, Clear Water Bay, K 62Department of Physics, Indiana University, Bloomington, Indiana, USA 63 63Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 64 64University of Iowa, Iowa City, Iowa, USA 65Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA 66 or Nuclear Research, JINR Dubna, Dubna, Russia High Energy Accelerator Research Organization, Tsukuba, Japan 68 gy Accelerator Research Organization, Tsukuba, Ja 68Graduate School of Science, Kobe University, Kobe, Japan 69 69Faculty of Science, Kyoto University, Kyoto, Japan 70 70Kyoto University of Education, Kyoto, Japan 71Department of Physics, Kyushu University, Fukuoka, Japan al de La Plata and CONICET, La Plata, Argentina Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 73 73Physics Department, Lancaster University, Lancaster, United Kingdom 74 Lancaster University, Lancaster, United Kingdom 4 P H Y S I C A L R E V I E W L E T T E R S Javakhishvili Tbilisi State University, Tbilisi, Georgia 53bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 54II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55SUPA—School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 57Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 58Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 59aKirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 59bPhysikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 59cZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 60Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 61aDepartment of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China 61bDepartment of Physics, The University of Hong Kong, Hong Kong, China 61cDepartment of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 62Department of Physics, Indiana University, Bloomington, Indiana, USA 63Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 64University of Iowa, Iowa City, Iowa, USA 65Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA 66Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 67KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 68Graduate School of Science, Kobe University, Kobe, Japan 69Faculty of Science, Kyoto University, Kyoto, Japan 70Kyoto University of Education, Kyoto, Japan 71Department of Physics, Kyushu University, Fukuoka, Japan 72Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 73Physics Department, Lancaster University, Lancaster, United Kingdom 74aINFN Sezione di Lecce, Italy 74bDipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 75Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 76Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slovenia 77School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 78Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 79Department of Physics and Astronomy, University College London, London, United Kingdom 80 PRL 117, 182002 (2016) P H Y S I C A L R E V I E W L E T T E R S 28 OCTOBER 2016 35dSchool of Physics, Shandong University, Shandong, China f y g y g 35eDepartment of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology, 35eDepartment of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai Jiao Tong University Shanghai; (also affiliated with PKU CHEP) China 35fPhysics Department, Tsinghua University, Beijing 100084, China de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) 82Fysiska institutionen, Lunds universitet, Lund, Sweden 83Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 84Institut für Physik, Universität Mainz, Mainz, Germany 85School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 86CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 87Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 88Department of Physics, McGill University, Montreal, QC, Canada 89School of Physics, University of Melbourne, Victoria, Australia 90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA 91Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 92aINFN Sezione di Milano, Italy 92bDipartimento di Fisica, Università di Milano, Milano, Italy 93B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 94National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republic of Belarus 95Group of Particle Physics, University of Montreal, Montreal, QC, Canada 96P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 97Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 98National Research Nuclear University MEPhI, Moscow, Russia 99D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 100Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 101Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 102Nagasaki Institute of Applied Science, Nagasaki, Japan 103Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 104aINFN Sezione di Napoli, Italy 104bDipartimento di Fisica, Università di Napoli, Napoli, Italy 105Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 106Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 107Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 108Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 109Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 110Department of Physics, New York University, New York, New York, USA 111Ohio State University, Columbus, Ohio, USA 112Faculty of Science, Okayama University, Okayama, Japan 113Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 114Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 115Palacký University, RCPTM, Olomouc, Czech Republic 116Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 117LAL, Univ. P H Y S I C A L R E V I E W L E T T E R S Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 118Graduate School of Science, Osaka University, Osaka, Japan 119Department of Physics, University of Oslo, Oslo, Norway 120Department of Physics, Oxford University, Oxford, United Kingdom 121aINFN Sezione di Pavia, Italy 121bDipartimento di Fisica, Università di Pavia, Pavia, Italy 122Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 123National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 124aINFN Sezione di Pisa, Italy 124bDipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 125Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 126aLaboratório de Instrumentação e Física Experimental de Partículas—LIP, Lisboa, Portugal 126bFaculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 126cDepartment of Physics, University of Coimbra, Coimbra, Portugal 126dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 126eDepartamento de Fisica, Universidade do Minho, Braga, Portugal 126fDepartamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 126gDep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 127Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 128Czech Technical University in Prague, Praha, Czech Republic 129Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 130St t R h C t I tit t f Hi h E Ph i (P t i ) NRC KI R i 101Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 102 102Nagasaki Institute of Applied Science, Nagasaki, Japan uate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 104 182002-16 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S 1aINFN Sezione di Pavia, Italy 121bDipartimento di Fisica, Università di Pavia, Pavia, Italy 123National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 124aINFN S i di Pi I l h Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersbu 124 urchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 124aINFN S i di Pi It l Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 124aINFN Sezione di Pisa, Italy 124b 04aINFN Sezione di Napoli, Italy 104bDipartimento di Fisica, Università di Napoli, Napoli, Italy 105Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 106 l Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 108Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 109 109Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 110 110Department of Physics, New York University, New York, New York, USA 111 111Ohio State University, Columbus, Ohio, USA y 112Faculty of Science, Okayama University, Okayama, Japan L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 114 114Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 115 115Palacký University, RCPTM, Olomouc, Czech Republic 16 cký University, RCPTM, Olomouc, Czech Republic 116Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 117 AL, Univ. Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 118 118Graduate School of Science, Osaka University, Osaka, Japan 119 119Department of Physics, University of Oslo, Oslo, Norway 120Department of Physics, Oxford University, Oxford, United Kingdom 121 21aINFN Sezione di Pavia, Italy 124aINFN Sezione di Pisa, Italy y 124bDipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy ment of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA atório de Instrumentação e Física Experimental de Partículas—LIP, Lisboa, Portugal 126b bFaculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 126cDepartment of Physics, University of Coimbra, Coimbra, Portugal 26d 126dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 126 126eDepartamento de Fisica, Universidade do Minho, Braga, Portugal 126eDepartamento de Fisica, Universidade do Minho, Braga, Portugal artamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain nto de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 126gDep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 127 128Czech Technical University in Prague, Praha, Czech Republic 129Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 130State Research Center Institute for High Energy Physics (Protvino), NRC KI, Russia 131 131Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 182002-17 week ending 28 OCTOBER 2016 59aTRIUMF, Vancouver, BC, Canada 159bDepartment of Physics and Astronomy, York University, Toronto, ON, Canada 160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan 160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan 161Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 161Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 162D f Ph i d A U i i f C lif i I i I i C lif i USA 161Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 162Department of Physics and Astronomy University of California Irvine Irvine California USA ment of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA t of Physics and Astronomy, University of California Irvine, Irvine, California, USA 163 163aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy 163b P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) 132aINFN Sezione di Roma, Italy 132bDipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 133aINFN Sezione di Roma Tor Vergata, Italy 133bDipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 134aINFN Sezione di Roma Tre, Italy 134bDipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy 135aFaculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies—Université Hassan II, Casablanca, Morocco 135bCentre National de l’Energie des Sciences Techniques Nucleaires, Rabat, Morocco 135cFaculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocco 135dFaculté des Sciences, Université Mohamed Premier and LPTPM, Oujda, Morocco 135eFaculté des sciences, Université Mohammed V, Rabat, Morocco 136DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique aux Energies Alternatives), Gif-sur-Yvette, France 137Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA 138Department of Physics, University of Washington, Seattle, Washington, USA 139Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 140Department of Physics, Shinshu University, Nagano, Japan 141Fachbereich Physik, Universität Siegen, Siegen, Germany 142Department of Physics, Simon Fraser University, Burnaby, BC, Canada 143SLAC National Accelerator Laboratory, Stanford, California, USA 144aFaculty of Mathematics, Physics & Informatics, Comenius University, Bratislava, Slovak Republic 144bDepartment of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republ 145aDepartment of Physics, University of Cape Town, Cape Town, South Africa 145bDepartment of Physics, University of Johannesburg, Johannesburg, South Africa 145cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa 146aDepartment of Physics, Stockholm University, Sweden 146bThe Oskar Klein Centre, Stockholm, Sweden 147Physics Department, Royal Institute of Technology, Stockholm, Sweden 148Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, New York, USA 149Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 150School of Physics, University of Sydney, Sydney, Australia 151Institute of Physics, Academia Sinica, Taipei, Taiwan 152Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 153Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 154Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 155International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 156Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 157Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 158Department of Physics, University of Toronto, Toronto, ON, Canada 159aTRIUMF, Vancouver, BC, Canada 159bDepartment of Physics and Astronomy, York University, Toronto, ON, Canada 160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan 161Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 162Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 163aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy 163bICTP, Trieste, Italy 163cDipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 164Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 165Department of Physics, University of Illinois, Urbana, Illinois, USA 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingenier Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 167Department of Physics, University of British Columbia, Vancouver, BC, Canada 168Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 169Department of Physics University of Warwick Coventry United Kingdom PRL 117, 182002 (2016) 28 OCTOBER 201 bDipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy ulté des Sciences, Université Mohamed Premier and LPTPM, Oujda, Morocco 135 135eFaculté des sciences, Université Mohammed V, Rabat, Morocco herches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France aux Energies Alternatives), Gif-sur-Yvette, France ute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA 138Department of Physics, University of Washington, Seattle, Washington, USA artment of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 140 139Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 140 140Department of Physics, Shinshu University, Nagano, Japan 141 141Fachbereich Physik, Universität Siegen, Siegen, Germany nt of Physics, Simon Fraser University, Burnaby, B 143SLAC National Accelerator Laboratory, Stanford, California, USA lty of Mathematics, Physics & Informatics, Comenius University, Bratislava, Slovak Republic ysics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 145aDepartment of Physics, University of Cape Town, Cape Town, South Africa 5b 145bDepartment of Physics, University of Johannesburg, Johannesburg, South Africa 145 ersity of the Witwatersrand, Johannesburg, South Africa 5cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa 146aDepartment of Physics, Stockholm University, Sweden 146bThe Oskar Klein Centre, Stockholm, Sweden 147Physics Department, Royal Institute of Technology, Stockholm, Sweden Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, New York, USA ts of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, New York, USA 149Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom artment of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 150 151Institute of Physics, Academia Sinica, Taipei, Taiwan 152Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 152Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel of Physics, Technion: Israel Institute of Technology, 153Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, I 154 ackler School of Physics and Astronomy, Tel Aviv U 154Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 155International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 156Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 156Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 157 157Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 158 158Department of Physics, University of Toronto, Toronto, ON, Canada 159 158Department of Physics, University of Toronto, Toronto, ON, Canada 159 163bICTP, Trieste, Italy 163cDipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 164Department of Physics and Astronomy University of Uppsala Uppsala Sweden 163cDipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 164D t t f Ph i d A t U i it f U l U l S d Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 164Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 165 p f y y y f pp pp 165Department of Physics, University of Illinois, Urbana, Illinois, USA p f y , y f , , , 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 167Department of Physics, University of British Columbia, Vancouver, BC, Canada 168Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 169Department of Physics, University of Warwick, Coventry, United Kingdom 170Waseda University, Tokyo, Japan 171Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 172D f Ph i U i i f Wi i M di Wi i USA p f y y f 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 167Department of Physics, University of British Columbia, Vancouver, BC, Canada 168Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 169Department of Physics University of Warwick Coventry United Kingdom 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain orpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Inge uscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingenier to de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valenc 167 167Department of Physics, University of British Columbia, Vancouver, BC, Canada 168 168Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 169 70Waseda University, Tokyo, Japan y y p 171Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 172 epartment of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 172 72Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 172Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 173Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 182002-18 182002-18 182002-18 week ending 28 OCTOBER 2016 P H Y S I C A L R E V I E W L E T T E R S P H Y S I C A L R E V I E W L E T T E R S uAlso at Department of Physics, National Tsing Hua University, Taiwan. uAlso at Department of Physics, National Tsing Hua University, Taiwan. vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. wAlso at Department of Physics, The University of Texas at Austin, Austin, TX, USA. wAlso at Department of Physics, The University of Texas at Austin, Austin, TX, USA. xAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. xAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. yAlso at CERN, Geneva, Switzerland. gian Technical University (GTU), Tbilisi, Georgia. zAlso at Georgian Technical University (GTU), Tbilisi, Georgia. aaAlso at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan bb aaAlso at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. bb so at Ochadai Academic Production, Ochanomizu U bbAlso at Manhattan College, New York, NY, USA. bbAlso at Manhattan College, New York, NY, USA. ccAlso at Hellenic Open University, Patras, Greece. dd p y, , ddAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan ee l h l f h i h d i i h d hi ddAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, ddAlso at Academia Sinica Grid Computing, Institute of Physics, Acad eeAlso at School of Physics, Shandong University, Shandong, China. ff eeAlso at School of Physics, Shandong University, Shandong, China. ff ffAlso at Moscow Institute of Physics and Technology State University, Dolgo ffAlso at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. o at Section de Physique, Université de Genève, Ge hhAlso at Eotvos Lorand University, Budapest, Hungary. ii iiAlso at Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook jj so at Departments of Physics & Astronomy and Ch iiAlso at Departments of Physics & Astronomy and Chemistry, Stony Brook University, Sto jj jjAlso at International School for Advanced Studies (SISSA), Trieste, Italy. kk jjAlso at International School for Advanced Studies (SISSA), Trieste, Italy. kk kkAlso at Department of Physics and Astronomy, University of South Carolina, Columbia, SC, USA. P H Y S I C A L R E V I E W L E T T E R S PRL 117, 182002 (2016) 174Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 175Department of Physics, Yale University, New Haven, Connecticut, USA 174Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 175Department of Physics, Yale University, New Haven, Connecticut, USA f y y 176Yerevan Physics Institute, Yerevan, Armenia 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France aDeceased. b bAlso at Department of Physics, King’s College London, London, United Kingdom. lso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. dAlso at Novosibirsk State University, Novosibirsk, Russia. eAlso at TRIUMF, Vancouver BC, Canada. f fAlso at Department of Physics & Astronom fAlso at Department of Physics & Astronomy, University of Louisville fAlso at Department of Physics & Astronomy, University of Louisville, Louisville, KY, USA. gAlso at Department of Physics, California State University, Fresno, CA, USA. h hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i iAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain j jAlso at Departamento de Fisica e Astronomia, Fa k kAlso at Tomsk State University, Tomsk, Russia. l lAlso at Universita di Napoli Parthenope, Napoli, Italy. lAlso at Universita di Napoli Parthenope, Napoli, Italy. mAlso at Institute of Particle Physics (IPP), Canada. nAlso at National Institute of Physics and Nuclear Engineering, Bucharest, Romania. nAlso at National Institute of Physics and Nuclear Engineering, Bucharest, Romania. oAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Pe p pAlso at Department of Physics, The University of Michigan, Ann Arbor, MI, USA. pAlso at Department of Physics, The University of Michigan, Ann Arbor, MI, USA. Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. qAlso at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Tow rAlso at Louisiana Tech University, Ruston, LA, USA. sAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. t sAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. t o at Institucio Catalana de Recerca i Estudis Avanca tAlso at Graduate School of Science, Osaka University, Osaka, Japan. tAlso at Graduate School of Science, Osaka University, Osaka, Japan. P H Y S I C A L R E V I E W L E T T E R S ll kkAlso at Department of Physics and Astronomy, University of South Carolina, Columbia, y g g y g mmAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Acad mmAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Sofia, Bulgaria. nnAlso at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia. nnAlso at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia. ooAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. ooAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. ppAlso at National Research Nuclear University MEPhI, Moscow, Russia. ppAlso at National Research Nuclear University MEPhI, Moscow, Russia. National Research Nuclear University MEPhI, Mos qqAlso at Department of Physics, Stanford University, Stanford, CA, USA. qqAlso at Department of Physics, Stanford University, Stanford, CA, USA. rrAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. rrAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. y g ssAlso at Flensburg University of Applied Sciences, Flensburg, Germany. at Flensburg University of Applied Sciences, Flensb ttAlso at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. ttAlso at University of Malaya, Department of Physics, Kuala Lumpur, Malaysi uuAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. uuAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. 182002-19
https://openalex.org/W4389291772	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/095D34719EE02CA2E91B3D2F9B0CE738/S1360674323000515a.pdf/div-class-title-language-change-is-span-class-italic-wicked-span-semantic-and-social-meaning-of-a-polysemous-adjective-div.pdf	English	null	Language change is <i>wicked</i>: semantic and social meaning of a polysemous adjective	English language and linguistics	2,023	cc-by	9,643	1 The authors would like to thank Justyna Robinson for sharing her wicked data with us and for engaging in discussions regarding her ﬁndings. We would also like to thank the anonymous reviewers and statistics consultant as well as Laurel Brinton for valuable and insightful comments on previous versions of this article. Any remaining shortcomings are the responsibilities of the named authors. English Language and Linguistics, 28.1: 135–156. © The Author(s), 2023. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. English Language and Linguistics, 28.1: 135–156. © The Author(s), 2023. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. Language change is wicked: semantic and social meaning of a polysemous adjective1 R H Y S J . S A N D O W Queen Mary University of London G E O R G E B A I L E Y University of York and N ATA L I E B R A B E R Nottingham Trent University (Received 16 December 2022; revised 21 August 2023) (Received 16 December 2022; revised 21 August 2023) As a result of an ameliorative shift-to-opposite, the polysemous adjective wicked is an auto- antonym, having two senses opposite in meaning, that is, ‘evil’ and ‘good’. We discuss two studies which explore the social life of this word, with the ﬁrst focusing on its production and the second on its perception. In the ﬁrst study, conducted in Cornwall, United Kingdom, we ﬁnd that young men are most advanced in the use of wicked ‘good’ while young women appear not to contribute to the incrementation, that is, the advancement, of this change. In the second study, conducted online across England, we ﬁnd wicked ‘good’, relative to its synonym good, to be perceived as less young and to be evaluated positively across disparate characteristics relating to status and solidarity, particularly by older men. We ﬁnd wicked ‘evil’, in contrast to its synonym evil, to be evaluated higher in status-type characteristics. This newly uncovered indexical ﬁeld of wicked presents a possible explanation for the observed changes in production, contributing to ongoing questions about the role of social meaning in driving the incrementation of change. More generally, this article adds to the growing yet limited literature which explores semantic variation through the lens of variationist sociolinguistics. Keywords: semantic change, language perception, language production, matched-guise technique, wicked 1 Introduction The social meaning of language variation has been prominent since the advent of the third wave of the variationist sociolinguistic paradigm (e.g. Eckert 2000; Kiesling 2004; https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 136 Campbell-Kibler 2007; Podesva 2007; Moore & Podesva 2009; Cole 2020; see also Labov 1963). Such works have shed light on the non-referential meanings of linguistic features, from words (e.g. Kiesling 2004; Bucholtz 2009) to morphosyntax (Moore & Podesva 2009), to phonetics and phonology (Foulkes & Doherty 2006; Eckert & Labov 2017). However, the study of semantic meaning in sociolinguistics has been somewhat neglected (although, see Robinson 2010a, 2010b, 2012a, 2012b, 2014; Beal & Burbano-Elizondo 2012; Bailey & Durham 2021; Sandow 2023a, 2023b, in press; for discussion see Durkin 2012; Robinson 2012a; Sandow 2021). This article seeks to redress the limited, but growing, body of literature on the socially mediated trajectory of semantic change in the context of social meaning by exploring the polysemous adjective wicked. Inthis articlewe report on the usage and perception of wicked, using evidence from two studies. The ﬁrst, conducted in Cornwall in 2017–18, focuses on the social distribution of the senses of this polysemous word, such as ‘evil’ and ‘good’. The second study, conducted online across England in 2022, employs the matched-guise technique and investigates the perception of two senses of wicked, ‘evil’ and ‘good’. We ﬁnd that while a change towards wicked ‘good’ is female-led among older speakers, younger women do not continue with the incrementation of this change; in contrast, their male counterparts rapidly accelerate this incrementation. In terms of perception, in study 2, we ﬁnd that older men perceive wicked ‘good’ to index both solidarity-type and status-type traits, while wicked ‘evil’ is perceived to index status-type traits, particularly ‘educated’. We conclude that the social meaning of the senses of wicked can, to a great extent, account for the socially mediated incrementation, and lack thereof, of this polysemous adjective. 2 We use the term semantic as opposed to semasiological throughout this article. https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 4 For an overview of social meaning in semantics and pragmatics see Beltrama (2020). 3 The perception aspect of this study did not distinguish between discourse marker and quotative like. 4 LANGUAGE CHANGE IS WICKED 137 cheeky ‘mildly illicit’, with younger speakers in Britain leading this shift from cheeky ‘impudent’ towards greater acceptance of the innovative variant. Such studies have demonstrated the structured heterogeneity of semantic change. However, this body of socio-semantic research literature remains somewhat limited. We argue that without a lexical perspective, our knowledge of language and society, and the subsequent theory, is necessarily abridged. By contributing to a developing lexis-oriented branch of sociolinguistic theory, we are testing the utility of sociolinguistic theory and working towards a more holistic knowledge of the social life of language and the mechanisms of linguistic change. In recent decades, a number of studies have investigated the social meaning of lexical items. Such studies typically either infer social meanings from usage patterns (e.g. Kiesling 2004; Bucholtz 2009; Snell 2018) or from metalinguistic discussion (e.g. Robinson 2010a; Braber 2022; Sandow 2022, 2023a, 2023b). While studies have used experimental methods to explore the social meaning of phonetic, phonological and morphosyntactic features, such as the matched-guise technique (Campbell-Kibler 2007; Johnstone & Kiesling 2008; Gilabert & Fuss 2018), Implicit Association Test (Campbell-Kibler 2012; McKenzie & Carrie 2018; Álvarez-Mosquera & Marín-Gutiérrez 2021) and the Social Category Association test (Llamas et al. 2016), limited research has employed such methods at the level of lexical variation. Pragmatic and discourse features have also been studied through the matched-guise technique (e.g. Dailey-O’Cain 2000; Buchstaller 2006; Maddeaux & Dinkin 2017; Davydova & Hazen 2021; Schleef 2022). For example, Dailey-O’Cain (2000) used the matched-guise technique to explore the social meanings of discourse marker like and quotative like. Dailey-O’Cain (2000) found that like3 is associated with speakers who are younger, attractive, cheerful, friendly and successful, but less educated. One study which does demonstrate the applicability of such experimental methods to lexical variation is Beltrama & Staum Casasantro (2017), who demonstrated that matched-guise techniques can highlight social meanings at the level of lexical variation.4 For example, totally, as an unbounded adjective, solicits stronger social meanings relating to solidarity (friendly, outgoing, excitable and cool) than its synonyms completely and really as well as a null (bare form) variant. This existing body of research tends to focus on either semantic production or perception. In this article we consider production and perception as two sides of the same coin and report on both the usage and social meanings of the polysemous adjective wicked. 3 Wicked 2 Background The study of semantic change is a developing area of sociolinguistics, a ﬁeld which traditionally eschewed lexis from its ‘theoretical and epistemological considerations’ (Robinson 2012a: 38; see also Durkin 2012). Most sociolinguistic studies that do consider lexis do so from the perspective of onomasiology, that is, variation in the word form (e.g. Meyerhoff 1993; Chambers 2000; Beeching 2011; Tagliamonte & Brooke 2014; Braber 2018; Sandow & Robinson 2018; Sandow 2020; Lafond & Moffett 2020; Tagliamonte & Pabst 2020), rather than semasiology,2 that is, variation in word meaning. Despite this, the fact that semantic change follows a socially mediated trajectory has been repeatedly attested in recent years (e.g. Robinson 2010a, 2010b; Beal & Burbano-Elizondo 2012; Bailey & Durham 2021; Sandow 2023b, in press). For example, using elicited data, Robinson (2012a) found the polysemous adjective gay to be undergoing change in apparent-time, with males leading the change from ‘happy’ towards the senses ‘homosexual’ and ‘lame’. Bailey & Durham (2021) used acceptability judgements to trace the development of the sense 323000515 Published online by Cambridge University Press https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 3 Wicked Due to an ameliorative semantic shift-to-opposite affecting the adjective wicked, the more traditional sense which we gloss here as ‘evil’ now exists alongside the sense we gloss as https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 138 ‘good’. The Oxford English Dictionary (OED online) ﬁrst attests the usage of the former sense in c.1275anddeﬁnes it as‘[b]ad in moralcharacter,disposition,orconduct; inclined or addicted to wilful wrongdoing; practicing or disposed to practice evil; morally depraved’. The second sense, attested by the OED (online) in 1920 in the USA and 1977 in the UK, is deﬁned as ‘[e]xcellent, splendid; remarkable’. Thus, wicked is now an auto-antonym, with the word having two senses which are opposite in meaning, that is, ‘evil’ and ‘good’. ‘good’. The Oxford English Dictionary (OED online) ﬁrst attests the usage of the former sense in c.1275anddeﬁnes it as‘[b]ad in moralcharacter,disposition,orconduct; inclined or addicted to wilful wrongdoing; practicing or disposed to practice evil; morally depraved’. The second sense, attested by the OED (online) in 1920 in the USA and 1977 in the UK, is deﬁned as ‘[e]xcellent, splendid; remarkable’. Thus, wicked is now an auto-antonym, with the word having two senses which are opposite in meaning, that is, ‘evil’ and ‘good’. One study which has investigated the semantic change in the usage of wicked is Robinson (2010a; see also Robinson 2014),5 conducted in Shefﬁeld, South Yorkshire, in 2006–7. Robinson found real-time evidence of the change towards ‘good’ from corpus evidence and in apparent-time using elicited data. The elicited data were analysed for sociolinguistic patterns of variation. Young people were more advanced in the use of wicked ‘good’, indicating a change in apparent-time. This change was also more advanced among females aged 19–60 (Robinson personal communication). Robinson (2010a: 267) does highlight that wicked ‘good’ is likely to have a ‘life expectancy’, suggesting that this change may be ephemeral or age-graded. Robinson (2010a) also provides some metalinguistic commentary from participants relating to the semantic change of wicked, which provides insight into its social meanings. 5 These publications refer to wicked as part of a cluster of variables, rather than in isolation. Thus, the speciﬁc usage-based results from this study relating to wicked are not published. Our insights into this data come from personal communication with Dr Justyna Robinson, Reader in English Language and Linguistics, University of Sussex. 3 Wicked For example, Robinson (2010a: 210) reports participants who observe that ‘wicked ‘good’ is [used in] speaking and wicked [‘evil’] in writing’ and it is ‘a question of class and education if you use wicked ‘good’’.6 While such comments provide speciﬁc insights into the indexical meaning of the senses of wicked, more broadly, they also evidence that this semantic change is occurring above the level of conscious awareness. In the present article, we explore three research questions: • How is the semantic change of wicked conditioned by social factors? • How are the two senses of wicked, ‘evil’ and ‘good’, perceived? • To what extent can perception data be used to explain production results? The ﬁrst study explores the ﬁrst question, while the second question is investigated in the second study. We synthesise the results of both studies to answer our third research question. With the implication being here that those of higher social class/education would be less likely to use wicked ‘good’ https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 4 Study 1– Semantic variation Study 1 is concerned with patterns of semantic variation and change in the usage of wicked. The method reported on here is part of a larger study conducted by the ﬁrst author of this article (see Sandow 2021). Eighty speakers from the Cornish towns of https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 139 Camborne and Redruth, and surrounding villages, were interviewed in 2017–18. The participants in this study were balanced for age (older than 40, N = 40, younger than 30, N = 40),7 gender (male, N = 40, female, N = 40),8 and socioeconomic class (middle- class, N = 40, working-class, N = 40),9 with each of these categories being conceived of as binary. Semasiological data were elicited by employing a methodology developed and ﬁrst executed by Robinson (2010a). Robinson’s who/what and why method consists of two adjacency pairs of which the ﬁrst is designed to elicit a reference of the investigated polysemous word and the second is designed to elicit a sense, such as: Q1: Who or what is wicked? A1: My uncle Q2: Why is your uncle wicked? A2: Because he’s so cool It is thought (see Robinson 2010a) that this method is less direct than simply asking ‘what does X mean’. This is advantageous as direct questioning, e.g. ‘what does wicked mean’, is not consistent with the elicitation of relatively unmonitored speech (e.g. Labov 1972, 1984). Using this method, the structured semantic variation of polysemous adjectives (Robinson 2010a, 2010b, 2012a, 2012b, 2014) and (using a slightly adapted elicitation prompt) nouns (e.g. Sandow 2023a, 2023b)10 has been well attested. Participants were asked about each word twice, with the stimuli in Q1 altering slightly in the second iteration to ‘who or what else is [polysemous adjective]’. By asking the participants the who/what questions twice, participants had the opportunity to provide two senses of the word under investigation. In the study, participants were asked to provide senses for ten words. Eight of these were adjectives, namely, sick, wicked, awesome, hot, gay, cool, ﬁt and hard, as well as two nouns, the Anglo-Cornish dialect words emmet ‘tourist/ant’ (see Sandow 2023b) and maid ‘woman/female servant or attendant’ (see Sandow 2023a). It is important to note that our conceptualisation of the semasiological variable here requires a departure from the more traditional deﬁnition of a sociolinguistic variable. 7 There were no speakers aged between 30 and 40 in the sample. 8 Participants were given the opportunity to select a gender other than ‘male’ and ‘female’ but none elected to do so. 9 Socioeconomic class was determined using an index consisting of information regarding occupation, education and place of domicile (see Sandow 2021). 10 Sandow (2023a, 2023b) focus on Anglo-Cornish dialect words. Thus, this is the ﬁrst output from this study which focuses on variation and change of more widespread semasiological forms (see Sandow et al. (in press) for a discussion of variation and change of more widespread onomasiological features). 7 There were no speakers aged between 30 and 40 in the sample. 8 7 There were no speakers aged between 30 and 40 in the sample. 8 11 This category consists of references to the hit musical Wicked, based on the 1995 novel by Gregory Maguire. An example of this variant is ‘Q: Who or what is wicked? A: A musical.’ 4 Study 1– Semantic variation While Labov (1978: 13) asserted that ‘we must not avoid the study of differences of meaning’, the study of semantic variation is not directly compatible with Labov’s (1972: 271) classic conceptualisation of the sociolinguistic variable as ‘two ways of saying the same thing’. Despite the centrality of this concept to the variationist programme, there is a relatively large body of research, particularly that which extends the envelope of variation beyond phonetics and phonology, that eschews this deﬁnition https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 140 in favour of functional equivalence (for discussion, see Dines 1980; Coupland 1983; Pichler 2010; Terkouraﬁ2011). However, the semasiological variable also cannot be deﬁned in terms of functional equivalence. Robinson (2010a: 275) proposes that a semasiological variable can be thought of as ‘saying different things in the same way’. Various studies using this deﬁnition of the semantic variable have demonstrated highly socially stratiﬁed patterns of variation (e.g. Robinson 2010a, 2010b, 2012a, 2012b, 2014; Sandow 2023a, 2023b, in press). It is this deﬁnition of the variable that we adopt in this study. From the elicitation procedure used in study 1, wicked ‘evil’, wicked ‘good’, wicked ‘musical’11 and wicked ‘good_reported’ are the four attested variants. The ‘reported’ variant refers to instances where participants demonstrated an awareness of the sense but made clear that they did not use this sense, such as ‘I know young people would use wicked when talking about something good but I don’t do that’ (see also Robinson 2010a). https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 4.1 Results However, the change towards wicked ‘good’ among female speakers actually peaks at an earlier stage and at a lower rate, with the most frequent users being women in their 40s (30 per cent, N = 3/10) before essentially plateauing for the very youngest female speakers (25 per cent, N = 10/40). Mixed-effects logistic regression was carried out in R to establish the statistical signiﬁcance of these effects. A model was ﬁtted to all tokens of the dependent variable (N = 160), coding each observation as 1 if wicked ‘good’ was produced and as 0 if any other sense was produced; the model included a by-speaker random intercept to account for the fact that individual speakers produce multiple observations. Step-wise regression was used to identify the best-ﬁtting set of independent variables as determined by the lowest Akaike Information Criterion (AIC), a commonly used method of quantifying the relative quality of a model balanced between its predictive power (the amount of variation in the data it explains) and its complexity (the number of predictors it includes). The best-ﬁtting model is one that contains only the predictors of age and gender and their interaction, where the former is operationalised as a tripartite split between younger (< 30), middle-aged (40s) and older (50+) age groups to capture the curvilinear effect described above. The full table of coefﬁcients is reported in table 1. Although the age×gender interaction does not quite reach the traditional threshold for statistical signiﬁcance ( p = 0.105), removing the interaction term leads to a slightly worse model, suggesting that it holds some explanatory power and that this effect would most likely be signiﬁcant with a slightly larger sample size. This cross-over trend, for women to have been leading the change towards wicked ‘good’ before an apparent plateau while men continue the incrementation of change, is shown clearly in the model prediction plot in ﬁgure 1. This result is also supported by the fact that when we operationalise the dependent variable slightly differently and ﬁt a model to individual speakers, coding wicked ‘good’ users as 1 and those who do not use that sense of the word at all in the production study as 0, the age×gender interaction term is statistically signiﬁcant (β = 2.82, p = 0.01). Table 1. Coefﬁcients table of the logistic regression model modelling use of wicked ‘good’. Intercept corresponds to middle-aged female speakers. 4.1 Results We focus our analysis on wicked ‘good’ (N = 37) and wicked ‘evil’ (N = 113), collapsing other uses (such as the musical) into a single wicked ‘other’ category due to their low frequencies (N = 10). It is important to note here that the number of tokens collected is relatively small but, despite this, they serve to reveal usage patterns which speak to the socially mediated distribution of the senses of wicked. Initial exploratory analysis reveals an overall change in apparent time towards increasing wicked ‘good’ usage among the younger generations; the use of this sense has increased from 11 per cent (N = 9/80) among older speakers to 35 per cent (N = 28/80) among younger speakers. Considering gendered patterns of variation also reveals an interesting interaction, with wicked ‘good’ increasing from 15 per cent (N = 6/40) among older women (aged 40+) to 25 per cent (N = 10/40) among women younger than 30, while for men the change has seen rates increase more dramatically from 7.5 per cent (N = 3/40) to 45 per cent (N = 18/40). This displays an apparent cross-over effect, with women previously leading the change towards wicked ‘good’ before being overtaken by men. Adopting a ternary division of age (coding 50+ as ‘older’, 40s as ‘middle’ and 20s and below as ‘younger’) lends further insight into the nature of this cross-over pattern, although the results should be interpreted with a degree of caution given that token counts decrease as more ﬁne-grained groupings of age are used. We ﬁnd that the change among male speakers is purely monotonic, with wicked ‘good’ becoming increasingly favourable across generations: it is almost completely absent from the older male group (4 per cent, N = 1/26), rising to 14 per cent (N = 2/14) for the middle-aged male group, before culminating in a rate of 45 per cent usage (N = 18/40) https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 141 among male speakers under the age of 30. However, the change towards wicked ‘good’ among female speakers actually peaks at an earlier stage and at a lower rate, with the most frequent users being women in their 40s (30 per cent, N = 3/10) before essentially plateauing for the very youngest female speakers (25 per cent, N = 10/40). among male speakers under the age of 30. 4.1 Results More positive estimates correspond to increased likelihood of wicked ‘good’ use; more negative estimates correspond to decreased likelihood (AIC: 163.72) Predictor level Estimate Standard error z-value p-value (intercept) –0.8473 0.6901 –1.228 0.2200 age: older –1.3499 0.9201 –1.467 0.142 age: younger –0.2513 0.7807 –0.322 0.748 gender: male –0.9445 1.0293 –0.918 0.359 agegender: oldermale –0.0772 1.5714 –0.049 0.961 agegender: youngermale 1.8424 1.1375 1.620 0.105 Table 1. Coefﬁcients table of the logistic regression model modelling use of wicked ‘good’. Intercept corresponds to middle-aged female speakers. More positive estimates correspond to increased likelihood of wicked ‘good’ use; more negative estimates correspond to decreased likelihood (AIC: 163.72) p ( ) Predictor level Estimate Standard error z-value p-value (intercept) –0.8473 0.6901 –1.228 0.2200 age: older –1.3499 0.9201 –1.467 0.142 age: younger –0.2513 0.7807 –0.322 0.748 gender: male –0.9445 1.0293 –0.918 0.359 agegender: oldermale –0.0772 1.5714 –0.049 0.961 agegender: youngermale 1.8424 1.1375 1.620 0.105 https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 142 Figure 1.Model prediction plotillustrating the interactionbetweenage group and gender for the use of wicked ‘good’ Figure 1.Model prediction plotillustrating the interactionbetweenage group and gender for the use of wicked ‘good’ While there appear to be age and gender differences, the effect of social class is completely absent. No measure of social class is included in the best-ﬁtting model, which is not surprising when one explores the data descriptively: ﬁgure 2 plots the distribution of wicked variants by the different factors measuring aspects of socioeconomic status (domicile deprivation, educational level and occupational level) and the results show no signiﬁcant differences across the levels of these various factors. The only exception to this is the apparent decreased use of wicked ‘good’ among those with the lowest educational level, but it should be noted that this is highly collinear with age: all but one of these speakers are above the age of 40, so this is actually reﬂecting the strong effect of age outlined earlier. The results from our production study are remarkably similar to those of Robinson (p.c.) with the exception of the youngest group of females whose counterparts in Shefﬁeld would have been too young to participate in the earlier study. This suggests that the sociolinguistic usage patterns discussed in this article are not particular to Cornwall but speak to a broader process of variation and change of wicked. https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 12 The guises employed here use real social media posts which have been minimally altered to protect the anonymity of the social media users who posted them. 13 We acknowledge that the synonyms are not ‘absolute synonyms’ as these are extremely rare or non-existent (see discussion in Cruse 1986; Murphy 2013), but they do share a functional equivalence (cf. Dines 1980; Beltrama & Staum Casasanto 2017). 4.1 Results 0515 Published online by Cambridge University Press https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 143 Figure 2. The distribution of all wicked variants used by three measures of socioeconomic status: domicile deprivation, level of education, and level of occupation (total number of observations denoted in parentheses) Figure 2. The distribution of all wicked variants used by three measures of socioeconomic status: domicile deprivation, level of education, and level of occupation (total number of observations denoted in parentheses) https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 5 Study 2 – Matched-guise technique To complement the usage-based study 1, we also conducted a study which explores the perception of the polysemous senses of wicked by employing the matched-guise technique (Lambert et al. 1960; Campbell-Kibler 2007). We presented participants with ‘social media posts’12 and they were asked to evaluate the posts along a range of persona traits, such as ‘professional’ or ‘posh’ (see ﬁgure 3 for an example page from the online experiment). Each post had two versions, or ‘guises’, which are identical with the exception of one lexical item. We explored the social meanings of wicked in two different senses, ‘evil’ and ‘good’, which were contrasted with synonyms.13 All participants saw the carrier phrase ‘I’m getting pretty decent at this baking thing, I just made some ___ cake mix this morning.’ While half saw the blank slot ﬁlled with wicked, the other half saw good. Similarly, for the carrier phrase ‘These people are inherently ___ and will do anything to stay in power’, half of the participants saw wicked for the blank slot while the other half saw evil. The carrier phrases were selected as they make it clear which sense is being employed. In the ﬁrst example, positive affect is evident, making it clear that the ‘good’ sense is being employed, https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 144 Figure 3. Example page from the online matched-guise experiment, showing the wicked ‘good’ stimulus Figure 3. Example page from the online matched-guise experiment, showing the wicked ‘good’ stimulus Figure 3. Example page from the online matched-guise experiment, showing the wicked ‘good’ stimulus whereas in the second example, negative affect is clear, which makes it apparent that the ‘evil’ sense is being employed. whereas in the second example, negative affect is clear, which makes it apparent that the ‘evil’ sense is being employed. Spoken stimuli could lead to accent features becoming confounding variables (see also Buchstaller 2006). The written nature of the guises used in this study removes this possibility and enables us to isolate lexical differences across the guises. Table 2 lists the persona traits that participants evaluated using the sliding scales illustrated in ﬁgure 3, which allowed whole-number ratings from 1 (low agreement) to https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 145 Table 2. 5 Study 2 – Matched-guise technique List of persona traits used in the matched-guise study ‘Status’ traits ‘Solidarity’ traits Other Educated Posh Formal Cool Friendly Honest Attractive Young Table 2. List of persona traits used in the matched-guise study Table 3. The sociodemographic composition of the 100-participant sample, by gender, age, occupational category and region Gender Male: N=30 Female: N=70 Age 18–29: N=33 30–49: N=52 >49: N=15 Occupation 1: N=7 2: N=43 3: N=32 4: N=6 5: N=12 Region of England North: N=24 Midlands: N=27 South: N=49 able 3. The sociodemographic composition of the 100-participant sample, by gender, age, occupational category and region 7 (high agreement). As well as the individual persona traits, for the purposes of our analysis we can additionally cluster these into traits that are broadly composite of ‘status’ and ‘solidarity’ (see also Brown 1965; Luhman 1990; Dailey-O’Cain 2000; Ng & Diskin-Holdaway 2021). We consider ‘educated’, ‘posh’ and ‘formal’ as ‘status’ traits. Conversely, we consider ‘cool’, ‘friendly’, ‘honest’ and ‘attractive’ as ‘solidarity’ traits (see, for example, Beltrama 2018; Clark & Schleef 2010). One hundred individuals from England took part in the study, which was administered through Proliﬁc. Information was collected regarding the age, gender and occupational type of each respondent. The sociodemographic composition of the 100-participant sample is displayed in table 3. Participants were asked to self-identify their gender, age, occupation and the region of England in which they were born and raised. In terms of occupation, participants were asked to state if their work was best categorised as ‘higher managerial and professional’ (1), ‘intermediate’ (2), ‘“white-collar” and lower managerial or clerical’ (3), ‘ “blue-collar” lower supervisory and technical’ (4) or ‘semi-routine or routine’ (5). We employed a between-subjects design, with each of the four variants being rated by a total of 50 participants. A control stimulus was seen by all participants. This served to ensure that there were no differences in how the four groups of participants responded to the same stimuli. Indeed, there were no statistically signiﬁcant effects for this guise. Thus, any differences in the perceptions between the stimuli can be attributed to the differences in social meaning between the synonyms. 5.1 Results The overall results from the perception task are presented in ﬁgure 4, plotted separately for the two pairs of guises (wicked ‘good’ vs good and wicked ‘evil’ vs evil), aggregated https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 146 Figure 4. Perceptions of wicked ‘good’ vs good (top) and wicked ‘evil’ vs evil (bottom); 1 = not at all, 7 = very much so. Diamonds/circles correspond to mean rating for that particular variant–scale pair Figure 4. Perceptions of wicked ‘good’ vs good (top) and wicked ‘evil’ vs evil (bottom); 1 = not at all, 7 = very much so. Diamonds/circles correspond to mean rating for that particular variant–scale pair across all listeners and split by each of the eight descriptive scales. Although the differences between guises are generally quite small, there are a number of interesting trends observable in the data. Wicked ‘good’ was evaluated as indexical of the solidarity trait ‘cool’, while it scored comparatively lower in the status characteristic of ‘formality’. On the other hand, wicked ‘evil’ was evaluated more positively on status-related dimensions, particularly ‘educated’ and ‘formal’, as opposed to its synonym evil. An interesting result emerges on the ‘young’ descriptive scale: both positive and negative senses of wicked are seen as less ‘young’ than their respective synonyms, suggesting that it may be the form wicked that is perceived as less young, rather than any particular sense. We can gain further insight into the perception of wicked by considering how the evaluative responses might interact with the gender or age of the listener. In ﬁgure 5 we combine the individual evaluative scales into broader status/solidarity dimensions and plot the perception of wicked ‘good’ by gender. It shows that while females penalise this use of wicked along status dimensions, males do not. In fact, males perceive wicked ‘good’ to be slightly more statusful than good. To establish the statistical signiﬁcance of these results, the data were modelled using cumulative link mixed models (CLMM), which are more appropriate than linear regression models in this case of ordinal data where the perceptual distances between points on the scale may vary across the scale and between listeners. A model was initially ﬁtted to the response data including an interaction between variant (e.g. wicked ‘good’ vs good) and scale, and a random intercept of listener. https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 5.1 Results To diagnose signiﬁcant differences between evaluations of the variants on individual descriptive scales, pairwise comparisons were conducted by comparing estimated https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 147 Figure 5.Perceptions of wicked‘good’ vs good splitby listenergender; 1 = notat all,7 = very much so. Diamonds/circles correspond to mean rating for that particular variant–scale pair Figure 5.Perceptions of wicked‘good’ vs good splitby listenergender; 1 = notat all,7 = very much so. Diamonds/circles correspond to mean rating for that particular variant–scale pair Figure 5.Perceptions of wicked‘good’ vs good splitby listenergender; 1 = notat all,7 = very much so. Diamonds/circles correspond to mean rating for that particular variant–scale pair marginal means using the emmeans package in R (Lenth 2021). The results reveal no signiﬁcant differences between the perception of wicked ‘good’ and good when we model all listeners together. However, signiﬁcant differences do emerge between wicked ‘evil’ and its synonym evil, with the former indexing status signiﬁcantly more than the latter (β = 0.635, p = 0.036); when investigating this in a ﬁner-grained manner looking at the individual descriptive scales, it seems this effect is driven primarily by the ‘educated’ trait (β = 1.228, p = 0.006), although the formal scale also nears signiﬁcance (β = 0.860, p = 0.063). Wicked ‘evil’ is also seen as signiﬁcantly more ‘honest’ than evil (β = 1.063, p = 0.022), but no other signiﬁcant indexicalities are found.14 More differences emerge when we consider how these perceptions might interact with the gender and age of the listener, and these largely parallel the gendered differences in production found in study 1 as discussed earlier. A second CLMM was ﬁtted to the data, this time with a four-way interaction between variant, scale type (status vs solidarity traits), and listener age group (younger [<40] vs older [40+]) and gender (male vs female). Signiﬁcant differences emerge for the older male group, who rate wicked ‘good’ as signiﬁcantly higher than good on measures of both status (β = 2.282, p = 0.033) and solidarity (β = 3.319, p < 0.001). 14 One possible reason for the ﬁnding that wicked ‘evil’ is perceived to index honesty is the association between this sense and religion. For example, when searching Twitter for stimuli, the majority of the clear uses of wicked ‘evil’ were religious in nature. 5.1 Results This is visualised in ﬁgures 5–6, which plot the predicted values from the regression model and visualise the probability of each rating on the 1–7 scale as a function of variant; positive values represent an increased likelihood of wicked ‘good’, rather than good, receiving that rating, whereas negative values for a rating represent a decreased likelihood of that rating being given to wicked ‘good’. Figure 6 clearly shows that this https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press s’ scales; positive values indicate higher rating for wicked ‘good’, negative values indic higher rating for good positi r ratin scales; p higher scales; p highe LANGUAGE CHANGE IS WICKED 149 status-oriented social meaning is only present for the older male respondents, with wicked ‘good’ signiﬁcantly more likely to receive mid-to-high ratings (particularly 4–5) and less likely to receive the lower ratings (particularly 1–2) Figure 7 plots the same information Figure 7. Predicted CLMM rating probabilities on the ‘solidarity’ scales; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good Figure 8. Predicted CLMM rating probabilities on the ‘young’scale; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good Figure 7. Predicted CLMM rating probabilities on the ‘solidarity’ scales; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good Figure 7. Predicted CLMM rating probabilities on the ‘solidarity’ scales; positive values indicate hi h ti f i k d ‘ d’ ti l i di t hi h ti f d Figure 7. Predicted CLMM rating probabilities on the ‘solidarity’ scales; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good Figure 7. Predicted CLMM rating probabilities on the ‘solidarity’ scales; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good Figure 8. Predicted CLMM rating probabilities on the ‘young’scale; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good Figure 8. Predicted CLMM rating probabilities on the ‘young’scale; positive values indicate higher rating for wicked ‘good’, negative values indicate higher rating for good status-oriented social meaning is only present for the older male respondents, with wicked ‘good’ signiﬁcantly more likely to receive mid-to-high ratings (particularly 4–5) and less likely to receive the lower ratings (particularly 1–2). 5.1 Results Figure 7 plots the same information for the solidarity scales, and shows a broadly similar pattern: older respondents evaluate wicked ‘good’ more positively than good with a higher probability of ratings 5–7 and https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 150 lower probability of ratings 1–4, but this is strongest (and only reaches statistical signiﬁcance) among older males speciﬁcally. lower probability of ratings 1–4, but this is strongest (and only reaches statistical signiﬁcance) among older males speciﬁcally. The focus of these results has thus far been on the status and solidarity ratings, but listeners were also asked to rate the guises based on how young they perceive the author to be. The model estimates for this scale are visualised in ﬁgure 8 in the same way those for status and solidarity were presented earlier. No signiﬁcant differences emerged between how wicked ‘good’ and its synonym good were rated on the ‘young’ scale, although the most common trend in the data sees wicked ‘good’ being perceived as less young than good (β = 1.223, p = 0.156 for older women; β = 0.720, p = 0.425 for younger men). Curiously, the older male respondents are the only ones for whom the non-signiﬁcant trend patterns in the opposite direction, with wicked ‘good’ possibly perceived as younger (β = 0.594, p = 0.694), and were also the only social group to perceive it signiﬁcantly more positively on the status and solidarity scales. https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press 6 Discussion Interpreting the results of study 1 in the context of Robinson’s (p.c.) ﬁndings, which showed a female-led change towards wicked ‘good’ in 2005–6, we too ﬁnd that this change is most advancedinwomen,withtheexceptionoftheyoungestgroup(seeﬁgure1).Itisthemalesin the youngest group who are most advanced in their usage of wicked ‘good’. One possible interpretation of this pattern is that young women are leading a shift away from this positive usage of wicked, or at the very least have stopped its incrementation in a way that is not at all evident for young men, who have continued to increasingly use wicked ‘good’. This interpretation is consistent with Labov’s (1990, 2001) principles of language change, with women leading in the initial change but also being at the forefront of this more recent shift away from it. It is worth noting that wicked ‘evil’ is typically seen to hold a higher degree of prestige than wicked ‘good’, as indicated by its status-type indices in study 2 and metalinguistic commentaries from Robinson (2010a: 210). The elicited data may show an incipient retrograde change at the level of the community, with young women shifting away from the newer sense. Citing evidence from her panel study conducted in 2015–16, Robinson (p.c.) also provides some evidence of retrograde change at the level of the individual, with participant metalinguistic commentary reﬂecting on their adoption and subsequent shedding of wicked ‘good’. This pattern is not age-grading proper, which refers to a cyclical process of community stability and individual instability (Chambers 2003; Boberg 2004; Sankoff & Blondeau 2007; Buchstaller 2015). The data do not seem to indicate community-wide stability here, but change across apparent-time. Thus, it is possible that the observed variation and change of wicked ‘good’ is indicative of retrograde lifespan change (see Wagner 2012; Sankoff 2019), with change at the community and individual levels coalescing around the loss of wicked ‘good’. However, further longitudinal analysis would be required to verify this interpretation. The ﬁndings of study 2 account, to a large extent, for the usage patterns in study 1. Given that young men are the most frequent users of wicked ‘good’ it is unsurprising https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 151 that males evaluate this sense more positively than its synonym evil, in both status and solidarity attributes. Conversely, the sense favoured most by young women, wicked ‘evil’, is rated positively for status-type characteristics. 6 Discussion The age pattern in our usage data, which suggests a nascent change away from wicked ‘good’ towards the older sense wicked ‘evil’, is consistent with the trend in the perception data, albeit not statistically signiﬁcant, that wicked ‘good’ is not perceived as young. This provides an interesting parallel to the observation that young women are not participating in the incrementation of the change towards wicked ‘good’ in the same way that women in older generations did, relative to their male counterparts. It is perhaps not surprising that the demographic group who rate wicked ‘good’ as the least young are the older women, who have been around long enough to observe both the original semantic change towards its positive sense and this more recent, female-driven lack of incrementation of it; that is, they are the most acutely aware of the zeitgeist-esque nature of the change and its relatively short-lived popularity in usage, and are therefore the least likely to associate the use of this sense with a youthful style of speech.15 The results presentedhere are testamentto the vigorous,yetsocially mediated,nature of lexical change. As lexis is less constrained by the critical period, it can react to perceptual changes more dynamically than more structural features such as phonetics, phonology and morphosyntax. The case study of wicked demonstrates the speed and dynamism of lexical change, shifting to and then, slightly away from wicked ‘good’. These results, speciﬁcally the parallels between patterns of variation in production and perception, also speak to the mechanisms of lexical change which can complement ongoing research into the role of social meaning and indexicality that has previously taken place primarily in relation to sound change (see Eckert & Labov 2017; Bermúdez-Otero 2020; Hall-Lew et al. 2021). Although it is not possible to draw a direct causal link between them, the indexical ﬁeld of wicked – for both its ‘good’ and ‘evil’ senses – does provide a possible explanation for the changes we observe in production. The role of social meaning in driving forward the incrementation of language change has been called into question in cases of sound change speciﬁcally (see e.g. Bailey 2019, and Bermúdez-Otero 2020 for a more general discussion of these issues), but it is possible that such causal relationships are more likely in the case of lexical/semantic changes for the reasons discussed above. 15 See Fruehwald (2017) for a detailed treatment of the ‘zeitgeist’ effects in language change and how, with the appropriate wealth of data, these can be disentangled from more typical instances of generational change and lifespan change. 6 Discussion It is important to acknowledge a number of limitations of the studies presented in this article and the subsequent discussion. Firstly, while we compare the results from study 1 with Robinson’s (2010a) earlier data, it is important to highlight that the localities in which the data were collected have non-trivial differences. While Robinson’s data were collected in Shefﬁeld, South Yorkshire, an urban post-industrial city, our study 1 was conducted in the (also post-industrial) Cornish towns of Camborne and Redruth. Although Camborne–Redruth is urban by Cornish standards, it is far less urban than https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press RHYS J. SANDOW ET AL. 152 Shefﬁeld. Thus, the nature of social network structures may differ between the communities, which could account for some degree of the variation between the two studies. Additionally, a key limitation of the matched-guise study is that each sense was tested in one context only. More robust conclusions could be drawn from a study which contrasts a polysemous adjective with synonyms in multiple carrier phrases, ranging in terms of conceptual domains, e.g. wicked vs good/evil in the context of food, sport, weather etc. Similarly, all of the guises were presented as ‘social media posts’. Further research may determine whether this context reduces the distinctions in social meanings between variants when compared with other contexts, such as formal writing. A further limitation in both studies is the relatively small data sets. While 80 participants in a variationist study (such as study 1) is relatively large, they each produced two variants of the investigated variables, which is much lower than is typically the case for variables at other levels of the grammar. In study 2, 100 participants is a relatively small number for a matched-guise study. This means that our results do not have a great deal of statistical power, and that larger data sets would therefore be needed to diagnose differences of this effect size at the conventional threshold for signiﬁcance. To conclude, in this article we have showcased the value of considering both production and perception data in order to understand processes of semantic change, using wicked as a case study. We have adopted Robinson’s (2010a: 275) deﬁnition of a semantic variable, that is, ‘saying different things in the same way’ and demonstrated the socially mediated trajectory of semantic change. 6 Discussion Speciﬁcally, we have shown that young women are no longer incrementing the change towards wicked ‘good’ and may be participating in an incipient change back towards the more traditional sense wicked ‘evil’. This ﬁnding speaks to a broader point about the utility of sociolinguistic theory. While Labov (1990, 2001) did not primarily have lexis in mind when discussing gender effects in sociolinguistic variation and change, we have demonstrated their applicability to the polysemous adjective wicked. We have also highlighted the potential to elicit rich information regarding the social meaning of lexical items through modiﬁed matched-guise stimuli, which in this case enables us to interpret usage data in the context of indexical meanings. Synthesising the usage and perception data in this study has enabled us to develop a more thorough understanding of the process of semantic change than would be possible by studying production or perception in isolation. Ultimately, this article makes a contribution to the growing yet still limited literature which explores semantic variation through a variationist sociolinguistic lens and, more particularly, considers the role of social meaning in semantic change. Authors’ addresses: Department of Linguistics Arts One Queen Mary University of London Mile End Road Department of Linguistics Arts One Queen Mary University of London Mile End Road https://doi.org/10.1017/S1360674323000515 Published online by Cambridge University Press LANGUAGE CHANGE IS WICKED 153 Bethnal Green London E1 4PA United Kingdom r.sandow@qmul.ac.uk Bethnal Green London E1 4PA United Kingdom r.sandow@qmul.ac.uk Department of Language and Linguistic Science Vanbrugh College University of York York YO10 5DD United Kingdom george.bailey@york.ac.uk School of Arts & Humanities Nottingham Trent University 50 Shakespeare Street Nottingham NG1 4FQ United Kingdom natalie.braber@ntu.ac.uk References Álvarez-Mosquera, Pedro & Alejandro Marín-Gutiérrez. 2021. Language attitudes in a lingua franca: The case of black South African college students. Journal of Language and Social Psychology 40(3), 378–97. Bailey, George. 2019. 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https://openalex.org/W3083395861	https://www.pure.ed.ac.uk/ws/files/162952894/jmse_901525.pdf	English	null	Numerical Modelling and Experimental Testing of the Hydrodynamic Characteristics for an Open-Frame Remotely Operated Vehicle	Journal of marine science and engineering	2,020	cc-by	9,811	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. Digital Object Identifier (DOI): 10.3390/jmse8090688 Published In: Journal of Marine Science and Engineering Edinburgh Research Explorer Numerical modelling and experimental testing of the hydrodynamic characteristics for an open-frame remotely operated vehicle Citation for published version: Li, Q, Cao, Y, Li, B, Ingram, D & Kiprakis, A 2020, 'Numerical modelling and experimental testing of the hydrodynamic characteristics for an open-frame remotely operated vehicle', Journal of Marine Science and Engineering, vol. 8, no. 9, 688. https://doi.org/10.3390/jmse8090688 Citation for published version: Li, Q, Cao, Y, Li, B, Ingram, D & Kiprakis, A 2020, 'Numerical modelling and experimental testing of the hydrodynamic characteristics for an open-frame remotely operated vehicle', Journal of Marine Science and Engineering, vol. 8, no. 9, 688. https://doi.org/10.3390/jmse8090688 Citation for published version: Li, Q, Cao, Y, Li, B, Ingram, D & Kiprakis, A 2020, 'Numerical modelling and experimental testing of the hydrodynamic characteristics for an open-frame remotely operated vehicle', Journal of Marine Science and Engineering, vol. 8, no. 9, 688. https://doi.org/10.3390/jmse8090688 Article Numerical modelling and experimental testing of the hydrodynamic characteristics for an open-frame remotely operated vehicle Qian Li * , Yu Cao , Boyang Li , David M. Ingram and Aristides Kiprakis School of Engineering, The University of Edinburgh, Sanderson Building, Robert Stevenson Road, Edinburgh EH9 3FB, UK; Qian.Li-2@ed.ac.uk (Q.L.); Yu.Cao@ed.ac.uk (Y.C.); Boyang.Li@ed.ac.uk (B.L.); David.Ingram@ed.ac.uk (D.M.I.); Aristides.Kiprakis@ed.ac.uk (A.K.) * Correspondence: Qian.Li-2@ed.ac.uk Qian Li * , Yu Cao , Boyang Li , David M. Ingram and Aristides Kiprakis School of Engineering, The University of Edinburgh, Sanderson Building, Robert Stevenson Road, Edinburgh EH9 3FB, UK; Qian.Li-2@ed.ac.uk (Q.L.); Yu.Cao@ed.ac.uk (Y.C.); Boyang.Li@ed.ac.uk (B.L.); David.Ingram@ed.ac.uk (D.M.I.); Aristides.Kiprakis@ed.ac.uk (A.K.) * Correspondence: Qian.Li-2@ed.ac.uk Version August 31, 2020 submitted to J. Mar. Sci. Eng. Abstract: The remotely operated vehicles (ROVs) are important to provide the technology support 1 for both the traditional offshore structures and rapidly-growing renewable energy facilities during 2 their full-lifecycles, such as site survey, installation, inspection, maintenance and repair. Regarding 3 the motion and performance of a ROV, the understanding of its hydrodynamic properties is essential 4 when exposing to the disturbances of wave and current. In this study, a numerical model is proposed 5 within the frame of an open-source platform OpenFOAM. The hydrodynamics of the adopted 6 ROV (BlueRov2) in its four principal degrees of freedoms (DOFs) is numerically simulated by a 7 Reynolds-Averaged Navier-Stokes (RANS) solver. Meanwhile, an experimental test is carried out 8 by using a novel technique on measuring the hydrodynamic forces and moments. To validate 9 the numerical prediction methodologies, a set of systematic simulations of the ROV subjected 10 to the disturbances caused by various ﬂow conditions are performed. Comparing to the model 11 test measurement, the numerical model proved to be reliable in offering a good estimation of the 12 hydrodynamic parameters. This also indicates that the presented numerical methodologies and 13 experimental techniques can be applied to other types of open-frame ROVs in quantifying the 14 hydrodynamic parameters, capturing the physics of the ﬂuid-structure interaction (FSI) and feature of 15 the turbulent vorticity which are all essential for the effective control of the ROVs under the nonlinear 16 ﬂow disturbances. 17 Keywords: Remotely operated vehicle; Hydrodynamic forces and moments; Numerical simulation; 18 Experimental test; Turbulent ﬂow modelling 19 Keywords: Remotely operated vehicle; Hydrodynamic forces and moments; Numerical simulation; 18 Experimental test; Turbulent ﬂow modelling 19 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 Submitted to J. Mar. Sci. Eng., pages 1 – 15 www.mdpi.com/journal/jmse 1. Introduction 20 The remotely operated vehicles are important to deliver the services like subsea survey, 21 underwater condition assessment, and data acquisition in a complex environment which are risky 22 and expensive to do by human divers. The fast development of the offshore renewable industry 23 also creates new demand for underwater data collection, damage and corrosion assessment for the 24 offshore wind farms and subsea renewable energy facilities. However, there are challenges for ROV 25 control when facing the unpredictable disturbances caused by the current and waves in its operating 26 environment [1]. The model-based controllers usually require the hydrodynamic parameters of the 27 ROV to build a precise dynamic model in predicting its behaviours. One of the common methods to 28 investigate the hydrodynamic parameters of the vehicle is the experimental test which can be classiﬁed 29 into two categories. Within the ﬁrst category, substantial researches have been conducted to extend the 30 towing tank principle from the ship models to the underwater vehicle identiﬁcation such as the planar 31 Submitted to J. Mar. Sci. Eng., pages 1 – 15 www.mdpi.com/journal/jmse Version August 31, 2020 submitted to J. Mar. Sci. Eng. 2 of 15 motion mechanism (PMM) which carried out in a towing tank for seakeeping tests and other tests 32 with free-running models in all degrees of freedom (DoFs) [2–5]. A free decay test is an alternative 33 method used for the ROV testing [6]. Besides, a modiﬁcation test method, based on the free decay test 34 applying a pendulum motion instead of the spring oscillation, is proposed in Eng et al. [7,8]. In the 35 second category, vehicles generate the forces and moments by their own propulsion system, rather 36 than by the externally forced motions. The parameters are identiﬁed by either the least square method 37 [9–11] or a grapho-analytical method [12]. A comparison between model tests employing methods 38 from the above two categories was carried out for the heave freedom of degree in [13]. Although 39 these methods are the most prevailing ones among all the experimental approaches, data obtained 40 from above tests is not completely reliable because of the facility limitations and errors, and they are 41 generally time-consuming and high cost. 1. Introduction 20 42 g y g g With the signiﬁcant growth of the computer hardware capability in the recent decades, the 43 applications of Computational-Fluid Dynamics (CFD) in the hydrodynamics study tend to become 44 prevailing [14–17]. Skorpa [18] studied the drag, lift and moment history for the Merlin WR200 ROV 45 model with different turbulent models in FLUENT. Numerical modelling was carried out to the RRC 46 ROV and validated by a free-decaying model testing [19,20]. Suzuki et al. [21] evaluated two kinds of 47 forced oscillation methods on PICASSO, in which both the steady-state and unsteady-state conditions 48 were simulated considering the wall effects [22]. Generally, the simulation of a six-DoF dynamics model 49 of the ROV is more challenging than that of a torpedo-shaped streamlined autonomous underwater 50 vehicle (AUV) which has an analytical solution. Theoretical models are not suitable for the open-frame 51 ROVs since the ﬂow-structure interaction through the vehicle is not considered. Although there are 52 consistent efforts to improve the algorithm efﬁciency and robust [23–26], due to its inherent complex 53 structure and FSI feature, the applications of CFD in the ROV modelling are still computational 54 costly and unaffordable, especially considering the modelling of thruster-hull and thruster-thruster 55 interaction effects. This also leads to certain kind of simpliﬁcations adopted in the simulation practice, 56 and the error discrepancies between the numerical simulation results and that of the experiment tests 57 are around 20%-30%. However, the tool of CFD still plays an important role considering the limitation 58 of the model test in the cost, test model scale and facility capability. 59 y p y As part of the ORCA Hub project [27,28], both experimental and numerical studies have been 60 carried out to investigate the hydrodynamic performances of the ROV. In the numerical modelling, a 61 CFD package OpenFOAM [29] is adopted to implement the methodology proposed in this paper. As 62 an open-source solver, it is a powerful ﬁeld manipulation tool offering versatile libraries and utilities 63 [30]. In terms of the user-friendly customizable solvers, the object-oriented techniques of C++ allow 64 the codes to closely resemble its mathematical expression and makes the top-level syntax amenable 65 to further development. 1. Introduction 20 All these features of OpenFOAM enable it to tackle the key issues posed 66 in this study like the dynamics mesh tracking and turbulent ﬂow modelling, making it a suitable 67 platform for the targeted numerical modelling. Besides, the experimental investigation of the vehicle 68 was conducted in the FloWave wave and current facility [31], located at the University of Edinburgh. 69 A novel test method was designed to match the requirement of the study and make the best usage of 70 the FloWave facility [32]. During the test, eight tethers were applied to hold the ROV in place without 71 introducing substantial interference, and each tether was equipped with a load cell to track the motions 72 and rotations which is integrating with an underwater video motion capture system. 73 2. Dynamic of the ROV 74 In this study, BlueROV2 (Blue Robotics, Torrance, USA), a commercially available ROV is used. The BlueROV2 depicted in Figure 1 has an open-frame structure with a dry weight of 10 kg and is 457 mm long, 338 mm wide, and 254 mm high. BlueROV2 is ideal for operations in shallow to moderate waters with a standard 100 m depth rating and up to 300 m tether, and comprised of six T200 thrusters together with a rugged frame and quick-swappable batteries. More details about BlueROV2 is given by [33]. The coordinate system used in the ROV analysis is illustrated in Figure 1. To describe the Version August 31, 2020 submitted to J. Mar. Sci. Eng. 3 of 15 6-DOF differential nonlinear equation of motion of an underwater vehicle, the equations given by Fossen [34] are applied and can be expressed as MRB ˙v + CRB (v) v + MA ˙vr + CA (vr) vr + D (vr) vr + g (η) = τ (1) (1) in which, MRB and CRB ∈R6×6 are the rigid body forces, MA, CA and D ∈R 6×6 represent the 75 hydrodynamic forces; g (η) is the hydrostatic forces. The right-hand term τ ∈R 6×1 is the external force 76 term. The hydrodynamics forces is the function of the relative velocity (vr) that between the ﬂow and 77 the vehicle. in which, MRB and CRB ∈R6×6 are the rigid body forces, MA, CA and D ∈R 6×6 represent the 75 hydrodynamic forces; g (η) is the hydrostatic forces. The right-hand term τ ∈R 6×1 is the external force 76 term. The hydrodynamics forces is the function of the relative velocity (vr) that between the ﬂow and 77 the vehicle. Figure 1. The coordinate system used in the ROV analysis Figure 1. The coordinate system used in the ROV analysis For the BlueROV2, the metacentric height provides adequate static stability which guarantee 79 the passive pitch and roll motions and leads to a small roll and pitch angle amplitude. Hence, the 80 nonlinear components of the forces and moments can be considered caused by the viscous effects of 81 the ﬂow, which becomes less important as the pitch angle is small [1]. Therefore, the hydrodynamics 82 behaviour in the surge, sway, heave and yaw are treated as the four principal degrees of freedoms of 83 BlueRov2. 84 3. Hydrodynamic Model 85 The ﬂuid dynamics model in this study is based on the Navier-Stokes equations and the continuity 86 equation. Considering an incompressible Newtonian ﬂuid, the momentum and continuity equations 87 can be written as 88 ∂ui ∂t + uj ∂ui ∂xj = −1 ρ ∂p ∂xi + ν ∂ui ∂xj∂xj (2) ∂uj ∂xj = 0 (i = 1, 2, 3) (3) ∂ui ∂t + uj ∂ui ∂xj = −1 ρ ∂p ∂xi + ν ∂ui ∂xj∂xj (2) (2) ∂uj ∂xj = 0 (i = 1, 2, 3) (3) (3) in which x is the Cartesian coordinate, t is the time, u is the velocity, p is the pressure, ν is the kinematic 89 viscosity and ρ is the ﬂuid density. Subscripts i and j are summation indexes, which represent relevant 90 Cartesian components and equal to 1, 2 and 3 for three-dimension issues in this study. It should 91 be noted that here and throughout this paper, a summation over the range of that index is implied 92 whenever the same index appears twice in any term. In the Reynolds-Averaged Navier-Stokes (RANS) 93 model employed in this study, an ensemble averaging method is applied for the unsteady turbulent 94 ﬂow modelling. The idea is that the unsteadiness in the ﬂow is ensemble-averaged out and regarded 95 as part of the turbulence. The ﬂow variables are represented as the sum of the average and ﬂuctuating 96 term: 97 Version August 31, 2020 submitted to J. Mar. Sci. Eng. 4 of 15 Version August 31, 2020 submitted to J. Mar. Sci. Eng. 4 of 15 ui(xi, t) = ui(xi) + ui′(xi, t) (4) (4) where the symbols (−) and (′) stand for the average and the ﬂuctuating component, respectively. 98 Repeating a series of measurement with the number of Nt samples, it can be described as 99 where the symbols (−) and (′) stand for the average and the ﬂuctuating component, respectively. 98 Repeating a series of measurement with the number of Nt samples, it can be described as 99 ui (xi, t) = 1 Nt Nt ∑ n=1 uni (xi, t) (5) (5) in which Nt represents the total number of independent trials, uni (xi, t) is u(xi, t) captured at the 100 nth series. 3. Hydrodynamic Model 85 Adopting it to the incompressible continuity equation and substituting Equation 4 to the 101 corresponding momentum equation, it eventually leads to RANS equation 102 in which Nt represents the total number of independent trials, uni (xi, t) is u(xi, t) captured at the 100 nth series. Adopting it to the incompressible continuity equation and substituting Equation 4 to the 101 corresponding momentum equation, it eventually leads to RANS equation 102 ∂ui ∂t + uj ∂ui ∂xj = ∂ ∂xj " −1 ρ Pδij + ν ∂ui ∂xj + ∂uj ∂xi ! −ui′uj′ # (6) (6) There are three stress terms on the right-hand side: −1 ρ Pδij is the mean pressure ﬁeld; δij is the Kronecker delta (δij = 1 if i = j and δij = 0 if i ̸= j) and ν ∂ui ∂xj + ∂uj ∂xi represents the viscous stress from the momentum transfer at the molecular level, ui′uj′ is the Reynolds stresses arising from the ﬂuctuating velocity ﬁeld. To close the system, following the Newton’s law of viscosity where the viscous stress is proportional to the velocity gradient, this leads to There are three stress terms on the right-hand side: −1 ρ Pδij is the mean pressure ﬁeld; δij is the Kronecker delta (δij = 1 if i = j and δij = 0 if i ̸= j) and ν ∂ui ∂xj + ∂uj ∂xi represents the viscous stress from the momentum transfer at the molecular level, ui′uj′ is the Reynolds stresses arising from the ﬂuctuating velocity ﬁeld. To close the system, following the Newton’s law of viscosity where the viscous stress is proportional to the velocity gradient, this leads to τij = µsij = µ ∂ui ∂xj + ∂uj ∂xi ! (7) (7) in which µ = νρ is the dynamic viscosity of the ﬂow. In the stress tensor matrix, the diagonal 103 components are the normal stresses, and the off-diagonal components are the shear stresses. Since the 104 turbulent kinetic energy k is the half trace of the Reynolds stress tensor, this gives 105 k = 1 2ρui′ui′ (8) (8) Since the isotropic stress is deﬁned as 3 2kδij. 3. Hydrodynamic Model 85 the deviatoric part of the stress can be found by aij = ui′uj′ −3 2kδij (9) (9) The turbulent-viscosity hypothesis is introduced by Boussinesy [35] which analogy to the 106 stress-strain relation for a Newtonian ﬂuid(see Equation 7), since the turbulent stresses increase 107 as the mean rate of deformation increase. Based on the turbulent-viscosity hypothesis, the turbulent 108 stress can be derived as 109 τij = −ui′uj′ = νT ∂ui ∂xj + ∂uj ∂xi ! −3 2kδij (10) (10) in which νT = νT(xi, t) refers as the turbulent or eddy viscosity. This hypothesis introduces the 110 macroscopic representations of the micro-scale ﬂuctuating ﬂow. It offers an access to model the 111 overall effects of small vortexes by correlations and meanwhile, resolve the larger eddies through 112 the numerical simulation. Therefore, the computational time is dramatically reduced compared to 113 the direct numerical simulation (DNS) in which the ﬂuctuating ﬂow and small eddies are directly 114 modelled. By substituting Equation 10 into Equation 6, it gives 115 ∂ui ∂t + uj ∂ui ∂xj = ∂ ∂xj " νe f f ∂ui ∂xj + ∂uj ∂xi !# −1 ρ ∂ ∂xj P + 2 3ρk (11) (11) Version August 31, 2020 submitted to J. Mar. Sci. Eng. 5 of 15 νe f f (xi, t) = ν + νT (xi, t) (12) νe f f (xi, t) = ν + νT (xi, t) (12) where ν is the constant molecular viscosity and νT(xi, t) is the spatial-temporal dependent 116 turbulent/eddy viscosity, and together they compose the effective viscosity νe f f (xi, t). 117 where ν is the constant molecular viscosity and νT(xi, t) is the spatial-temporal dependent 116 turbulent/eddy viscosity, and together they compose the effective viscosity νe f f (xi, t). 117 f f The Equation 2 -12 are targeted at solving ﬁxed mesh (Eulerian mesh) issues. However, if a 118 moving structure is involved, as in this study, the computational mesh may need to move to conform 119 to the motion of the rigid body. The alternative is introducing an additional treatment, e.g. treat 120 the structure as an additional phase in the modelling system as in the immersed boundary method 121 (IBM). 3. Hydrodynamic Model 85 In this study, the ﬂow distribution in the computational domain and the mesh are updated 122 following the motion of the structure and satisfying the adopted non-slip boundary condition on the 123 structure surface. Meanwhile, the body motion is calculated based on the Newton’s 2nd law in which 124 the force due to the ﬂuid distribution variation on the structure is modelled by the pressure updated by 125 Equation 11 on the rigid body surface. This indicated that the above equations require the accompany 126 of a computational mesh which can cope with both the ﬁxed Eulerian mesh and mesh following the 127 body motion. This leads to the Arbitrary Lagrangian-Eulerian (ALE) form equations, which can be 128 written as 129 ∂uTj ∂xj = 0 (13) ∂uTj ∂t + uTj −ubj ∂uTj ∂xj = ∂ ∂xj " νe f f ∂uTj ∂xj + ∂uTj ∂xi !# −1 ρ ∂pT ∂xi (14) νe f f (xi, t) = ν + νT (xi, t) (15) (13) ∂uTj ∂t + uTj −ubj ∂uTj ∂xj = ∂ ∂xj " νe f f ∂uTj ∂xj + ∂uTj ∂xi !# −1 ρ ∂pT ∂xi (14) (14) νe f f (xi, t) = ν + νT (xi, t) (15) νe f f (xi, t) = ν + νT (xi, t) (15) in which, uT and pT are the ensemble-averaged ﬂow velocity and pressure, respectively. An additional 130 term, ubj, is introduced in the convective term to accommodate the movement of meshes when ﬂow 131 subjecting to the motion of the body. If the nodal velocity is following the ﬂuid velocity, i.e. ubj = uTj, 132 Equation 14 is transformed to the corresponding Lagrangian form ; whereas if a static body is involved 133 with ﬁxed mesh, i.e. ub = 0 , Equation 14 convert to a Eulerian form which is same as Equation 11. 134 3.1. OpenFOAM solver Validation 135 In this section, the feasibility and the reliability of the OpenFOAM solver are examined at prior. 136 Flow past a circular cylinder frequently serves as a classic example and benchmark in terms of ﬂow 137 separation and vortex shedding physics [36]. Besides, the ﬂow disturbances caused by the interaction 138 between a circular cylinder and the ROV will be one of the main focuses of ORCA project in following 139 next stage. Therefore, the validation is carried out by using a circular cylinder subjected to the uniform 140 current. In the validation, the drag coefﬁcient from the experimental data for 40 < Re < 5 × 105 and 141 Schewe [37] for Re > 105, and corresponding numerical results from Stringer et al. [38] are compared 142 to that predicted by the OpenFOAM solver. An appropriate turbulent model is desired in calculating 143 the turbulent viscosity νT (xi, t) in Equation 15 . Hence, two classic turbulent models, i.e. k −ϵ and 144 k −ω SST turbulent models are employed and evaluated, in which the main issues concerned is the 145 drag/lift coefﬁcient (see Equation 16 and 17) and vortex shedding frequencies that reﬂected by the 146 Strouhal number (St). 147 CD = 1 2 FD ρu2A (16) CL = 1 2 FL ρu2A (17) (16) (17) in which, u is the ﬂow velocity; ρ is the ﬂuid density; FD and FL are the drag force and the lift force, 148 respectively; FD and FL is the force component in the direction of the ﬂow velocity and the cross-ﬂow 149 direction, respectively and A is the cylinder cross-sectional area. 150 6 of 15 Version August 31, 2020 submitted to J. Mar. Sci. Eng. In the ﬂow past a circular cylinder case, it is well understood that after the Reynolds number 151 excess 40, the wake becomes unstable and eventually leads to a set of vortex street shedding alternately 152 on either side of the cylinder at a certain frequency. This also results in the oscillation of the drag force 153 together with the unsymmetrical distribution of the turbulent viscosity νT (xi, t) and vorticity (see 154 Figure 2). More details about this physics can be seen in [24]. 3.1. OpenFOAM solver Validation 135 Figure 3 demonstrates the comparisons 155 of CD, from which it is found that CD predicted by OpenFOAM with k −ω SST turbulent model 156 generally agrees well with that of the experimental results, and the maximum relative discrepancy 157 (around 13 %) is observed at Re = 5 × 105. Since the drag force is the main concern in this study, the 158 details of lift force comparison is not given here but can be seen in [24]. It should be pointed out that 159 the success of RANS on modelling the turbulent ﬂow largely relying on achieving the desired accuracy 160 of the eddy viscosity. Since the eddy viscosity captured by k −ω SST model can satisfactorily reﬂect 161 the macroscopic representation of the ﬂuctuating ﬂow ﬁeld, one may agree that with the presence 162 of an adverse pressure gradient, the performance of the k −ω SST is superior to that of k −ϵ model. 163 Based on the fact that RANS solver with k −ω SST turbulent model can provide predictions fairly 164 close to the experimental data within a large range of Re, the same numerical conﬁguration will be 165 employed in the following simulations. 166 Figure 2. Instantaneous spatial distribution of the fully developed turbulent viscosity and vorticity around the cylinder at Re = 106 [24] Figure 2. Instantaneous spatial distribution of the fully developed turbulent viscosity and vorticity around the cylinder at Re = 106 [24] Figure 3. Validation of the mean drag coefﬁcient which is the function of Re [24] Figure 3. Validation of the mean drag coefﬁcient which is the function of Re [24] 4. Numerical Simulation Conﬁgurations 167 4. Numerical Simulation Conﬁgurations 167 In the numerical simulation, a rectangular computational domain is adopted. The length and the 168 width of the computational domain are 28B and 16B respectively, where B is the characteristic scale 169 of the ROV. The 3D and 2D views of the computational domains are given in Figure 4 and Figure 5, 170 respectively. The CAD geometry of the vehicle is shown in Figure 6 (a) and the computational mesh is 171 generated by OpenFOAM internal utility snappyHexMesh (see Figure 6(b)). A series of numerical 172 Version August 31, 2020 submitted to J. Mar. Sci. Eng. 7 of 15 simulations target on the hydrodynamics performances of the four principal motions (surge, sway, 173 heave and yaw) are conducted with the boundary conditions including:(1) a Neumann zero-gradient 174 velocity boundary condition is implemented at the outlet boundary; (2) a slip boundary condition is 175 applied at the top, bottom, front and back boundaries and (3) a non-slip condition is used on the body 176 surface. 177 Figure 4. Sketch of the 3D computational domain Figure 5. 2D view of the domain (XY-plane) with the inlet and outlet boundaries Figure 4. Sketch of the 3D computational domain Figure 4. Sketch of the 3D computational domain Figure 4. Sketch of the 3D computational domain Figure 5. 2D view of the domain (XY-plane) with the inlet and outlet boundaries Figure 5. 2D view of the domain (XY-plane) with the inlet and outlet boundaries Figure 5. 2D view of the domain (XY-plane) with the inlet and outlet boundaries Figure 6. (a) The computer-aided design (CAD) model of the ROV applied in the numerical simulation created by software SolidWorksTM; (b) Sketch of the computational domain with inlet and back boundries Figure 6. (a) The computer-aided design (CAD) model of the ROV applied in the numerical simulation created by software SolidWorksTM; (b) Sketch of the computational domain with inlet and back boundries The investigations are performed at the Reynolds number ranging from 6.76 × 104 to 3.38 × 105 178 which corresponds to an incoming current velocity between 0.2m/s to 1.0m/s, with ρ = 1025kg/m3, 179 ν = 1 × 10−6m2/s and the characteristic length is 0.338 m. 4. Numerical Simulation Conﬁgurations 167 One may agree that all CFD work is highly 180 The investigations are performed at the Reynolds number ranging from 6.76 × 104 to 3.38 × 105 178 which corresponds to an incoming current velocity between 0.2m/s to 1.0m/s, with ρ = 1025kg/m3, 179 ν = 1 × 10−6m2/s and the characteristic length is 0.338 m. One may agree that all CFD work is highly 180 Version August 31, 2020 submitted to J. Mar. Sci. Eng. 8 of 15 dependent on the mesh resolution. Therefore, for each of the four degree of freedoms, the convergence 181 test against mesh resolution is performed to identify the suitable mesh conﬁguration with a minimal 182 computational cost. Wall treatment is always one of the biggest challenges raised in the turbulent 183 ﬂow simulation, which can be classiﬁed into two categories: the low-Reynolds-number (LR) models 184 and high-Reynolds-number (HR) models. The low-Reynolds-number (LR) approach accompanied 185 by a wall functions is targeting at the sublayer where exists a local low turbulent Reynolds number. 186 One alternative to wall functions is to adopt a ﬁne-grid conﬁguration that allows the application of a 187 laminar ﬂow boundary condition. To reach the viscous sublayer, the normalized distance (y+) from 188 the ﬁrst mesh cell centre to body surface is supposed to be around 1, where y+ = u∗yw/νe f f . In the 189 numerical practice, the desired y+ is usually obtained through consistent trials. However, the HR 190 model can cope with a much larger y+ ( around 30) which integrates with a log law to estimate the 191 gradient approaching the body wall. It should be noted that the ﬁrst computational mesh should 192 be placed either in the log-layer or the viscous sublayer but not in-between [39], since none of the 193 categories can deal with the buffer layer where both viscous and Reynolds stresses are signiﬁcant. 194 Within certain mesh conﬁguration, the time step size ∆t is automatically determined by using the ﬁxed 195 Courant number C0 (C0 = (u∆x) /∆t, where ∆x is the mesh size). 196 5. Experiment Setup 197 In this study, a new test technique was designed to quantify the hydrodynamic forces on a ROV 198 in the FloWave facility. FloWave is a 25 m diameter circular tank with a total water depth of 2 m. The 199 ﬂoor of the tank is buoyant and can be raised out of the water for model installation and the water 200 currents can be generated from any direction of the tank(see Figure 7). More details of the FloWave 201 current generation are provided in [31]. During the test, the ROV was connected to eight tethers 202 to the frame at the height of 1 m from the ﬂoor (see Figure 7 (a)). The conﬁgurations of the frame 203 and tethers are given by Gabl et al. [32]. The measurement instrumentation used were: (1) motion 204 capturing system (MoCAP) to record the motion and rotation of the different structures, (2) load cells 205 to measure the forces along the eight tethers. The MoCAP worked together with four underwater 206 cameras provided by Qualisys. Knowing the position of the ROV, the mounting points (connection of 207 the tether to the ROV) can be calculated as the virtual points. This allowed the direction of the force 208 vector can be accurately determined and three-dimensional force components to be resolved. The 209 working conditions tested in the model test can be seen in Table 1. For the surge drag measurement, 210 the velocities examined was ranging from 0.2m/s to 1.0 m/s with a increment of 0.2m/s, and in both 211 the forward and backwards surge directions. For the sway drag, a smaller velocity range which up to 212 0.6m/s was tested, and also in the forward and backwards sway directions. 213 Table 1. Experimental test working conditions Surge Sway Flow velocity [m/s] [0.2 - 1.0] [0.2 - 0.6] Direction [degree] 180/-180 -90/90 Capture time [s] 512 512 Table 1. Experimental test working conditions 9 of 15 Version August 31, 2020 submitted to J. Mar. Sci. Eng. Figure 7. (a) Set up of the experiment in the FloWave circular tank; (b) Tethers equipped with load cells attached to the ROV Figure 7. (a) Set up of the experiment in the FloWave circular tank; (b) Tethers equipped with load cells attached to the ROV 6. Results Discussion 214 The physics and quantiﬁed hydrodynamic forces on a ROV from the numerical simulation and 215 experimental test are analysed and compared in this section. Figure 8 (a) reveals the instantaneous 216 pressure distribution on the vehicle and the streamlines around the vehicle. Higher pressure is 217 observed at the front of ROV while the wake at the rear creates a low-pressure region. Correspondingly, 218 a low velocity area at the front of the ROV is captured which can be seen from Figure 8 (b). 219 Figure 8. (a) Velocity streamlines around the vehicle and pressure on the vehicle; (b) Velocity ﬁeld of the vehicle from the numerical simulation Figure 8. (a) Velocity streamlines around the vehicle and pressure on the vehicle; (b) Velocity ﬁeld of the vehicle from the numerical simulation The ﬂow separations and ﬂow interactions between different parts of the vehicle are exhibited. 220 There are three individual shedding ﬁrst generated by the left, right frame and centre structure of the 221 vehicle, respectively. Strong interactions among them are observed with the development of the ﬂow 222 which eventually results in a single shedding moving towards the outlet. The development of the 223 turbulent vortices is captured which is triggered by the ﬂow separation at the wake of the vehicle 224 (see Figure 9 and Figure 10). The isosurfaces in Figure 9 are visualized vortices using Q criterion and 225 coloured with stream-wise velocities. The separated ﬂow and the corresponding shedding signiﬁcantly 226 alters the ﬂow pattern at the wake of the ROV which leads to the non-linear and ﬂuctuating drag 227 forces acting on the ROV. 228 10 of 15 10 of 15 Version August 31, 2020 submitted to J. Mar. Sci. Eng. Figure 9. The isosurfaces vorticites structures coloured with stream-wise velocities Figure 9. The isosurfaces vorticites structures coloured with stream-wise velocities Figure 9. The isosurfaces vorticites structures coloured with stream-wise velocities Figure 10. The interactions between ﬂows generated by different parts of the vehicle Figure 10. The interactions between ﬂows generated by different parts of the vehicle The instantaneous velocity ﬁeld of the vehicle under the yaw motion is demonstrated in Figure 11. 6. Results Discussion 214 229 Three sets of individual vortex shedding are formed at the rear of the vehicle, but due to the inlet ﬂow 230 direction is not aligned with the vehicle movement direction in the rotational motion, the interactions 231 between the three sets of the shedding are not as strong as that in the translational motion. 232 Figure 11. Top view of the instantaneous velocity ﬂow under the yaw motion of the ROV Figure 11. Top view of the instantaneous velocity ﬂow under the yaw motion of the ROV Figure 12 demonstrates the time series of surge drag force exerting on the ROV under the ﬂow 233 velocities ranging from 0.2 m/s to 1.0 m/s. The surge and sway drag forces measured by the test 234 are compared to the numerical results in Figure 13 and Figure 14, respectively. For the surge drag, 235 it can be observed that a good agreement is achieved throughout the velocity range. However, the 236 discrepancy between the numerical and experimental result is increasing with the increase of the 237 velocity acting on the ROV. Similarly, the same trend is exhibited in the sway drag comparison, with 238 the maximum discrepancy appears at the largest velocity tested (0.6m/s). The major sources of errors 239 Version August 31, 2020 submitted to J. Mar. Sci. Eng. 11 of 15 in the numerical simulations include the neglect of the geometry details, such as attached propellers 240 and tether. Other error sources may the differences between the turbulent ﬂow generated by the 241 turbulent model and the reality in the FloWave. 242 The damping coefﬁcients for each direction are obtained by using a second-order polynomial ﬁt 243 (see Figure 15), and the resulting drag coefﬁcients of the vehicle in its four principal DOFs are given in 244 Table 2. As exhibited in Figure 15, the largest drag is observed in the heave motion due to its largest 245 frontal area in the X-Y plane. Meanwhile, the drag force in the sway motion is slightly larger than that 246 in the surge motion since the frontal area in the Y-Z plane is smaller than that of X-Z plane. 247 0 5 10 15 20 25 0 20 40 60 Drag force (N) Time (s) 0.2 m/s 0.4 m/s 0.6 m/s 0.8 m/s 1.0 m/s Figure 12. 6. Results Discussion 214 Surge force time series under the current velocity ranging from 0.2m/s to 1.0m/s 0 5 10 15 20 25 0 20 40 60 Drag force (N) Time (s) 0.2 m/s 0.4 m/s 0.6 m/s 0.8 m/s 1.0 m/s Figure 12. Surge force time series under the current velocity ranging from 0.2m/s to 1.0m/s 0 5 10 15 20 25 0 20 40 60 Time (s) 0.2 m/s 0.4 m/s 0.6 m/s 0.8 m/s 1.0 m/s Figure 12. Surge force time series under the current velocity ranging from 0.2m/s to 1.0m/s 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 Experimental results Numerical results Velocity (m/s) Figure 13. Comparison of surge force between the numerical and experimental results 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 Experimental results Numerical results Sway drag force (N) Velocity (m/s) Figure 12. Surge force time series under the current velocity ranging from 0.2m/s to 1.0m/s 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 Experimental results Numerical results Velocity (m/s) Figure 13. Comparison of surge force between the numerical and experimental results 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 Experimental results Numerical results Velocity (m/s) Figure 13. Comparison of surge force between the numerical and experimental results Figure 13. Comparison of surge force between the numerical and experimental results 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 Experimental results Numerical results Sway drag force (N) Velocity (m/s) Figure 14. Comparison of sway force between the numerical and experimental results 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 Experimental results Numerical results Sway drag force (N) Velocity (m/s) Figure 14. Comparison of sway force between the numerical and experimental results Version August 31, 2020 submitted to J. Mar. Sci. Eng. 6. Results Discussion 214 Surge, sway and heave drag force under various velocities Table 2. Table to test captions and labels Damping coefﬁcient Surge Sway Heave Yaw KL KQ KL KQ KL KQ KL KQ Values 1.3125 38.169 9.1435 129.6607 2.015 243.25 0 4.86 Table 2. Table to test captions and labels 6. Results Discussion 214 12 of 15 12 of 15 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 surge drag (N) velocity (m/s) surge second order polynomial fit of surge y=38.169x2+1.3125x sway second order polynomial fit of sway heave drag (N) velocity (m/s) heave second order polynomial fit of heave sway drag (N) velocity (m/s) y=129.6607x2+9.1435x y=243.25x2+2.015x Figure 15. Surge, sway and heave drag force under various velocities 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 surge drag (N) velocity (m/s) surge second order polynomial fit of surge y=38.169x2+1.3125x 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 0.0 0.2 0.4 0.6 0.8 1.0 0 40 80 120 160 surge dra velocity (m/s) sway second order polynomial fit of sway heave drag (N) velocity (m/s) heave second order polynomial fit of heave sway drag (N) velocity (m/s) y=129.6607x2+9.1435x y=243.25x2+2.015x Figure 15. Surge, sway and heave drag force under various velocities Table 2. Table to test captions and labels Damping coefﬁcient Surge Sway Heave Yaw KL KQ KL KQ KL KQ KL KQ Values 1.3125 38.169 9.1435 129.6607 2.015 243.25 0 4.86 Conclusion In this study, a numerical model within the frame of OpenFOAM is proposed, which is capable simulating multi DOFs motions and turbulent ﬂow problems. We investigated the hydrodynamic haviour of the BlueROV2 which is complex with an open frame structure. The accuracy and reliability the numerical model are validated by the experiential test, in which a new test method targeted at e force and moment measurement of the vehicle is designed. With the hydrodynamic coefﬁcients und by the numerical simulation, a more robust and stability control system can be designed in the namic positioning of ROV when facing the combined effect of current and turbulence. Besides, the drodynamic disturbances acting on the vehicle can be treated as external forces within the nonlinear OV dynamic and propulsion model to improve its disturbance rejection performance. The good reement with the experimental result builds the conﬁdence of applying the proposed methodologies more complex working scenarios. For instance, the marine renewable energy facilities are typically ployed in the shallow water environments where is characterised by strong hydrodynamic forces Figure 15. 7. Conclusion 248 271 Abbreviations 272 The following nomenclature and abbreviations are used in this paper: 273 274 D Cylinder diameter U Incoming ﬂow velocity C0 Courant number CL,CD Drag and lift coefﬁcients FL, FD Fluid drag and lift force Nt Number of independent samples Re Reynolds number St Strouhal number U Assemble-average velocity of the ﬂow fs Vortex-shedding frequency u(xi, t) Velocity at the nth series y+ Normalised distance yw Distance from the centre of the ﬁrst mesh cell to the wall d Water depth ui′uj′ Reynolds stresses u∗ Friction velocity νT(xi, t) Turbulent or eddy viscosity τw Wall shear stress △x Mesh size k Turbulent kinetic energy ϵ Turbulent dissipation κ Von Karman constant µ Dynamic viscosity ν Constant molecular viscosity ρ Flow density ω Speciﬁc turbulent dissipation rate ALE Arbitrary Lagrangian-Eulerian AUV Autonomous Underwater Vehicle DNS Direct Numerical Simulation DOF Degree of freedom FSI Fluid-Structure Interaction FVM Finite Volume Method HR High Reynolds number wall treatment LR Low Reynolds number wall treatment MoCAP Motion capturing system QALE Quasi-Arbitrary-Lagrangian-Eulerian RANS Reynolds-Averaged Navier-Stokes equations ROV Remotely Operated Vehicle SST Shear-Stress-Transport model VIV Vortex Induced Vibration Version August 31, 2020 submitted to J. Mar. Sci. Eng. involving both wave and current. In future work, the disturba 261 ﬂow surface should be tracked where a two-phase solver is req 262 the nonlinear disturbances considering the direction between t 263 Author Contributions: conceptualization, Q.L.; methodology, Q.L.; v 264 draft preparation, Q.L.; writing–review and editing, Y.C., B.L., A.K. a 265 administration, A.K. and D.I. 266 Funding: This research is funded by the EPSRC as part of EP/R0 267 Intelligence Hub for Offshore Energy Asset Integrity Management (O 268 Acknowledgments: The authors are extremely grateful to the staff at t 269 the FloWave facility, for making this research possible. 270 Conﬂicts of Interest: The authors declare no conﬂict of interest. 7. Conclusion 248 In this study, a numerical model within the frame of OpenFOAM is proposed, which is capable 249 of simulating multi DOFs motions and turbulent ﬂow problems. We investigated the hydrodynamic 250 behaviour of the BlueROV2 which is complex with an open frame structure. The accuracy and reliability 251 of the numerical model are validated by the experiential test, in which a new test method targeted at 252 the force and moment measurement of the vehicle is designed. With the hydrodynamic coefﬁcients 253 found by the numerical simulation, a more robust and stability control system can be designed in the 254 dynamic positioning of ROV when facing the combined effect of current and turbulence. Besides, the 255 hydrodynamic disturbances acting on the vehicle can be treated as external forces within the nonlinear 256 ROV dynamic and propulsion model to improve its disturbance rejection performance. The good 257 agreement with the experimental result builds the conﬁdence of applying the proposed methodologies 258 to more complex working scenarios. For instance, the marine renewable energy facilities are typically 259 deployed in the shallow water environments where is characterised by strong hydrodynamic forces 260 Version August 31, 2020 submitted to J. Mar. Sci. Eng. involving both wave and current. In future work, the disturbances triggered by the presen 261 ﬂow surface should be tracked where a two-phase solver is required. Furthermore, the mod 262 the nonlinear disturbances considering the direction between the wave and current is desire 263 Author Contributions: conceptualization, Q.L.; methodology, Q.L.; validation, Q.L., Y.C., B.L.; writin 264 draft preparation, Q.L.; writing–review and editing, Y.C., B.L., A.K. and D.I; supervision, A.K. and D 265 administration, A.K. and D.I. 266 Funding: This research is funded by the EPSRC as part of EP/R026173/1, the UK Robotics and 267 Intelligence Hub for Offshore Energy Asset Integrity Management (ORCA-Hub). 268 Acknowledgments: The authors are extremely grateful to the staff at the ORCA-Hub project team, in a 269 the FloWave facility, for making this research possible. 270 Conﬂicts of Interest: The authors declare no conﬂict of interest. References 275 AIP Conference Proceedings 2009, 1089, 11–22. doi:10.1063/1.3078117. 290 8. Eng, Y.H.; Chin, C.S.; Lau, M.W.S. 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Conclusion 248 271 Abbreviations 272 The following nomenclature and abbreviations are used in this paper: 273 274 D Cylinder diameter U Incoming ﬂow velocity C0 Courant number CL,CD Drag and lift coefﬁcients FL, FD Fluid drag and lift force Nt Number of independent samples Re Reynolds number St Strouhal number U Assemble-average velocity of the ﬂow fs Vortex-shedding frequency u(xi, t) Velocity at the nth series y+ Normalised distance yw Distance from the centre of the ﬁrst mesh cell to the wall d Water depth ui′uj′ Reynolds stresses u∗ Friction velocity νT(xi, t) Turbulent or eddy viscosity τw Wall shear stress △x Mesh size k Turbulent kinetic energy ϵ Turbulent dissipation κ Von Karman constant µ Dynamic viscosity ν Constant molecular viscosity ρ Flow density ω Speciﬁc turbulent dissipation rate ALE Arbitrary Lagrangian-Eulerian AUV Autonomous Underwater Vehicle DNS Direct Numerical Simulation DOF Degree of freedom FSI Fluid-Structure Interaction FVM Finite Volume Method HR High Reynolds number wall treatment LR Low Reynolds number wall treatment MoCAP Motion capturing system QALE Quasi-Arbitrary-Lagrangian-Eulerian RANS Reynolds-Averaged Navier-Stokes equations ROV Remotely Operated Vehicle SST Shear-Stress-Transport model VIV Vortex Induced Vibration 13 of 15 Version August 31, 2020 submitted to J. Mar. Sci. Eng. involving both wave and current. In future work, the disturbances triggered by the presence of the 261 ﬂow surface should be tracked where a two-phase solver is required. Furthermore, the modelling of 262 the nonlinear disturbances considering the direction between the wave and current is desired too. 263 Author Contributions: conceptualization, Q.L.; methodology, Q.L.; validation, Q.L., Y.C., B.L.; writing–original 264 draft preparation, Q.L.; writing–review and editing, Y.C., B.L., A.K. and D.I; supervision, A.K. and D.I; project 265 administration, A.K. and D.I. 266 Author Contributions: conceptualization, Q.L.; methodology, Q.L.; validation, Q.L., Y.C., B.L.; writing–original 264 draft preparation, Q.L.; writing–review and editing, Y.C., B.L., A.K. and D.I; supervision, A.K. and D.I; project 265 administration, A.K. and D.I. 266 Funding: This research is funded by the EPSRC as part of EP/R026173/1, the UK Robotics and Artiﬁcial 267 Intelligence Hub for Offshore Energy Asset Integrity Management (ORCA-Hub). 268 Acknowledgments: The authors are extremely grateful to the staff at the ORCA-Hub project team, in addition to 269 the FloWave facility, for making this research possible. 270 14 of 15 14 of 15 Version August 31, 2020 submitted to J. 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https://openalex.org/W4390886243	https://jurnal.polgan.ac.id/index.php/jmp/article/download/13337/2255	English	null	Implications of Technological Evolution on Human Resource Management Strategies and Legal Compliance	Jurnal Minfo Polgan	2,024	cc-by	4,593	Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 Terbit : 03 Januari 2024 Terbit : 03 Januari 2024 ABSTRACT Technological evolution, particularly in the realms of digitization and automation, has had a profound impact on how companies conduct their business. The use of artificial intelligence systems, data analytics, and collaborative platforms has transformed work processes and expedited business operations. This research aims to analyze the implications of technological evolution on human resource management strategies and legal compliance. The study utilizes a comprehensive analysis of previously published literature, employing a qualitative analysis approach to gain a thorough understanding of the issues at hand. The research period spans from 2000 to 2023. The study's findings indicate that technological evolution has significantly affected human resource management strategies and legal compliance, especially within the context of Indonesia's Law Number 13 of 2003 concerning Manpower. The integration of technology into human resource management processes has enhanced efficiency and productivity but has also posed new challenges related to employee privacy, data protection, and changes in work dynamics. Keywords: Technological Evolution, Human Resources, Legal Compliance This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Implications of Technological Evolution on Human Resource Management Strategies and Legal Compliance 1Yuspika Yuliana Purba 1Program Studi Ilmu Hukum, Fakultas Hukum, Universitas Simalungun, Indonesia INTRODUCTION Consequently, it becomes imperative to conduct an exhaustive study on the legal compliance aspects of deploying this technology in the context of labor management. This is not solely to ensure regulatory adherence but also to guarantee that the use of technology in recruitment and selection remains fair, cognizant of diversity, and upholds integrity and trust from both employee and legal perspectives. Thus, meticulous research and analysis are pivotal to ensuring that the technology adopted by companies in HR management results in accurate and equitable decisions. The application of technology in human resource management (HRM) brings not only benefits but also significant challenges related to worker data privacy and security. Alongside the integration of technology solutions such as cloud-based HR management systems and collaborative platforms, companies bear the responsibility of safeguarding workers' personal information in compliance with applicable data security standards (Dwivedi et al., 2023). Consequently, it is essential for companies to formulate and implement policies guaranteeing not only the privacy of workers but also compliance with rapidly evolving data protection regulations. Involving workers in this process, ensuring transparency regarding data collection and use, and embracing advanced security technologies are vital steps in preserving integrity and trust in technology-enabled HR management. Thus, awareness and commitment to the privacy and security aspects of data form foundational elements in embracing the positive potential of technology in the context of HR management. Law No. 13 Year 2003, governing various aspects of employment encompassing workers' rights and obligations, wage policies, and dispute resolution, stands as a crucial legal foundation defining the labor framework in Indonesia (Kemenperin, 2003). While this law provides a robust foundation, the rapid evolution in technology poses new challenges to its implementation and compliance. Changes in the way business is conducted, such as the utilization of artificial intelligence, automation, and digital platforms, can create disparities with the provisions of the law. Consequently, a thorough analysis is increasingly important to determine how companies can adapt their strategies to ensure full compliance with existing regulations. This involves integrating applicable labor principles with technological advancements and developing internal policies that respond to new dynamics in the labor context. Proactive and adaptive efforts are thus required to keep companies abreast of technological developments and maintain compliance with the applicable labor law framework. INTRODUCTION The swift evolution of technology, particularly in the domains of digitalization and automation, has profoundly reshaped the business landscape for companies. The adoption of artificial intelligence systems, data analytics, and collaborative platforms not only transforms the fundamental nature of work but also expedites overall business processes (Agustian, Mubarok, et al., 2023). These changes necessitate adaptations in human resource management (HRM) and compliance with labor laws. Faced with these challenges, companies are compelled to overhaul their HR management strategies, aligning them with the novel dynamics introduced by technological evolution. Additionally, adherence to labor regulations assumes paramount importance, given the substantial shifts in operational paradigms. Hence, conformity with today's technological transformation is not merely essential but imperative for sustaining competitiveness and efficiency in this digital era. The continual progression of technology has ushered in new opportunities in terms of work flexibility and the mobility of human resources (HR) (Sutrisno et al., 2023; Wahyoedi et al., 2023). Phenomena such as remote work, the utilization of smart devices, and collaborative platforms have become pervasive in the contemporary work environment. The ramifications of this transformation underscore the need for a comprehensive review of company labor policies, particularly concerning critical aspects like working hours, rest periods, and workers' rights. In an era where technological innovation consistently pushes traditional boundaries, companies must adopt a forward-thinking and adaptive approach (Wanof, 2023). This ensures that their internal policies are not only in This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. 2686 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 harmony with these new dynamics but also facilitate the implementation of inclusive working practices responsive to individual needs and the latest technological developments. harmony with these new dynamics but also facilitate the implementation of inclusive working practices responsive to individual needs and the latest technological developments. Today's corporate landscape exhibits an increasing reliance on technology for the recruitment and selection processes of human resources (HR). The integration of artificial intelligence algorithms into these processes can significantly impact crucial facets such as fairness and transparency in employee selection (Wibowo et al., 2023). INTRODUCTION The research envisaged aims to make a substantial contribution to deepening the understanding of the impact of technological evolution on human resource management (HRM) and labor law compliance in Indonesia. By focusing attention on the intricate dynamics between technological developments and HRM, the research aims to identify practical implications that may arise for companies. Through detailed analysis of specific aspects related to technological change within the ambit of HR management, this research also seeks to develop a practical guide. Such a guide could serve as a source of guidance for companies in effectively navigating this transformation. By providing in-depth insights and practical solutions, this research holds the potential to become a foundational resource for stakeholders in academia and industry. It will empower them to make informed and contextualized decisions in addressing the dynamics of technological evolution in the Indonesian labor context. This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Legal Compliance Legal compliance refers to the adherence of an individual, organization, or institution to the regulations, norms, and legal provisions applicable within a jurisdiction (Simmons, 2000). It involves understanding, implementing, and adjusting to regulations that govern their activities and operations. Legal compliance becomes a critical aspect in maintaining social and economic stability, as well as ensuring the protection of rights and obligations for all parties involved (Ratner, 2001). In a business context, legal compliance includes various aspects such as labor rights, environmental regulations, product safety, and tax regulations. Organizations operating with good legal compliance principles not only avoid legal risks and sanctions but also create a positive reputation and trust from stakeholders. Therefore, awareness and commitment to legal compliance are essential in conducting business activities or other endeavors ethically and in accordance with applicable legal provisions. Human Resources (HR) Human Resources (HR) refer to the human elements utilized by an organization or company to achieve its goals and ensure operational sustainability. HR involves all aspects related to the workforce, including employees, leaders, and management systems that support productivity and organizational development (Gadzali, Ausat, et al., 2023). The main components of HR include recruitment, selection, training, development, performance management, and labor policy regulation (Agustian, Pohan, et al., 2023). Organizations effective in managing their HR can achieve optimal productivity and create a healthy work environment (Tusriyanto et al., 2023). Human Resources not only involve operational aspects but also focus on individual potential development, ensuring diversity, and promoting fairness within the workplace (Gadzali, Gazalin, et al., 2023). By recognizing the key value of HR as a primary asset, organizations can achieve competitive advantages and long-term sustainability. Technological Evolution g Technological evolution refers to the development and changes in technological systems over time, encompassing innovation, improvement, and transformation in how humans utilize 2687 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 knowledge and tools to meet their needs (Suherlan & Okombo, 2023). This phenomenon is not limited to the advancement of hardware such as computers, phones, or vehicles but also involves progress in software, artificial intelligence, and ever-growing connectivity (Rijal & Saranani, 2023). In this process of evolution, technology undergoes not only individual changes but also interacts with various sectors of society, including the economy, education, health, and communication. Paradigm shifts in technological evolution often create significant impacts on how we work, communicate, and live daily, presenting both new challenges and opportunities in various aspects of human life (Beer & Mulder, 2020). Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 knowledge and tools to meet their needs (Suherlan & Okombo, 2023). This phenomenon is not limited to the advancement of hardware such as computers, phones, or vehicles but also involves progress in software, artificial intelligence, and ever-growing connectivity (Rijal & Saranani, 2023). In this process of evolution, technology undergoes not only individual changes but also interacts with various sectors of society, including the economy, education, health, and communication. Paradigm shifts in technological evolution often create significant impacts on how we work, communicate, and live daily, presenting both new challenges and opportunities in various aspects of human life (Beer & Mulder, 2020). This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. RESEARCH METHOD In this research, we employed a qualitative approach to thoroughly investigate the literature with the aim of exploring the impact of technological evolution on human resource management strategies and legal compliance. The critical time frame for this study was from 2000 to 2023, utilizing a methodology that involved the exploration of academic literature, conference papers, and reliable sources through Google Scholar. Our specific search criteria, designed to encompass relevant keywords such as technological evolution, human resources, and legal compliance, guided the selection of sources based on their relevance to the research objectives. We explicitly acknowledge all limitations, such as language constraints or publication bias. The research proceeded through a series of stages, starting with the identification of a specific and relevant research topic and the formation of a comprehensive understanding of the background and objectives of the study. Explicit search criteria guided the exploration of literature on Google Scholar, covering the period from 2000 to 2023. After obtaining search results, a careful literature selection process was conducted, involving the examination of abstracts and summaries for each identified article or source. Literature deemed less relevant or failing to meet the research criteria was excluded from the analysis. y The selected literature then underwent a thorough evaluation, focusing on identifying key 2688 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 findings, concepts, theories, and trends within the literature. A qualitative approach was chosen to achieve a comprehensive understanding of the research subject. Findings from the literature review were subsequently synthesized and further elucidated by the researchers to build a comprehensive d t di f th h t i Th fi di t d i d t il d h t Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 findings, concepts, theories, and trends within the literature. A qualitative approach was chosen to achieve a comprehensive understanding of the research subject. Findings from the literature review were subsequently synthesized and further elucidated by the researchers to build a comprehensive understanding of the research topic. These findings were presented in a detailed research report with a structured and coherent format, encompassing key findings, analysis, and in-depth interpretation. RESULTS AND DISCUSSION In navigating a rapidly evolving era, the technological revolution is not only a phenomenon, but also a transformational force that significantly changes the landscape of life, especially in the realm of human resource management and legal compliance in the world of work. This enormous impact cannot be separated from the increasingly complex dynamics of society and the global changes that occur continuously. In the context of Indonesian labour law, Law Number 13 Year 2003 is the legal foundation that comprehensively regulates the relationship between workers and employers. However, new challenges arising from the rapid pace of technological development have forced a review of existing legal provisions in order to maintain the relevance and sustainability of the labour system in the midst of ongoing transformation. First of all, it should be noted that the technological revolution has changed the traditional paradigm of human resource management, shaping an increasingly integrated and sophisticated landscape. The integration of information systems and artificial intelligence in employee recruitment, selection and performance evaluation processes is not just an innovation, but has become a necessity in many organisations. The application of these technologies, which include big data analytics, machine learning, and intelligent algorithms, can not only improve operational efficiency, but also increase accuracy and objectivity in human resource decision-making. However, the presence of these positive impacts also raises critical questions related to employee privacy and potential discrimination in the context of automated decision-making. Therefore, there is a need for deep reflection to harmonise technological advancements with ethical values and fairness in the context of human resource management (Davidescu et al., 2020). In addition, the evolution of technology has also changed the dynamics of work itself, shaping a new paradigm fuelled by digital advancements (Kraus et al., 2021). Flexible remote working is no longer just an option, but has become an increasingly popular choice, especially with the rise of digital collaboration platforms. This change not only creates flexibility for workers, but also has a significant impact on regulations related to working hours, leave entitlements, and employee welfare protection. Critical questions arise regarding the extent to which existing labour regulations are able to accommodate increasingly sophisticated and connected work dynamics (Kozlowski & Ilgen, 2006). RESEARCH METHOD Based on the reviewed literature, this methodology is expected to make a significant contribution to the development of theories, problem-solving, or decision-making in various academic disciplines, especially in the context of the impact of technological evolution on human resource management strategies and legal compliance. The structured methodology section provides clear and cohesive explanations for each stage of the research process, with subsections enhancing readability and ensuring a transparent and robust approach in conducting this research. This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. RESULTS AND DISCUSSION Meanwhile, from a legal compliance perspective, the use of technology in human resource management requires companies to not only understand, but also adapt themselves to follow regulations relating to data protection, privacy rights, and information security. The increasing reliance of companies on technology brings serious repercussions related to the risk of breaching these legal provisions. Such breaches not only have the potential to harm a company's reputation, but can also result in serious legal sanctions, including large fines and even revocation of business licences. Therefore, compliance with regulations relating to technology and data management is an absolute must for modern companies that want to maintain their integrity, ensure operational sustainability, and provide legal certainty for all parties involved. Thus, the implementation of technology must be accompanied by a strong legal compliance strategy, so that companies can run their operations efficiently and within the limits of applicable law. In the Indonesian context, given the rapidly evolving dynamics of work and technology, a revision of Law No. 13/2003 is likely necessary to address the new challenges and opportunities that arise. The government and relevant stakeholders must synergise and work together proactively to formulate regulations that are not only adequate but also relevant to the latest technological developments. This revision process is not only a legal necessity, but also an opportunity to create a legal foundation that is more inclusive and adaptive to the increasingly sophisticated work environment. In addition, it is important to ensure that in the revision process, employee rights are strengthened and remain substantially protected. These steps are key in building a responsive legal framework, creating a sustainable business climate, and providing guarantees for the healthy and fair development of the world of work in the modern technological era. To conclude, the evolution of technology has permeated various layers of human resource management strategies and legal compliance arrangements in the modern era with impacts that are not only broad but also profound. While technology opens the door to tantalising new opportunities, the complex challenge of maintaining a balance between innovative advancements and the protection of employee rights demands serious attention from all stakeholders. There needs to be a commitment to engage in open dialogue, conduct in-depth studies on the implications of new technologies, and make appropriate regulatory changes to accommodate the shifting work paradigm. RESULTS AND DISCUSSION Therefore, an in-depth study is needed to evaluate and modernise labour policies to address the new challenges that arise along with this paradigm shift, and ensure that the protection and rights of employees are maintained in line with technological advancements. On the other hand, along with technological advancements come increasingly complex issues related to digital security and employee data protection. In an effort to create a safe and trusted work environment, companies are not only faced with the challenge of data leaks that can jeopardise employee rights and privacy (Javaid et al., 2023), but also with the growing risks associated with cyberattacks and unethical use of technology. Facing these dynamics, there is a need for labour regulations that are not only responsive but also proactive in regulating companies' obligations in protecting employees' personal information (Irawan et al., 2022). Therefore, the importance of an up-to-date and sophisticated legal framework is becoming increasingly apparent, This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. 2689 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 to ensure that companies not only meet high digital security standards but are also responsible for to ensure that companies not only meet high digital security standards but are also responsible for managing employee data in a fair and transparent manner. to ensure that companies not only meet high digital security standards but are also responsible for managing employee data in a fair and transparent manner. However, these changes do not always go hand in hand with the development of labour law, creating an ever-widening gap between the speed of technological development and the law's ability to adjust to the evolving dynamics of work. As a concrete example, essential questions arise about the extent to which employee rights in a digital work environment are adequately addressed in Law No. 13 Year 2003, or whether substantive amendments are needed to accommodate these new dynamics. This challenge involves deep consideration of the rights to privacy, security and fairness in the face of an increasingly connected and technology-dependent work reality. Hence, there is a need for a thorough evaluation of the labour law framework to ensure that existing regulations are not only relevant but also proactive in anticipating the impacts that may arise due to the ongoing shift in the work paradigm. RESULTS AND DISCUSSION By doing so, society can ensure that the evolution of technology in the world of work not only brings balanced positive benefits, but also creates a fair environment for all parties, safeguards employee rights, and upholds the values of justice in shaping a smart and inclusive future of work. This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. CONCLUSION In conclusion, it can be inferred that technological evolution has significantly impacted human resource management strategies and legal compliance, particularly within the context of Indonesia's Law Number 13 of 2003 on Manpower. The integration of technology into HRM processes has enhanced efficiency and productivity but has simultaneously posed new challenges related to employee privacy, data protection, and changes in work dynamics. From the perspective of labor law, these changes require a prompt and sustained response from the authorities. A revision This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. 2690 Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 of Law Number 13 of 2003 may be necessary to accommodate the increasingly sophisticated work environment. This update should consider employee privacy rights, digital security, and compliance with international standards on data protection. Jurnal Minfo Polgan Volume 12, Nomor 2, Januari 2024 DOI : https://doi.org/10.33395/jmp.v12i2.13337 e-ISSN : 2797-3298 p-ISSN : 2089-9424 of Law Number 13 of 2003 may be necessary to accommodate the increasingly sophisticated work environment. This update should consider employee privacy rights, digital security, and compliance with international standards on data protection. of Law Number 13 of 2003 may be necessary to accommodate the increasingly sophisticated work environment. This update should consider employee privacy rights, digital security, and compliance with international standards on data protection. p p Recommendations involve collaboration between the government, businesses, and other stakeholders. Education and training on the legal implications and ethics of technology implementation need to be enhanced to ensure that all involved parties understand and comply with applicable regulations. Additionally, authorities need to actively monitor technological developments to respond quickly and appropriately to changes. Companies are expected to adopt internal policies that prioritize employee rights, maintain data security, and ensure compliance with regulations. The implementation of technology should be accompanied by transparency and accountability, creating a digital work environment where employees feel secure and respected. In the long term, the development of progressive and adaptive regulations can help create a work environment aligned with technological advancements. Through open and collaborative dialogue among the government, businesses, academics, and the public, Indonesia can take steps toward policies that support innovation while safeguarding the fundamental rights of workers. Thus, the technological evolution in the workplace can contribute positively to sustainable and inclusive development. Commons Attribution-NonCommercial 4.0 International License. This is an Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. 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https://openalex.org/W2947312239	https://pure.eur.nl/ws/files/48203668/Repub_116831_O-A.pdf	English	null	Risks and clinical predictors of cirrhosis and hepatocellular carcinoma diagnoses in adults with diagnosed NAFLD: real-world study of 18 million patients in four European cohorts	BMC medicine	2,019	cc-by	7,092	Alexander et al. BMC Medicine (2019) 17:95 https://doi.org/10.1186/s12916-019-1321-x Alexander et al. BMC Medicine (2019) 17:95 https://doi.org/10.1186/s12916-019-1321-x Open Access Risks and clinical predictors of cirrhosis and hepatocellular carcinoma diagnoses in adults with diagnosed NAFLD: real-world study of 18 million patients in four European cohorts Myriam Alexander1, A. Katrina Loomis2, Johan van der Lei3, Talita Duarte-Salles4, Daniel Prieto-Alhambra5, David Ansell6, Alessandro Pasqua7, Francesco Lapi7, Peter Rijnbeek3, Mees Mosseveld3, Dawn M. Waterworth8, Stuart Kendrick9, Naveed Sattar10† and William Alazawi11† Abstract Background: Non-alcoholic fatty liver disease (NAFLD) is a common condition that progresses in some patients to steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Here we used healthcare records of 18 million adults to estimate risk of acquiring advanced liver disease diagnoses in patients with NAFLD or NASH compared to individually matched controls. Methods: Data were extracted from four European primary care databases representing the UK, Netherlands, Italy and Spain. Patients with a recorded diagnosis of NAFLD or NASH (NAFLD/NASH) were followed up for incident cirrhosis and HCC diagnoses. Each coded NAFLD/NASH patient was matched to up to 100 “non-NAFLD” patients by practice site, gender, age ± 5 years and visit recorded within ± 6 months. Hazard ratios (HR) were estimated using Cox models adjusted for age and smoking status and pooled across databases by random effects meta-analyses. Results: Out of 18,782,281 adults, we identified 136,703 patients with coded NAFLD/NASH. Coded NAFLD/NASH patients were more likely to have diabetes, hypertension and obesity than matched controls. HR for cirrhosis in patients compared to controls was 4.73 (95% CI 2.43–9.19) and for HCC, 3.51 (95% CI 1.72–7.16). HR for either outcome was higher in patients with NASH and those with high-risk Fib-4 scores. The strongest independent predictor of a diagnosis of HCC or cirrhosis was baseline diagnosis of diabetes. Conclusions: Real-world population data show that recorded diagnosis of NAFLD/NASH increases risk of life- threatening liver outcomes. Diabetes is an independent predictor of advanced liver disease diagnosis, emphasising the need to identify specific groups of patients at highest risk. Keywords: Cirrhosis, Hepatocellular cancer, NAFLD, NASH, Population Correspondence: w.alazawi@qmul.ac.uk p @q †Naveed Sattar and William Alazawi contributed equally to this work. 11Barts Liver Centre, Blizard Institute, Queen Mary, University of London London, UK Full list of author information is available at the end of the article p q †Naveed Sattar and William Alazawi contributed equally to this work. 11Barts Liver Centre, Blizard Institute, Queen Mary, University of London, London, UK Full list of author information is available at the end of the article Methods Databases Databases were accessed via the European Medical In- formation Framework (EMIF) network: The Health Search Database (HSD) in Italy [13], The Integrated Pri- mary Care Information (IPCI) in the Netherlands [14], the Information System for the Development of Research in Primary Care (SIDIAP) in Spain [15] and The Health Information Network (THIN) in the UK [16] (Additional file 1: Table S1). HSD collects electronic medical record data from a network of over 800 Italian GPs who are members of the Italian College of General Practitioners. IPCI is a longitudinal collection of elec- tronic patient records from over 750 Dutch general practitioners, containing data from over 2 million pa- tients. SIDIAP collects data from 274 primary care prac- tices comprising 3414 basic care units [17], and THIN contains the electronic medical records of 11.1 million patients from 562 general practices in the UK, covering 6.2% of the UK population [18]. The data custodians for each database provided approval that the protocol of the study complied with local privacy laws. Anonymised data were extracted locally by each data custodian liaising with the EMIF Platform and using a data transformation tool called Jerboa Reloaded [10]. The data were then uploaded onto a secure remote server maintained by an independent academic centre (Erasmus Medical Centre Private Research Environment, Netherlands) and ana- lysed centrally. Our current understanding of NAFLD epidemiology and progression largely derives from single-centre stud- ies of small- or medium-sized cohorts and meta-analyses of these [5–7]. These studies, together with emerging data from placebo arms of therapeutic trials [8], have taught us that patients with existing evidence of progres- sive disease (e.g., fibrosis) are at risk of further progres- sion to HCC and decompensated cirrhosis, albeit this may reflect a degree of lead-time bias. Such studies often involve formal assessment of well-phenotyped patients at inclusion but are, by design, selective and may not represent the ‘real-world’ situation for the majority of patients with NAFLD. Paired biopsy data have been reported, although the second biopsy is often performed because of clinical suspicion and not per study protocol, which may bias estimates of pro- gression [9]. Real-world patients are socially and eth- nically diverse, have comorbidities and concomitant medications or simply cannot commit to long-term studies or trials and therefore may not be represented by any of these study designs. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Alexander et al. BMC Medicine (2019) 17:95 Alexander et al. BMC Medicine (2019) 17:95 Page 2 of 9 Methods Databases Increasingly, real-world data derived from primary care electronic health records (EHR) of a sizeable pro- portion of the general population [10, 11] are being used to address these issues. In many European countries, where healthcare is largely state-funded and there are low or absent primary care co-payments, the population has unrestricted access to healthcare via primary care physicians who act as gatekeepers for referral to second- ary care [12]. People register with primary care centres at birth or when they move to an area in order to access healthcare; therefore, primary care EHR represent data that are as close to the ‘general’ population as possible. If a practice joins the database, all the patients at that practice are registered in the database and, although there is an option for individual patients to opt out, this is minimal (< 1%). Background In order to gain insights into the NAFLD spectrum of diseases in real-world patients, we extracted data from four large European primary care databases and identi- fied a cohort of patients with a diagnosis of NAFLD or of NASH. Our aim in this study was to estimate the risk for patients with diagnoses of NAFLD or NASH to ac- quire a new diagnosis of cirrhosis and HCC and to understand the main predictors for this. g Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. NAFLD represents a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH) and fi- brosis [1]. The numbers of individuals presenting with end-stage complications of NASH, namely decompen- sated cirrhosis and hepatocellular carcinoma (HCC), are rising [2, 3], and NASH is rapidly becoming the most common indication for liver transplantation [4]. Yet not all patients within the NAFLD spectrum pro- gress, and for the majority, NAFLD is a benign condi- tion [1]. A key clinical challenge is to identify the proportion of patients who are at high risk of devel- oping advanced liver disease, so that interventions, in- cluding the many novel therapies in development, can be targeted to those at greatest need. Study design d y g We conducted a matched cohort study. All patients with a diagnosis of NAFLD or NASH (termed NAFLD/ NASH) prior to 01/01/2016 were identified in the four databases using harmonisation methods previously de- scribed [10]. Patients were included in the analysis if they were aged ≥18 at diagnosis and had medical re- cords available for ≥12 months from registration with the practice. Exclusion criteria were missing informa- tion on age and sex, a record of alcohol abuse at any time prior to diagnosis and a history of liver morbid- ity within the 12 months prior to diagnosis [10] (see Additional file 1: Supplementary Methods for exclusion diagnoses). Page 3 of 9 Page 3 of 9 Alexander et al. BMC Medicine (2019) 17:95 Alexander et al. BMC Medicine (2019) 17:95 Page 3 of 9 Each NAFLD/NASH patient was matched with up to 100 ‘non-exposed’ controls who did not have a NAFLD or NASH diagnosis at or prior to the index date (defined as the date of diagnosis of the matched NAFLD/NASH patient). Matching was done by practice site, age at index date ± 5 years, sex and a visit at the practice within ± 6 months of the index date. were available, ALT and AST values were highest in THIN, and the proportion of obese patients highest in SIDIAP. Sufficient data were available to calculate the non-invasive fibrosis Fib-4 score (age, AST, ALT and platelets) in 46.7% of patients (range 12.6–62.6%, Table 2). THIN (UK) had the smallest proportion of patients with Fib-4 data (12.6%), in whom the propor- tion of patients with high-risk scores was 10.5%, high- est among the four databases. were available, ALT and AST values were highest in THIN, and the proportion of obese patients highest in SIDIAP. Sufficient data were available to calculate the non-invasive fibrosis Fib-4 score (age, AST, ALT and platelets) in 46.7% of patients (range 12.6–62.6%, Table 2). THIN (UK) had the smallest proportion of patients with Fib-4 data (12.6%), in whom the propor- tion of patients with high-risk scores was 10.5%, high- est among the four databases. In the THIN and SIDIAP databases, the terminology of the database (Read code and International Classifica- tion of Disease version 10, ICD10, respectively) allowed NAFLD and NASH diagnoses to be distinguished from each other. Study design d Therefore, in these databases, a matched control cohort was constructed for each of the diagno- ses: NAFLD, NASH and, to enable comparison between all databases, NAFLD/NASH. If a patient had both NAFLD and NASH diagnoses recorded, the earliest event was used to define index date of NAFLD/NASH diagnosis, and the NASH diagnosis deemed an inci- dent event. In HSD (ICD 9) and IPCI (IPCI Dutch), where NAFLD and NASH could not be distinguished, only one cohort (NAFLD/NASH) was defined and controls matched to this. Patients with a coded diagnosis of NAFLD/NASH had comparable age and sex distribution, smoking rates and duration of follow-up as matched controls (Table 1). As expected, however, controls had lower BMI; lower rates of obesity, hypertension or diabetes; and lower serum levels of ALT and AST. Results Similarly, the risk of incident HCC diagnosis was sig- nificantly higher in coded NAFLD/NASH patients com- pared to controls. The pooled HR across the four databases for an incident diagnosis of HCC was 3.51 (95%CI 1.72–7.16 Fig. 2). There were no significant dif- ferences in the HRs when categorising patients into those with and without obesity, smoking, diabetes or hypertension; male sex and older age (Additional file 1: Figure S1). There were no significant differences in the HRs for cirrhosis and HCC diagnoses following adjust- ment for age and smoking alone in all coded NAFLD/ NASH patients compared to patients with available BMI data (Additional file 1: Figures S2 and S3). This is des- pite the fact that patients with BMI data were more likely to be smokers (19.5% vs 11.2%), diabetic (26.9% vs 7.0%) and hypertensive (50.1% vs 27.9%, Additional file 1: Table S5). Out of 18,782,281 eligible individuals in the four databases, we identified 136,703 (0.7%) who had a recorded diagnosis of either NAFLD or NASH (coded NAFLD/NASH) and who met the inclusion criteria (Additional file 1: Table S1). The Spanish (SIDIAP) and UK (THIN) databases contrib- uted 71% of all cases; the remaining 29% of coded NAFLD/ NASH cases were from the Dutch (IPCI) and Italian (HSD) databases. In SIDIAP, 2.5% of all coded NAFLD/NASH pa- tients (n = 1880) had NASH, and in THIN, this was 4.7% (n = 1212). Due to the coding, NAFLD and NASH could not be distinguished in IPCI and HSD. Therefore, in the initial phase of analysis, we combined all NAFLD and NASH codes from all four databases as coded NAFLD/ NASH. Comparing coded NAFLD/NASH patients across the four databases, there were minor differences between da- tabases in mean age, BMI and proportion with diabetes (Table 1 and Additional file 1: Table S2). BMI data were available in 64.6% of patients with coded NAFLD/NASH and in 45.9% of matched controls (Additional file 1: Table S3). In the subset of patients for whom data Risk of incident cirrhosis and HCC is higher in NAFLD/ NASH patients compared to controls Combining all four databases, the median duration of follow-up was 3.3 years (IQR 1.8–5.3) totalling 531,452 person-years for patients with coded NAFLD/NASH and 43,385,495 person-years for controls. Among all coded NAFLD/NASH patients, the incidence of cirrhosis diagnosis was 0.76 per 1000 person-years, (95% confi- dence interval (CI) 0.46 to 2.32), and the incidence of hepatocellular carcinoma diagnosis was 0.3 per 1000 person-years, (0.26 to 0.60; Additional file 1: Table S4). Patients with coded NAFLD/NASH were at significantly higher risk of acquiring a new diagnosis of cirrhosis compared to controls with a pooled HR of 4.73 (95%CI 2.43–9.19) after adjustment for age, smoking status and BMI (Fig. 1). Patients were followed up from the index date until the earliest of occurrence of cirrhosis, hepatocellular car- cinoma or NASH (where this could be identified), end of the study period (31/12/2015) and loss of follow-up due to exit out of the database or death. Events of interest were incident diagnosis of cirrhosis, hepatocellular car- cinoma or NASH, where this could be identified. See Additional file 1: Supplementary Methods for variable extraction and data analysis. Fib-4 predicts disease progression in patients with NAFLD/NASH In the subset of coded NAFLD/NASH patients in whom we could calculate Fib-4 (n = 63,971, Additional file 1: Table S3), Page 4 of 9 Alexander et al. BMC Medicine (2019) 17:95 Alexander et al. BMC Medicine Table 1 Descriptive characteristics of coded NAFLD/NASH patients and matched unexposed cohorts Baseline characteristics HSD - Italy IPCI - UK SIDIAP - Spain THIN - UK Total population NAFLD/NASH Matched non- NAFLD/NASH NAFLD/NASH Matched non- NAFLD/NASH NAFLD/NASH Matched non- NAFLD/NASH NAFLD/NASH Matched non- NAFLD/NASH NAFLD/NASH Matched non- NAFLD/NASH Follow-up years prior to index date: median (IQR) 7.5 (4.66–10.5) 7.7 (4.8–10.6) 2.2 (0.9–3.8) 2.2 (1–3.8) 5.2 (3.1–7.1) 5.2 (3.1–7.1) 13.4 (5.5–23.4) 13.9 (5.8–23.4) 5.4 (2.8–8.0) 5.5 (2.9–8.3) Follow-up years post index date: median (IQR) 5.3 (2.7–8.1) 5.2 (2.7–7.9) 1.8 (0.8–3.2) 1.8 (0.8–3.3) 3.5 (1.7–5.6) 3.5 (1.7–5.7) 3.1 (1.3–5.9) 3.0 (1.3–5.7) 3.3 (1.5–5.8) 3.2 (1.5–5.7) Age in years, mean (SD) 56.1 (14.4) 55.0 (13.7) 56.8 (13.9) 56.1 (13.5) 55.9 (13.4) 54.5 (13.1) 54.4 (13.4) 53.5 (13.5) 55.8 (13.6) 54.6 (13.3) Gender, % of Males 57.3 54.7 49.2 48.5 52.6 48.8 51.5 51.0 52.7 50.1 Current smokers, % 11.3 9.0 17.2 11.5 17.6 15.4 17.8 19.0 16.5 14.4 Body mass index in kg/m2, mean (SD) 29.8 (5.0) 27.5 (5.0) 30.8 (5.3) 28.3 (5.2) 31.4 (5.1) 28.7 (5.1) 32.5 (6.0) 28.5 (5.9) 31.3 (5.3) 28.5 (5.3) Obesity (%) 21.5 9.0 23.6 9.0 39.5 18.1 49.6 18.2 36.0 15.4 History of type 2 diabetes (%) 17.6 11.0 20.4 9.2 19.9 10 21.5 7.1 19.8 9.6 History of hypertension (%) 47.2 36.6 36.0 26.2 42.6 29.3 41.2 25.9 42.2 29.6 Statin use (%) 22.0 15.8 33.2 22.0 6.9 6.8 32.6 18.5 16.9 12.1 Aspartate transaminase (IU/L), median (IQR) 24 (19–32) 20 (17–25) 29 (22–40) 23 (20–28) 29 (22–40) 22 (18–27) 31 (23–45) 22 (19–27) 28 (21–39) 21 (18–26) Alanine transaminase (IU/L), median (IQR) 30 (20–48) 21 (16–29) 37 (25–55) 25 (18–33) 34 (22–53) 20 (15–28) 42 (26–65) 22 (17–31) 35 (23–54) 21 (16–29) After imputation of missing as non-smokers. N number of individuals. For laboratory values, we exclude outlier values greater than mean + 3 × SD (mean and SD computed separately in NAFLD and non-NAFLD separately) Alexander et al. BMC Medicine (2019) 17:95 Alexander et al. Fib-4 predicts disease progression in patients with NAFLD/NASH BMC Medicine Page 5 of 9 Table 2 Distribution of Fib-4 scores in coded NAFLD/NASH patients shown for each country database Risk category HSD Italy (%) IPCI Netherlands (%) SIDIAP Spain (%) THIN UK (%) Total population (%) Low (< 1.30) 64.7 69.7 65.3 63.4 65.4 Indeterminate (1.30–2.67) 31.0 26.6 30.1 26.2 29.8 High (> 2.67) 4.3 3.7 4.6 10.5 4.7 In the subset of patients with a coded diagnosis of NASH, the incidence of diagnoses of liver outcomes was higher than in those with NAFLD albeit confidence intervals overlapped: 3.25 per 1000 person-years (95%CI 2.41–4.10) for cirrhosis and 1.16 per 1000 person-years (95%CI 0.67– 1.65) for HCC (Figs. 1 and 2). the incidence of a new diagnosis of cirrhosis was significantly higher for the high-risk compared to low-risk category (HR 33.24, 95%CI 8.82–125.34), adjust- ing for age and smoking status and more modest, albeit still significant, for the intermediate compared to low-risk group (HR 5.04, 95%CI 2.30–11.04 Additional file 1: Figure S4A). Similarly, compared to patients with low-risk scores, the incidence of an HCC diagnosis was higher in patients with indeterminate (HR 3.74, 95%CI 1.76–7.96) or high-risk scores (HR 25.2, 95%CI 7.83–80.66, Additional file 1: Figure S4B). Short time interval to cirrhosis diagnosis in patients with NAFLD and NASH In SIDIAP, 174 out of 75,415 patients with coded NAFLD were coded as having cirrhosis (incidence rate 0.66 per 1000 person-years (95%CI 0.56–0.76) with a median time to the new diagnosis of 2.9 years whereas 38 out of 1880 patients with NASH acquired a diagnosis of cirrhosis (incidence rate 2.83 per 1000 person-years (95%CI 2.0–3.88, Additional file 1: Table S4) with a simi- lar median time to diagnosis of 3.0 years (Additional file 1: Table S6). In THIN, the incidence of cirrhosis was higher and the interval between diagnoses was shorter Distinguishing NAFLD from NASH diagnoses when estimating risk of cirrhosis and HCC The pooled HR for incident NASH diagnosis in patients with a coded diagnosis of NAFLD compared to controls was 7.75 (95%CI 2.56–23.51, p = 0.008) although this estimate is based on a very small number of individuals (n = 130 of whom only seven were in SIDIAP, Additional file 1: Figure S5). NAFLD/NASH HSD IPCI SIDIAP THIN Subtotal (I-squared = 96.6%, p = 0.000) NAFLD SIDIAP THIN Subtotal (I-squared = 98.6%, p = 0.000) NASH SIDIAP THIN Subtotal (I-squared = 90.2%, p = 0.001) 2.45 (1.68, 3.58) 5.20 (3.64, 7.42) 3.62 (3.03, 4.32) 10.47 (8.76, 12.52) 4.73 (2.43, 9.19) 3.26 (2.70, 3.94) 10.40 (8.62, 12.54) 5.83 (1.87, 18.13) 11.56 (6.63, 20.15) 45.19 (24.14, 84.59) 22.67 (5.96, 86.23) 2.45 (1.68, 3.58) 5.20 (3.64, 7.42) 3.62 (3.03, 4.32) 10.47 (8.76, 12.52) 4.73 (2.43, 9.19) 3.26 (2.70, 3.94) 10.40 (8.62, 12.54) 5.83 (1.87, 18.13) 11.56 (6.63, 20.15) 45.19 (24.14, 84.59) 22.67 (5.96, 86.23) 1 .6 1 2 4 8 16 32 64 128 252 Hazard ratio (95% CI) Database HR (95% CI) Exposure Fig. 1 Association of coded NAFLD/NASH, NAFLD and NASH with cirrhosis. Hazard ratios and 95% confidence interval for acquiring a new diagnosis of cirrhosis in each database and combined across databases (subtotal) Fig. 1 Association of coded NAFLD/NASH, NAFLD and NASH with cirrhosis. Hazard ratios and 95% confidence interval for acquiring a new diagnosis of cirrhosis in each database and combined across databases (subtotal) Alexander et al. BMC Medicine (2019) 17:95 Page 6 of 9 Alexander et al. BMC Medicine NAFLD/NASH HSD IPCI SIDIAP THIN Subtotal (I-squared = 92.0%, p = 0.000) NAFLD SIDIAP THIN Subtotal (I-squared = 94.8%, p = 0.000) NASH SIDIAP THIN Subtotal (I-squared = 0.0%, p = 0.506) 1.63 (1.00, 2.65) 7.92 (4.02, 15.57) 2.11 (1.59, 2.80) 6.07 (4.38, 8.43) 3.51 (1.72, 7.16) 1.90 (1.39, 2.58) 5.26 (3.76, 7.36) 3.15 (1.16, 8.56) 6.99 (3.18, 15.38) 11.75 (3.16, 43.64) 8.02 (4.08, 15.77) 1.63 (1.00, 2.65) 7.92 (4.02, 15.57) 2.11 (1.59, 2.80) 6.07 (4.38, 8.43) 3.51 (1.72, 7.16) 1.90 (1.39, 2.58) 5.26 (3.76, 7.36) 3.15 (1.16, 8.56) 6.99 (3.18, 15.38) 11.75 (3.16, 43.64) 8.02 (4.08, 15.77) 1 .1 .3 .6 1 2 4 8 16 32 64 128 252 Hazard ratio (95% CI) Database HR (95% CI) Exposure Fig. 2 Association of coded NAFLD/NASH, NAFLD and NASH with hepatocellular carcinoma (HCC). Diabetes predicts disease progression In coded NAFLD/NASH patients, the strongest associ- ation with incident liver outcomes was observed in pa- tients who also had a past diagnosis of diabetes at baseline (HR 2.3, 95% CI 1.9–2.78). In matched controls without coded NAFLD/NASH, smoking was also associ- ated with liver outcome (HR 1.5, 95% CI 1.41–1.6) in addition to the independent risk attributed to diabetes, which was higher than in patients with coded NAFLD/ NASH (HR 2.92, 95% CI 2.76–3.08, Table 3). We applied minimal selection criteria and therefore were able to include over 78% of all adults registered in the databases, hence the ‘real-world’ nature of the study. The overall proportion of people with coded NAFLD/ NASH diagnoses is lower than expected as reported pre- viously [10], is in keeping with other primary care work [19] and may reflect levels of awareness of NAFLD/ NASH in primary care [20, 21]. Hence, our data, by def- inition, can only represent the visible part of the clinical iceberg. Despite this, we find that patients with coded NAFLD/NASH acquire diagnoses of life-threatening liver disease within a relatively short follow-up period (median 3.3 years). Distinguishing NAFLD from NASH diagnoses when estimating risk of cirrhosis and HCC Hazard ratios and 95% confidence interval for acquiring a new diagnosis of HCC in each database and combined across databases (subtotal) Fig. 2 Association of coded NAFLD/NASH, NAFLD and NASH with hepatocellular carcinoma (HCC). Hazard ratios and 95% confidence interval for acquiring a new diagnosis of HCC in each database and combined across databases (subtotal) patients were well-matched to a very large number of controls according to sex, age, GP practice and most re- cent visit, thus limiting bias due to geographical and so- cioeconomic diversity and behaviours relating to health service utilisation. Patients with coded NAFLD/NASH are at significantly increased risk of acquiring a diagnosis of cirrhosis or HCC, compared to matched controls. The risk is greater in patients with a coded diagnosis of NASH compared to NAFLD and in those with high-risk Fib-4 fibrosis scores compared to indeterminate or low- risk scores. Diabetes is an independent risk factor for pro- gression to either HCC or cirrhosis diagnoses in both coded NAFLD/NASH patients and matched controls. for both stages of disease. One hundred three out of 24,743 patients with coded NAFLD acquired a cirrhosis diagnostic code (incidence rate 2.17 per 1000 person- years (95%CI 1.86–2.51) with median time to diagnosis of 2.0 years, compared to 26 out of 1212 patients with coded NASH (incidence rate 5.81 per 1000 person-years (95% CI 3.8–8.52) with median time to diagnosis of 0.5 years. Discussion NAFLD/NASH are consistent with the diagnostic codes, although if patients with NAFLD/NASH do exist in the control group then the effect sizes reported here are un- derestimates of the real risk. This means that there are individuals living with diabetes in primary care who have not been diagnosed with NAFLD/NASH but are at significantly increased risk of developing liver cirrho- sis and cancer. The estimated size of the NAFLD problem has raised fears of large unmanageable patient numbers who are not at immediate threat of disease. Notwithstanding our expectation that many cases have not been identified in this study, we have shown that 0.6% of patients with an existing coded diagnosis of NAFLD/NASH acquire a diagnosis of cirrhosis and/or HCC within a 3-year follow-up period. This gives us insight into the rate at which advanced disease is discovered, even if this is not the natural history in the general population. The clin- ical impact of our data is that they highlight the large gaps in diagnosis and risk assessment of NAFLD and NASH with variable rates of risk stratification, staging of disease and seemingly late diagnosis. p y g p py In the databases where NAFLD/NASH codes could not be distinguished (HSD and IPCI), even those with low-risk Fib-4 scores were at increased risk of cirrhosis and HCC compared to controls. This further suggests that primary care records under-estimate disease severity and that some patients with NAFLD/NASH diagnoses actually have advanced fibrosis or cirrhosis already. Apart from a diagnosis of NAFLD/NASH, diabetes was the strongest independent risk factor for acquiring a diagnosis of cirrhosis or HCC. In the matched control population, the HR for diabetes was even higher than the coded NAFLD/NASH cohort, which may reflect a significant number of individuals with undiagnosed NAFLD/NASH among the controls. The importance of diabetes is consistent with a review of patients who had undergone more than one biopsy in the course of their routine clinical care in the UK, which showed that dia- betes was a risk factor for progression of fibrosis [9]. Obesity is an important risk factor for many cancers in- cluding HCC [30], but we did not find that in our study. If patients are diagnosed late in the disease spectrum, it is unlikely that patients will have undergone surveillance and HCC may be diagnosed at late stages when symp- toms including weight loss are manifest. Discussion To our knowledge, this is the largest study to date that has used EHR data to investigate rates of new diagnoses of advanced liver disease in patients with NAFLD. Our Table 3 Association between covariates and risk of liver outcomes: cirrhosis or hepatocellular carcinoma. Using a 1-step Cox model stratified by database NAFLD/NASH HR (95% CI) Matched control HR (95% CI) Smoking status (current/not current) 1.19 (0.94; 1.51) 1.50 (1.41; 1.60) Age (years) 1.04 (1.03; 1.05) 1.04 (1.03; 1.04) History of diabetes (yes/no) 2.30 (1.90; 2.78) 2.92 (2.76; 3.08) History of hypertension (yes/no) 0.92 (0.76; 1.12) 1.07 (1.01; 1.13) BMI (kg/m2) 1.01 (1.00; 1.03) 1.04 (1.03; 1.04) It is not feasible that the short time intervals between coded diagnosis of NAFLD/NASH and advanced liver dis- ease reflect true rates of disease progression, estimated to be one fibrosis stage per 7 years [22]. The acquisition of a new code in the healthcare record does not necessarily mean that pathological progression has occurred at that time, nor that the stage did not exist at baseline. Our inter- pretation of these data is that patients in Europe are being diagnosed at the later stages of disease, which are associated with greater risk of liver-related mortality [23–25]. Page 7 of 9 Page 7 of 9 Alexander et al. BMC Medicine (2019) 17:95 Alexander et al. BMC Medicine (2019) 17:95 Less than 50% of patients had sufficient data to calcu- late Fib-4, the components of which are also needed to calculate many other non-invasive fibrosis scores [26]. There was marked national variation in fibrosis assess- ment; 73.1% of patients in whom we could calculate Fib-4 were from the Spanish database. We have no way of determining whether these scores were actually calcu- lated by clinicians and whether they influenced decision- making. This is despite the fact that such risk stratification is central to most guidelines [27–29], used to determine clinical management, select patients for clinical trials and probably triage patients for future therapy. NAFLD/NASH are consistent with the diagnostic codes, although if patients with NAFLD/NASH do exist in the control group then the effect sizes reported here are un- derestimates of the real risk. This means that there are individuals living with diabetes in primary care who have not been diagnosed with NAFLD/NASH but are at significantly increased risk of developing liver cirrho- sis and cancer. Conclusions k l d Our knowledge of NAFLD/NASH is being based on small, highly selected cohort studies. These have been accurate in telling us the potential scale of the preva- lence and progression of disease, but the reality for many in the general population is some way from that. In order to affect population health and make an impact on the overall health burden of advanced liver disease, we cannot simply rely on introducing effective therapies to the small number of people with established diagnoses. The current approach to opportunistically investigate those in whom abnormalities in liver tests arise is clearly not working. While better biomarkers are needed that identify those at risk more precisely, the current tools are not being used, leaving many patients unclear as to the stage of their disease and its significance to their health. Therefore, making an impact on advanced liver disease will need co-ordinated efforts to identify those with NAFLD, to stage their disease and target those at risk of progression. This study is subject to limitations. The nature of real-world data is such that we cannot ascertain the ori- gin of codes nor the motivation for adding diagnoses to the patient record. Although the study is based in pri- mary care, it is likely that a large proportion of diagnoses will have been made with some involvement of second- ary care. It would be inaccurate to assume that all pa- tients who carry the code ‘NASH’ have had a liver biopsy and histological assessment and it might be that the diagnosis was assumed and recorded based on, for example, ultrasound evidence of fatty liver and elevated serum transaminases or increased stiffness on transient elastography. Similarly, it was not possible to confirm that the matched controls did not have NAFLD/NASH. However, the clinical features of patients with coded Discussion Taken together, these findings emphasise the need to recognise risk fac- tors for progressive disease and to detect disease at early stages when interventions can be more effective. Additional file 1: Supplementary Methods. Table S1. Attrition table showing patients with recorded diagnoses of NAFLD or NASH and matched unexposed controls. Table S2. Descriptive characteristics of coded NAFLD or NASH patients and matched unexposed cohorts in SIDIAP and THIN. Table S3. Number of patients with data available in coded NAFLD/NASH and matched unexposed cohorts. Table S4. Incidence rate of liver outcomes in four primary care databases. Table S5. Descriptive characteristics of coded NAFLD/NASH patients and matched non-NAFLD/NASH in a sample of patients with and without BMI data Acknowledgements The European Medical Information Framework (EMIF) is a collaboration between industry and academic partners that aims to develop common technical and governance solutions to facilitate access to diverse electronic medical and research data sources. These analyses were supported by the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement n° 115372, resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. The authors would like to acknowledge Nicholas Galwey for his advice on the statistical methods, Alba Jene for her administrative support and support during submission to ethical review boards and Derek Nunez for support early on a protocol design stage. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Availability of data and materials 3. Mittal S, et al. Hepatocellular carcinoma in the absence of cirrhosis in United States veterans is associated with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2016;14(1):124–31 e1. This work uses data provided by patients and collected by the different healthcare systems involved as port of their care and support. All data relevant to the study purpose are within the paper and its Supporting Information files. Original, individual-level data are in custody to local partners, and the possibility to access them may vary depending on local governance rules. Local restrictions on publicly sharing original study data may vary on a case-by-case basis and depend on institutional review board, ethics committee or law. Further information on data request and access should be sent individually to the authors of this paper responsible for the data provided by the relevant organisations: SIDIAP (tduarte@idiapjgol. org), HSD (lapi.francesco@simg.it), THIN (d.ansell@bham.ac.uk), IPCI (j.van derlei@erasmusmc.nl). 4. Wong RJ, et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology. 2015;148(3):547–55. 5. Koehler EM, et al. Prevalence and risk factors of non-alcoholic fatty liver disease in the elderly: results from the Rotterdam study. J Hepatol. 2012; 57(6):1305–11. 6. Söderberg C, et al. Decreased survival of subjects with elevated liver function tests during a 28-year follow-up. Hepatology. 2010;51(2):595–602. 7. Younossi ZM, et al. Global epidemiology of nonalcoholic fatty liver disease- meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64(1):73–84. References 1. Friedman SL, et al. Mechanisms of NAFLD development and therapeutic strategies. Nat Med. 2018;24(7):908–22. 1. Friedman SL, et al. Mechanisms of NAFLD development and therapeutic strategies. Nat Med. 2018;24(7):908–22. 2. Dyson J, et al. Hepatocellular cancer: the impact of obesity, type 2 diabetes and a multidisciplinary team. J Hepatol. 2014;60(1):110–7. Abbreviations ALT: Alanine transaminase; AST: Aspartate transaminase; BMI: Body mass index; CI: Confidence interval; EHR: Electronic Health Record; EMIF: European Medical Information Framework; GP: General Practitioner; HCC: Hepatocellular carcinoma; HSD: Health Search Database; IPCI: Information System for Research in Primary Care; LFT: Liver function tests; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; SIDIAP: Information System for Research in Primary Care; THIN: The Health Improvement Network; UK: United Kingdom; US: United States Competing interests MA was contracted to work at and SK and DMW are employees of GlaxoSmithKline which has conducted clinical research including trials of therapeutic agents in NAFLD. AKL is an employee of Pfizer which is conducting clinical research including trials of therapeutic agents in NAFLD. TDS: none to declare. DP-A: unrestricted research grants from UCB, Amgen, Servier, and consultancy fees (paid to his department/ research group) from UCB Pharma. DA: consultancy and advice to many pharmaceutical companies on undertaking outcomes studies using real-world evidence. FL: consultancy for AlfaSigma, Bayer and Abbvie. SK: Employee and stock holder, GlaxoSmithKline. NS: consulted for Boehringer Ingelheim, Eli Lilly, Novo Nordisk, Janssen, and grants from Astrazeneca and BI. WA: Consultant and sponsored lectures: UCB Pharma, Gilead, Intercept and Medimmune. Author details 1 1Real World Data, GlaxoSmithKline, Uxbridge, UK. 2Worldwide Research and Development, Pfizer, Genome Sciences and Technologies, New York, USA. 3Erasmus Universitair Medisch Centrum, Rotterdam, Netherlands. 4Fundació Institut Universitari per a la Recerca a l’Atenció Primària de Salut Jordi Gol i Gurina, Barcelona, Spain. 5Centre for Statistics in Medicine, NDORMS, University of Oxford, Oxford, UK. 6Quintile IMS, London, UK. 7Health Search, Italian College of General Practitioners and Primary Care, Firenze, Italy. 8Genetics, GlaxoSmithKline, Collegeville, PA, USA. 9GlaxoSmithKline, Medicines Research Centre Cambridge UK 10University of Glasgow Additional file Additional file 1: Supplementary Methods. Table S1. Attrition table showing patients with recorded diagnoses of NAFLD or NASH and matched unexposed controls. Table S2. Descriptive characteristics of coded NAFLD or NASH patients and matched unexposed cohorts in SIDIAP and THIN. Table S3. Number of patients with data available in coded NAFLD/NASH and matched unexposed cohorts. Table S4. Incidence rate of liver outcomes in four primary care databases. Table S5. Descriptive characteristics of coded NAFLD/NASH patients and matched non-NAFLD/NASH in a sample of patients with and without BMI data Page 8 of 9 Page 8 of 9 Page 8 of 9 Alexander et al. BMC Medicine (2019) 17:95 Page 8 of 9 Alexander et al. BMC Medicine Ethics approval and consent to participate Ethics approval and consent to participate We followed local data laws in all four territories from which data were obtained, and in all countries, specific ethical approval was not required for this study that used anonymised data. However, approval was sought and obtained from the scientific research committee for THIN, the IPCI Governing Board (ref 2015/18) and the IDIAP Ethics Committee (Reference P15/167) and the scientific committee of the Italian College of General Practitioners and Primary Care. available, all datasets combined. Table S6. Median and interquartile range (in years) for time to event in coded NAFLD and matched non-NAFLD who experience a cirrhosis or hepatocellular carcinoma event during follow-up. Figure S1. Subgroup analysis of the association between coded NAFLD/ NASH and incident (A) cirrhosis and (B) hepatocellular carcinoma events by medical history and demographics. Figure S2. Hazard ratio (HR) for cirrhosis (A) adjusted for age and smoking in all patients and (B) adjusted for age and smoking in patients with BMI. Figure S3. Hazard ratio (HR) for HCC (A) adjusted for age and smoking in all patients and (B) adjusted for age and smoking in patients with BMI. Figure S4. Fib-4 Association with (A) cirrhosis or (B) HCC. Figure S5. Risk of coded NASH in patients with coded NAFLD. (DOCX 540 kb) pp p p We followed local data laws in all four territories from which data were obtained, and in all countries, specific ethical approval was not required for this study that used anonymised data. However, approval was sought and obtained from the scientific research committee for THIN, the IPCI Governing Board (ref 2015/18) and the IDIAP Ethics Committee (Reference P15/167) and h i ifi i f h I li C ll f G l P i i d Funding g FP7 Ideas: European Research Council Award Number 115372. ERC had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. DPA is funded by a National Institute for Health Research Clinician Scientist award (CS-2013-13-012). This article presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. This work was partially supported by the NIHR Biomedical Research Centre, Oxford. WA is in receipt of a Medical Research Council New Investigator Award. Glasgow, UK. 11Barts Liver Centre, Blizard Institute, Queen Mary, University of London, London, UK. Glasgow, UK. 11Barts Liver Centre, Blizard Institute, Queen Mary, University of London, London, UK. Received: 17 January 2019 Accepted: 10 April 2019 Authors’ contributions d 8. Nunez, D.J., et al., Factors influencing longitudinal changes of circulating liver enzyme concentrations in subjects randomized to placebo in four clinical trials. Am J Physiol Gastrointest Liver Physiol. 2019;316(3):G372–86. 8. Nunez, D.J., et al., Factors influencing longitudinal changes of circulating liver enzyme concentrations in subjects randomized to placebo in four clinical trials. Am J Physiol Gastrointest Liver Physiol. 2019;316(3):G372–86. 9. McPherson S, et al. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis d li i l t J H t l 2015 62(5) 1148 55 MA, AKL, JvdL, PR, DW, SK, NS and WA contributed to the study design. TDS, DP-A, DA, AP, FL, PR (data transformation and federated data analysis) and MM extracted the data. MA analysed the data. All authors interpreted the results. MA, NS and WA wrote the manuscript. All authors edited the manuscript. All authors read and approved the final manuscript for submission. liver enzyme concentrations in subjects randomized to placebo in four clinical trials. Am J Physiol Gastrointest Liver Physiol. 2019;316(3):G372–86. 9. McPherson S, et al. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis and clinical management. J Hepatol. 2015;62(5):1148–55. 9. McPherson S, et al. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis and clinical management. J Hepatol. 2015;62(5):1148–55. Page 9 of 9 Alexander et al. BMC Medicine (2019) 17:95 Alexander et al. BMC Medicine (2019) 17:95 10. Alexander M, et al. Real-world data reveal a diagnostic gap in non-alcoholic fatty liver disease. BMC Med. 2018;16(1):130. 11. Vargas-Santos AB, et al. Association of Chronic Kidney Disease with Allopurinol use in gout treatment. JAMA Intern Med. 2018;178(11):1526–33. use in gout treatment. JAMA Intern Med. 2018;178(11):1526–33. 12. Kringos D, et al. The strength of primary care in Europe: an international comparative study. Br J Gen Pract. 2013;63(616):e742–50. 13. Gini R, et al. Chronic disease prevalence from Italian administrative databases in the VALORE project: a validation through comparison of population estimates with general practice databases and national survey. BMC Public Health. 2013;13:15. 14. Vlug AE, et al. Postmarketing surveillance based on electronic patient records: the IPCI project. Methods Inf Med. 1999;38(4–5):339–44. 15. Garcia-Gil Mdel M, et al. Construction and validation of a scoring system for the selection of high-quality data in a Spanish population primary care database (SIDIAP). Inform Prim Care. 2011;19(3):135–45. 16. Blak BT, et al. Authors’ contributions d Generalisability of The Health Improvement Network (THIN) database: demographics, chronic disease prevalence and mortality rates. Inform Prim Care. 2011;19(4):251–5. 17. Filippi A, et al. Computerized general practice databases provide quick and cost-effective information on the prevalence of angina pectoris. Ital Heart J. 2005;6(1):49–51. 18. IQVIA. The Health Improvement Network. 18th March, 2019]; Available from: https://www.iqvia.com/locations/uk-and-ireland/thin-hes-data. Accessed 30 Apr 2019. 19. Alazawi W, et al. Ethnicity and the diagnosis gap in liver disease: a population-based study. Br J Gen Pract. 2014;64(628):e694–702. 20. Patel PJ, et al. Underappreciation of non-alcoholic fatty liver disease by primary care clinicians: limited awareness of surrogate markers of fibrosis. Intern Med J. 2018;48(2):144–51. 21. Standing HC, et al. GPs' experiences and perceptions of early detection of liver disease: a qualitative study in primary care. Br J Gen Pract. 2018;68(676): e743–9. 22. Singh S, et al. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies. Clin Gastroenterol Hepatol. 2015;13(4):643–54 e9. 22. Singh S, et al. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies. Clin Gastroenterol Hepatol. 2015;13(4):643–54 e9. 23. Boursier J, et al. Diagnostic accuracy and prognostic significance of blood fibrosis tests and liver stiffness measurement by FibroScan in non-alcoholic fatty liver disease. J Hepatol. 2016;65(3):570–8. 24. Ekstedt M, et al. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology. 2015; 61(5):1547–54. 25. Younossi ZM, et al. Nonalcoholic steatofibrosis independently predicts mortality in nonalcoholic fatty liver disease. Hepatol Commun. 2017;1(5):421–8. 26. De Silva S, et al. Non-invasive markers of liver fibrosis in fatty liver disease are unreliable in people of south Asian descent. Frontline Gastroenterol. 2018;9(2):115–21. 27. Chalasani N, et al. The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology. 2018;67(1):328–57. 28. European Association for the Study of the Liver European Association for the Study of Diabetes European Association for the Study of, O. EASL–EASD–EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016;64(6):1388–402. 29. Newsome PN, et al. Guidelines on the management of abnormal liver blood tests. Gut. 2018;67(1):6–19. 29. Newsome PN, et al. Guidelines on the management of abnormal liver blood tests. Gut. 2018;67(1):6–19. 30. Hassan MM, et al. Authors’ contributions d Obesity early in adulthood increases risk but does not affect outcomes of hepatocellular carcinoma. Gastroenterology. 2015;149(1):119–29.
https://openalex.org/W3159610101	https://cris.unibo.it/bitstream/11585/820603/1/A%20Design%20Of%20Experiment%20Approach%20To%203D-printed%20mouthpieces%20sound%20analysis.pdf	English	null	A design of experiment approach to 3D-printed mouthpieces sound analysis	Progress in additive manufacturing	2,021	cc-by	9,559	Abstract Nowadays additive manufacturing is affected by a rapid expansion of possible applications. It is defined as a set of technolo- gies that allow the production of components from 3D digital models in a short time by adding material layer by layer. It shows enormous potential to support wind musical instruments manufacturing because the design of complex shapes could produce unexplored and unconventional sounds, together with external customization capabilities. The change in the pro- duction process, material and shape could affect the resulting sound. This work aims to compare the music performances of 3D-printed trombone mouthpieces using both Fused Deposition Modelling and Stereolithography techniques, compared to the commercial brass one. The quantitative comparison is made applying a Design of Experiment methodology, to detect the main additive manufacturing parameters that affect the sound quality. Digital audio processing techniques, such as spectral analysis, cross-correlation and psychoacoustic analysis in terms of loudness, roughness and fluctuation strength have been applied to evaluate sounds. The methodology herein applied could be used as a standard for future studies on additively manufactured musical instruments. Keywords Additive manufacturing · Sound analysis · Musical instruments · Design of experiment · Stereolithography Progress in Additive Manufacturing https://doi.org/10.1007/s40964-021-00183-5 Progress in Additive Manufacturing https://doi.org/10.1007/s40964-021-00183-5 FULL RESEARCH ARTICLE A design of experiment approach to 3D‑printed mouthpieces sound analysis Antonio Bacciaglia1 · Alessandro Ceruti1 · Alfredo Liverani1 Received: 26 June 2020 / Accepted: 10 April 2021 © The Author(s) 2021 Received: 26 June 2020 / Accepted: 10 April 2021 © The Author(s) 2021 * Antonio Bacciaglia antonio.bacciaglia2@unibo.it 1 Department of Industrial Engineering‑DIN, University of Bologna, Bologna, Italy 1 Introduction and understanding of ancient instruments for historical con- servation reasons. Ancient musical instrument components (i.e. wooden mouthpieces) are very rare, sensitive and sus- ceptible to damages. For that reason, it is important to rep- licate their 3D models using powerful technological instru- ments as X-ray tomography and CT scans and then use the models to manufacture a faithful copy in an easy way using, for instance, AM. Sometimes, ancient musical instrument parts can be either recreated from manufacturers’ technical drawings instead of 3D reconstructed models, as described in [5]. Several AM techniques (FDM, polyjet, Digital Light Processing and Digital Light Synthesis) are used to reach satisfactory accuracy. Nowadays, AM is used to produce prototypes, mostly for validation tests instead of taking advantage of its incredible design freedom capability [1]. Nevertheless, AM is becom- ing to be used also in the musical field for final product generation due to two main reasons: (1) reconstruction and replication of ancient musical instruments for conservation reasons [2]; (2) design with optimization of new musical instruments for innovative shape research to produce the desired sounds [3]. There is an increasing interest in the research commu- nity about these topics. As an example, the 3D model of an ancient instrument is reconstructed by Computed Tomogra- phy (CT) scans and subsequently manufactured using AM technology [4], for example, employing nylon with Selective Laser Sintering (SLS) technology [2]. Musical instrument reconstruction can be motivated by a thirst for knowledge On the other hand, the research of innovative shapes use- ful to obtain unexplored acoustic capabilities is described in [3] with a discussion of the AM techniques which can be used. Indeed, AM gives the possibility to customize the musical instrument according to the musician’s needs, producing innovative shapes that are optimized iteratively, thanks to musician feedbacks together with sound analy- sis in a fast design-to-manufacturing cycle by the Digital Manufacturing concept employment [6]. As an example, [7] describes the design of an end-user-oriented component that (0121 3456789) 3 Progress in Additive Manufacturing compare traditional and additively manufactured music instruments. This methodology could be extended to other kinds of musical instruments. The scope of this paper is to apply a design methodology usually used in an industrial engineering context to a highly customized field represented by the production of musical instruments. 1 Introduction In particular, addi- tive manufacturing has been evaluated as a potential candi- date for the manufacturing of musical instruments due to the superior design flexibility and customization capabilities assured. An approach based on the Design of Experiment (DOE) methodology and sound quality check in terms of conventional and psychoacoustic analysis has been followed. As a case study, different trombone mouthpieces produced with SLA and FDM additive manufacturing techniques have been investigated to demonstrate that it is possible to obtain almost comparable sound performances compared to commercial metallic components and to search which addi- tively manufactured piece has better sound performances compared with the brass one. is manufactured in AM to create an efficient clarinet mouth- piece customized for a bell’s palsy patient. A musician-tai- lored design approach is used to design and manufacture saxophone mouthpiece according to players’ needs [8]. To design new shapes to be able to produce unexplored acoustics, the musical instrument design must consider several key factors. The strength and stiffness of musical instruments must be guaranteed to avoid deformation which reflects on sound modifications. Furthermore, in the case of wind instruments, additional constraints must be taken into mind. Designers must consider the moisture that can be produced when the air flows inside the instrument and the material, that must be biocompatible, used to produce the mouthpiece because the component is close or in contact with the mouth [3]. Looking at the available researches about new design frontiers in musical instruments, [9] describes the digital optimization of an acoustic guitar using technological tools leading to the uniqueness of the acoustic sound that is cre- ated. Indeed, players can customize their sounds and their acoustic guitar substituting the large chamber with different small ones. [10] describes the design and manufacture of a flute with soft and rigid regions, using AM. Polyjet technol- ogy [11] has been selected to manufacture different parts made by different materials. The multi-material capability is employed in the valve areas and exploited to change the air pressure inside the channels to get different pitches (namely the human perception of a sound that allows the ordering on a frequency-related scale [12]). This work is organized as follows: after this brief intro- duction of AM technology and some examples found in the research community of its employment in the music sector, Sect. 1 Introduction 2 will describe the methodology used to compare dif- ferent mouthpieces with both DOE and sound analysis tools along with the trombone mouthpiece geometry description and the AM technologies employed to manufacture the trom- bone components. In Sect. 3, the paper contains the results and discussion on how the sound is recorded and how its quality can be evaluated. Sound analysis in both time and frequency domain are discussed to have a confirmation from a numerical point of view that the conclusions drawn are consistent. Section 4 highlights some conclusions and future developments. q y From this brief introduction, it can be noticed that AM has great possibilities with wind musical instruments: com- plex shapes could produce unexplored and unconventional sounds; the customization allows adding personal symbols and comfortable shapes for the single individual; biomate- rials can be used [13]; problems related to lip freezing in winter could be avoided using plastics instead of metals. Using AM technologies instead of traditional manufactur- ing processes, designers can exploit the advantages of addi- tive technology. This is especially true for small batches of production, as it happens in custom musical instruments design. Acknowledged advantages of AM are the absence of manufacturing constraints, no shape limitation which perfectly fit the high customization, opening new frontiers in the musical instrument design process. Several contribu- tions in the literature focus on the design and manufacturing of woodwind mouthpieces for saxophone or clarinet [4, 5, 8, 14]. The source [15] focuses on the production of brass instruments such as the trombone’s mouthpiece through AM. Indeed, this work can be seen as a continuation of the research carried out in [15]. This article suggests a methodology useful to evalu- ate the influence of design parameters of musical instru- ment’s parts—such as material and cup geometry—on the sound performances. This approach has been applied and tested using a case study where a trombone mouthpiece built through additive manufacturing techniques has been analysed. 3 2 Methodology for performance comparison of instrumental components made in additive manufacturing The proposed methodology is based on the following proce- dure. At first, a professional musician plays a short musical track with different mouthpieces, having different character- istics, in a professional recording room. The recorded tracks are then evaluated by different experts in classical music and a score is given for each mouthpiece depending on their impressions. In the following, a mathematical analysis is To contribute to the research topic of musical instrument customization and production with AM, this work aims to develop a methodology, not available in literature yet, to 1 3 3 Progress in Additive Manufacturing analysis considers only some of all the possible combina- tions of factors and levels. carried out to understand how each design characteristic affects the produced sound: this is carried out based on an objective data analysis procedure relying on factorial analy- sis using the Design of Experiment (DOE) methodology [16]. Moreover, the recorded tracks are analysed with some mathematical tools [17], such as Fast Fourier Transform, Cross-correlation function, and Spectrogram, to check a cor- relation between the results of the DOE analysis and music tracks. Psychoacoustic parameters, like loudness, roughness and sharpness are evaluated to distinguish features of music acoustics too [18]. All the previously cited mathematical tools are described in more details in the following for a bet- ter understanding of the methodology. The overall methodol- ogy phases are depicted in the flow chart included in Fig. 1. The more common DOE analysis is called 2k-factorial design, where all the k factors have only two levels and all the possible combinations are 2k . Without the DOE meth- odology application, it could be possible to understand, with a trial and error approach, the contribution of each factor to the output. However, in this latter case, it would be hardly understandable the interaction effect among the factors. In the following, a 2k-factorial design approach will be used to investigate the influence of material and cup dimensions (k = 2) of a trombone mouthpiece on the sound quality. In this research, the results of the DOE analysis are computed exploiting the capabilities of the Minitab™ software. 2.1 Design of experiment methodology To analyse the produced sound from a musical instrument, it is common to examine the recorded data in the time and frequency domain. Usually, the sound amplitude envelop is at first plotted in time to visualize how the sound amplitude changes along the span of the recorded track. The sound amplitude represents the maximum displacement from the equilibrium point of the air particles when the sound wave travels through the air itself. An increase of the sound ampli- tude means an increase of the force applied to the human eardrum and it grows the perception of the sound intensity. The cross-correlation MATLAB function xcorr can be a good tool in the time domain to compare the sound. Thanks to this tool it is possible to measure the similarity of a signal as a function of the temporal translation: this function has been applied in this work on music tracks to capture pos- sible similarities in time of the sound behaviour of different mouthpieces. The design of experiment methodology can be defined as a statistically rigorous approach developed to assess the influ- ence of some parameters (called factors) which have some values inside a range (called level) affecting the output of a certain process. DOE helps the understanding of a process and suggests how the factors may affect it. The DOE statisti- cal analysis is based on the comparison of some design input of an experiment, aiming to improve the process knowledge by as few as possible runs, thus reducing the need for numer- ous tests which may be expensive both in terms of time and costs.if This methodology is used to find cause-and-effect rela- tionships to optimize the output ( yi ), by knowing the process inputs ( x1, x2, x3,…xi ) and their interactions (see Fig. 2). , Thanks to the DOE approach, production and design costs can be easily reduced, by reducing the process variance and increasing its understanding. There are different types of DOE analysis, such as full factorial, where all the factors and levels are considered and fractional factorial, where the To transform the sound amplitude envelop in time to the frequency domain. It is possible to apply a mathematical operation called Discrete Fourier Transform (DFT). Thanks 1 3 Fig. 1 Applied methodology for objective comparison of addi- tively manufactured trombone mouthpieces Fig. 2 Simplified process model in the absence of external disturbances Fig. 2.1 Design of experiment methodology 1 Applied methodology for objective comparison of addi- tively manufactured trombone mouthpieces Fig. 1 Applied methodology for objective comparison of addi- tively manufactured trombone mouthpieces Fig. 2 Simplified process model in the absence of external disturbances 3 3 Progress in Additive Manufacturing used the MATLAB function Y = fft (X) that computes the discrete Fourier transform (DFT) of an array X using a fast Fourier transform (FFT) algorithm, where X is the sound signal recording of the musical instrument under analysis. The MATLAB Spectrogram function is used to evaluate the frequency domain variation in time of the produced sound of all the mouthpiece alternatives. to this function, the sound is digitalized when stored in a computer by a sampling procedure, returning a vector that contains sound intensity values for each frequency fn , once a given sampling frequency fs [19] is set by the experimenter. This mathematical tool allows extrapolating individual frequency components of a given signal in time [20]. Analy- sis in the frequency domain is preferred compared to the time domain because parameters, as pitch and sound bright- ness, necessary for sound analysis, are easier to detect and evaluate in comparison with a sound temporal representa- tion, in which the intensity or amplitude of the sound in time is known. The spectral centroid, which can be associated with the barycentre of the spectrum [24] is an important parameter in audio signal analysis to characterize the frequency spec- trum. In practice, it is often associated with the brightness of a sound which increases as the spectral centroid increases [25]. The spectral centroid can be evaluated with the follow- ing equation (Eq. 1): The Fast Fourier Transform (FFT) speeds up the process- ing speed of the Fourier Transform [21], by producing an array of complex numbers which is often used to calculate the behaviour of magnitude or power versus frequency to represent it as a 2D graph [14]. (1) C = ∑N∕2 k=1 f(k)A(k) ∑N∕2 k=1 A(k) (1) However, the FFT is a signal analysis where the tem- poral dimension of the signal is lost, namely the informa- tion content about how the frequency domain changes in time are lost. This is the reason for the wide use in music of the spectrogram, which is a complete temporal mapping of how the frequency domain changes. 2.1 Design of experiment methodology A spectrogram is obtained as a set of single analysis at regular intervals of a small signal time window. The signal batch involved in the analysis is isolated from the whole signal and multiplied by a bell’s function window to avoid that the truncation opera- tion affects the result. The ‘windowing procedure’ is used to obtain the signal spectrum with higher accuracy. There are different types of window but in the present case study, the default window implemented in MATLAB, that is the Hamming, is used [22]. It is important to underline that the window length choice is crucial and based on a trade-off analysis. A better temporal resolution (small window size) is paid back with a worse frequency resolution: it is worth citing the uncertainty principle stating that it is impossible to evaluate simultaneously with arbitrarily precision both temporal and frequency parameters of a signal. In the case study presented in this paper, the window size was chosen after an iterative process to find a good compromise in terms of temporal and frequency resolution. Once available a sin- gle recorded track, a spectrogram analysis is applied to it, using different window lengths inside a certain range. In the following, the spectrograms are visually compared and the interval is narrowed until the spectrogram is satisfac- tory from a visual point of view; the figures of these spec- trograms are not included for brevity. However, the result coming from this study shows that too small or too large window length is detrimental for sound analysis because several details are lost, confirming what expected. This is the reason why a trade-off value has been set for this study. For this work, according to the literature contribution where N is the total number of the Fast Fourier Transform (FFT) points, \|A(k)\| is the spectral value corresponding to the k-th bin and f(k) is the frequency at FFT k-th bin [24]. The fundamental frequency [26] of the sound can be con- sidered as another interesting parameter to see if new mate- rials can substitute the commercial metallic (brass mainly) used for wind musical instruments. This characteristic can be evaluated in MATLAB using the pitch function. Further information about the implementation of this and similar functions can be found in the MATLAB software user man- ual [27–29]. To distinguish musical acoustic features, conventional sound signal parameters are not enough. 3 2.1 Design of experiment methodology For this reason, additional psychoacoustic parameters are introduced: loud- ness focuses the attention on the distribution of critical bands and masking properties in the hearing, describing the human perception of sound volume. This parameter is evalu- ated with the acousticLoudness function, according to the Zwicker loudness definition (ISO 532-1) [30]. Sharpness expresses the quality of the sound and it is strongly related to how pleasant an auditor feels with the sound. If there is high- frequency energy in a sound, then the sound will be sharper: this psychoacoustic parameter can be computed with the acousticSharpness MATLAB function, according to DIN 45692 and ISO 532-1 [31]. Finally, fluctuation strength and roughness analyse the time structures of the sound signal. On one hand, fluctuation strength indicates the perception of low-frequency modulations that are discernible individually (computed with acousticFluctuation function [32]). On the other hand, roughness indicates the rough-sounding percep- tion of stimuli related to modulations at frequencies too high to be discerned separately. This last psychoacoustic parame- ter is computed using an open-source function [33], based on the Daniel and Weber algorithm [34]. The authors are aware that this parameter list is not exhaustive and other temporal, f For this work, according to the literature contribution regarding musical instruments sound analysis [23], authors 1 3 Progress in Additive Manufacturing harmonic spectral and perceptual audio descriptors should be considered for a complete comparative study and timbre estimation: other factors could be investigated in the fol- lowing studies. It is worth noting that all these evaluation parameters provide a similar ranking for the manufacturing technologies considered in this paper.f depending on the component geometry. Indeed, the expert musicians know that the produced sound depends also on the geometry of the mouthpiece and there are some geom- etry parameters stalwartly affecting the sound. Just to men- tion, cup, rim, throat and backbore dimensions influence the emitted sound. However, in literature, there is no men- tion of whether AM technology can be used to produce mouthpieces with equivalent performances compared to the traditional in brass. The analysis of the produced sound and the effects of multiple musical instruments design parameters, such as geometry, material and AM technique used to manufacture the instrument, can be investigated thanks to these math- ematical tools. The classification of the tools used in this paper is shown in Fig. 3. 2.1 Design of experiment methodology The comparison which follows involves the commer- cial metallic (brass) mouthpiece and alternative versions that are additively manufactured using stereolithography (SLA) [36] and Fused Deposition Modelling (FDM) [37] printing strategies. These two different technologies were selected to evaluate the effect of the change in the mate- rial due to their suitability to the case study application and investigate how the acoustical characteristics would be affected by material properties [15]. Indeed, the selected AM technologies use economical affordable raw materi- als (respectively, photo-sensible resins and thermoplastic polymers) and are largely diffused in labs and the hobby field. A large number of practitioners could access these two technologies. On the other hand, AM technologies based on metal powders, such as Selective Laser Melting (SLM) and Electron Beam Melting (EBM), are used only in advanced industrial contexts, and the manufacturing costs could be so high to reduce the interest for this kind of AM technologies for musical instruments. Moreover, the overwhelming majority of experimenters and practition- ers can’t access metal AM machines for tests and custom realizations. This is the reason why SLA and FDM tech- nologies are selected for this research. 2.3 Case study: trombone mouthpiece geometry and material The trombone is a wind instrument belonging to the brass instrument family. There are different types of trombone, such as soprano, alto, tenor, bass, and contrabass. While the other brass family members have valves to change notes, the trombone has a slide mechanism to change the produced note. From literature [35], the technique to play the trombone consists of lip vibration and in the change of the airflow direction exiting from the mouth. The mouthpiece helps the lips vibration and canalizes the airflow coming from the mouth towards the instrument itself. The flow impacts the inner part of the mouthpiece then flows through its throat section and goes into the instrument itself, which can be compared to a sounding board. There are several mouthpiece models: some of them help to reach high registers, while others, the lower ones Fig. 3 Applied methodology for sound analysis by the employment of several MATLAB functions (in bracket) Fig. 3 Applied methodology for sound analysis by the employment of several MATLAB functions (in bracket) 1 3 Progress in Additive Manufacturing Fig. 4 Technical drawing of the mouthpiece with two variable dimen- sions affecting the cup volume: a cup depth, b cup diameter technique (#2 and #3) and two components made in FDM (#4 and #5). The mouthpiece characteristics are collected in Table 1. According to design requirements, the #3 com- ponent was designed with a smaller cup volume (decreased diameter and depth) to fulfil the player needs, namely to have high-pitched and shrill tones. The labels contained in the table will be used in the following as a reference to make the discussion clearer. Two mouthpiece versions have been manufactured using the SLA technique since an external high-quality finishing can be achieved, together with a smoothness in the inner channel and in the region of mouth contact useful to increase the comfort and reduce the staircase effect. To make the component biocompatible, the Dental SG Resin by Formlabs is chosen. This material is often used for prostheses that are in direct contact with human tissues, to avoid the possibility of contact between toxic materials and the player mouth. For its applications, this resin is studied to be resistant in a moist environment as the mouth still maintaining high stiffness and rigidity. The SLA machine used for this study is Form 2 by Formlabs which allows a layer thickness of 25 ÷ 300 micron. Fig. 2.3 Case study: trombone mouthpiece geometry and material 4 Technical drawing of the mouthpiece with two variable dimen- sions affecting the cup volume: a cup depth, b cup diameter Fig. 5 Additively manufactured trombone mouthpieces: the SLA ver- sion in orange on the left and the FDM one in white on the right On the other hand, the other two mouthpiece kinds have been manufactured using the Creality 3D CR 10s5 machine, based on FDM technology. Indeed, many literature contribu- tions stress the fact that FDM suffers from staircase effect and high porosity, rough surfaces, air leakages and over- all low accuracy which may affect the sound [3]. However, FDM technologies have been tested to prove with experi- mental tests that FDM is not the best technology to be used in musical instrument manufacturing. Fig. 5 Additively manufactured trombone mouthpieces: the SLA ver- sion in orange on the left and the FDM one in white on the right Moreover, the cup dimension has been changed in terms of cup depth (a) and cup diameter (b) in the mouthpiece 3D models, according to Fig. 4. As the last variable, the mouth- pieces are manufactured using SLA and FDM to investigate their influence on the sound changes: in this way both mate- rial and geometry changes are investigated. All the alterna- tives were designed according to feedback coming from an expert trombone player, which customized the mouthpiece according to his needs. Two of the manufactured compo- nents are represented in Fig. 5. 3 Sound recording and quality evaluation After the manufacturing process has been completed, all the five mouthpieces have been tested by an expert musician and professional trombone player. To obtain high-quality record- ings, the sound has been acquired in a professional recording studio using a certified Rode NTK microphone, a Motu 8pre USB amplifier system and a 44100 Hz sampling frequency for better sound post-processing (Fig. 6). All the recorded tracks have been saved in.WAV format. The musician played the same short track, of almost 1-min duration, repeating it for all the five available mouthpieces. At the beginning of The rigorous objective analysis that follows, involves the commercial brass mouthpiece (label #1 will be used to refer to it), two versions manufactured in AM using the SLA Table 1 Alternative mouthpieces tested Mouthpiece labels Description of its characteristics #1 Commercial brass mouthpiece (a = 32.5 mm, b = 33.8 mm) #2 Shape of commercial mouthpiece (a = 32.5 mm, b = 33.8 mm), SLA technique #3 Decreased cup volume (a = 25.1 mm, b = 29.6 mm), SLA technique #4 Shape of commercial mouthpiece (a = 32.5 mm, b = 33.8 mm), FDM technique #5 Decreased cup volume (a = 25.1 mm, b = 29.6 mm), FDM technique 1 3 3 Progress in Additive Manufacturing Fig. 6 Recording studio of Music) have been interviewed to evaluate the sound qual- ity of the recorded five tracks, played within a high-quality stereo. Each expert listened to the five tracks three times and gave a score from 1 to 10 for each mouthpiece test, where 1 stands for insufficient sound quality, while 10 stands for extremely good sound performance. The results of the Com- parative Mean Opinion Score (CMOS) are shown in Fig. 7 for each test and each expert. The mean score value for each mouthpiece and its standard deviation have been evaluated. 3.1 DOE study: 2k factorial analysis In this section, the application of the DOE methodology to the case study is described, and all the results are reported. The DOE analysis aims to understand which factor is more relevant to the sound quality produced by trombone mouth- pieces made by AM techniques. Such an approach is useful to understand if some correlation exists between material and cup dimensions to get good sound quality and to select the best additively manufactured component to be compared with the brass one in the following stage, specifically the sound analysis.l Fig. 6 Recording studio Fig. 6 Recording studio each track, the musician played a musical extemporization characterized by six long notes for better sound analysis. Being available the.WAV music file for each mouthpiece, all the tracks have been analyzed in detail. There are some variables or external disturbances which will be not taken into account in this study for simplicity such as how hard the performer was blowing air into the mouthpiece. It can be a challenging task for a performer who is changing rapidly between components to maintain consist- ency of approach in his performance. This is why, in certain other studies, machines have been constructed to replicate the human blowing process, so that more consistent results can be achieved. However, since the scope of this work is to investigate the possibility to use additively manufactured components in an innovative and unusual sector as the musi- cal one, for simplicity this variable will be neglected in the following. Applying the general flowchart shown in Fig. 2 to this case study, the factors xi are the AM technology and the cup dimension, while the output y1 was associated with the sound quality evaluation of three music experts. Even if the expert’s subjectivity may affect the output value, this choice is made to associate to each mouthpiece a single numerical value that is directly linked with the sound quality. Fig. 7 Comparative mean opin- ion score of the sound quality of five mouthpieces 3.1.2 Statistical analysis Fig. 9 Pareto chart of material and cup dimension factor effects on the output; only the factors which reach the red line are relevant in the analyzed process The DOE analysis is performed in the Minitab software. 3 replicates were selected to respect the number of experts who evaluated the sound quality. Minitab returns a work- sheet with all the 12 possible combinations, and the user must fill the output column by himself. Pareto graph shows that the material factor is more impor- tant and relevant to the sound quality compared to the cup geometry dimensions (Fig. 9) (Pareto graph detailed treat- ment can be found in [38]). i After the statistical DOE testing is set, the methodology shown in the flow chart in Fig. 8 is used.i After the detection of more relevant factors, Minitab allows understanding which is the output value (the pre- dicted sound quality) depending on the chosen levels of the two factors in a graphic way, thanks to the factorial plots, which are reported in Fig. 10. l A first iteration where both the factors [material (A) and cup dimension (B)] as well as their interaction AB has been considered. Then, thanks to the factorial regression study, we found that AB interaction doesn’t affect strongly the out- put: this can be noticed in the Pareto graph or with the P value-coded coefficients. If a factor has a P value lower than 0.05, its contribution to the output is not significant for the process under analysis. Neglecting the interaction term, the analysis becomes more straightforward, and the results are easier to understand. From the results shown on the left in Fig. 10, it can be easily seen that the material factor is more relevant com- pared to the cup geometry on the sound quality, which agrees with the Pareto graph. Moreover, the SLA material gives a higher output value compared to the FDM and a bigger cup volume may be a better design choice to achieve high sound quality. From the cube plot (see Fig. 10 on the right), the best combination seems to be a mouthpiece made with SLA technology with big cup dimensions, while the worst combination is the selection of FDM technique and small 3.1.1 Design degrees of freedom The input parameters in the DOE analysis are called factors and, referring to this case study, they are the material and the cup geometry. Since a 2k factorial analysis is applied, where In the following, based on the methodology shown in Fig. 1, three experts in music (a musician and two Professors 1 3 Fig. 7 Comparative mean opin- ion score of the sound quality of five mouthpieces 1 3 3 Progress in Additive Manufacturing k = 2, the possible combinations of inputs are 4. Such kind of analysis can be applied when for each factor only two levels are present. In this case, the levels are: Fig. 9 Pareto chart of material and cup dimension factor effects on the output; only the factors which reach the red line are relevant in the analyzed process • AM technology: SLA/FDM • Cup dimension: big/small As previously mentioned, the output of the analyzed pro- cess is the sound quality evaluated by three experts in music, with a score from 1 to 10. More complex analyses involving more than 2 design parameters could be carried out extend- ing the methodology herein described. 3.1.3 Results Neglecting the mixed term, the statistical DOE analysis is run again, and the regression analysis is performed. The Fig. 8 Statistical DOE analysis methodology applied for the specific case study Fig. 8 Statistical DOE analysis methodology applied for the specific case study Fig. 8 Statistical DOE analysis methodology applied for the specific case study 1 3 Progress in Additive Manufacturing Fig. 10 Statistical DOE analysis results: output prediction based on level values of each factor: the main effect plot, where mean sound quality is plotted for each factor level connected by a line (left); the cube plot, used to show the relationship between factors on the mean sound quality score (right) cube plot, used to show the relationship between factors on the mean sound quality score (right) Fig. 10 Statistical DOE analysis results: output prediction based on level values of each factor: the main effect plot, where mean sound quality is plotted for each factor level connected by a line (left); the cube plot, used to show the relationship between factors on the mean sound quality score (right) Fig. 10 Statistical DOE analysis results: output prediction based on level values of each factor: the main effect plot, where mean sound quality is plotted for each factor level connected by a line (left); the After the analysis of the sound amplitude plot in time (Fig. 11), it is possible to see in the second half of the track that the highest amplitude values are reached with the com- mercial brass version. In the case of SLA manufacturing, a 28% decrease compared to the brass mouthpiece in the A(t) plot can be noticed. On the other hand, the FDM shows a sound with a lower amplitude in time, suggesting a low- intensity sound (referring to the track portion highlighted with the red circle), with a 67% peak amplitude reduction. cup volume. However, looking carefully at the results, the coupling FDM-big cup dimension gives a satisfying value in terms of sound performances. Therefore, for sake of the fol- lowing methodology stage (additively manufactured mouth- pieces compared to the brass one), both SLA and FDM ver- sions with big cup dimensions will be considered. The small cup design, whatever is the material, leads to poor sound performances according to the expert’s assessment. 3.1.3 Results If the statistical analyses are carried out in a good way, the sound analysis confirms that the sound differences from brass and FDM versions are higher compared to the SLA and com- mercial brass comparison. Moving to the frequency domain and looking at the fre- quency spectrum diagram (Fig. 12), the reader can visually notice that the spectra are similar qualitatively. This means that the sound performances are consistent when AM is cho- sen as the production process for musical instrument com- ponents. Moreover, extracting the amplitude peaks visible in Fig. 12 in the frequency spectrum (taking as lower threshold an amplitude value of 1800), it can be said that these peaks are located at the same frequency values for the brass and SLA mouthpiece (a non-inclusive list is: 234, 353, 468, 707, 886 Hz). On the other hand, some of these frequencies con- tribute to a lower wave amplitude in the FDM mouthpiece, especially at 234, 353 and 886 Hz. This behaviour can be attributed to the out of tuning of the FDM component due to the difficult management of the mouthpiece by the player which was perceived by the experts in music who evaluated the tracks. 3.2 Sound and psychoacoustic analysis Sound analysis has been performed in MATLAB, thanks to its Audio Toolbox, developed to post-process and com- pare sounds both in terms of conventional and psychoacous- tic parameters. The mouthpieces #1, #2 and #4 have been analyzed, being available the.WAV sound files from studio recordings. At first, a lowpass filter with a cut-off frequency of 15 KHz has been implemented in the code to delete pos- sible high-frequency bias and noise. Such frequency value is chosen according to the upper limit of the audible range of an adult human ear, according to what suggested by lit- erature [39]. However, above the 2 KHz frequency signals, the energy distribution into frequency components of the signal con- tained in the spectrum (the subplot area) is lower for both the SLA and FDM mouthpieces compared to the commer- cial one. This issue is more evident considering the Power Spectral Density (PSD) which describes the signal’s power as a function of frequency per unit frequency. For contribu- tions higher than 2 KHz, the energy and therefore the power contained in the waves is higher for the brass mouthpiece Then, a qualitative comparison in the time domain has been carried out to evaluate the track repeatability along with all the tests. The recorded audio files are of the ‘dou- ble’ type format with 64 bits-per-sample. Using the audi- oread MATLAB function to read the recorded signal, the output vector refers to the normalized amplitude with values between − 1 and 1. 1 3 Progress in Additive Manufacturing Fig. 11 Sound amplitude in the time domain. From the top: brass, SLA and FDM mouthpieces Fig. 12 Frequency spectrum: from top to down: brass, SLA and FDM mouthpieces Progress in Additive Manufacturing Fig. 11 Sound amplitude in the time domain. From the top: brass, SLA and FDM mouthpieces Fig. 11 Sound amplitude in the time domain. From the top: brass, SLA and FDM mouthpieces Fig. 11 Sound amplitude in the time domain. From the top: brass, SLA and FDM mouthpieces Fig. 12 Frequency spectrum: from top to down: brass, SLA and FDM mouthpieces Fig. 12 Frequency spectrum: from top to down: brass, SLA and FDM mouthpieces Fig. 12 Frequency spectrum: from top to down: brass, SLA and FDM mouthpieces 1 3 Progress in Additive Manufacturing temporal amplitude envelop compared to the brass piece: this is in agreement with the DOE approach. Fig. 3.2 Sound and psychoacoustic analysis 13 Power spectral density distribution as a function of frequency In the frequency spectrum, the time domain is completely lost: a better and complete sound overview is shown in the spectrogram (Fig. 14). From a visual comparison, it can be noticed that spectrogram features are repeatable between the tests using different mouthpieces in terms of temporal events but less in power content. Indeed, it can be detected a lower power content in the additively manufactured mouthpieces (already highlighted with the PSD plot), with a more rel- evant difference at the high frequencies (fewer portions of spectrum in yellow colour). However, once again, this dif- ference is more evident in the second half of the FDM spec- trogram highlighted with a red circle in Fig. 14. Moreover, it is possible evaluating the harmonics occurring at whole- number multiples of the fundamental frequency. Fig. 13 Power spectral density distribution as a function of frequency Similar results can be obtained from the spectral cen- troid plot, which is evaluated in time for the played tracks. The centroid behaviour has several similitudes among the three versions under investigation, as can be seen in Fig. 15. However, a slight difference can be noted: for instance, the peak value at around 37 s in the track for the FDM mouthpiece is lower than brass and SLA com- ponents (12% of difference of the FDM one compared to 0.7% of the SLA version). The spectral centroid is usually associated with the brightness of a sound; therefore, it is frequently used in digital audio processing as an automatic index of musical timbre. The higher is the spectral centroid (Fig. 13), while lower (and similar) for SLA and FDM components. As previously stated, a mathematical tool to evaluate the “distance” between two signals is the cross-correlation func- tion. It has been evaluated that the maximum of the cross- correlation function is located at almost 0 time-shift for both brass-SLA and brass-FDM cases. However, the brass-SLA correlation value is slightly higher compared to the brass- FDM (304 vs 289) meaning that the test’s reproducibility is generally good for both additively manufactured components but the mouthpiece produced in SLA shows a more similar 1 3 Fig. 14 Spectrograms: from the top, mouthpieces #1, #2 and #4 Fig. 14 Spectrograms: from the top, mouthpieces #1, #2 and #4 3 3 Progress in Additive Manufacturing Fig. 3.2 Sound and psychoacoustic analysis 15 Spectral centroids in time: from the top, mouthpieces #1, #2 and #4 Fig. 16 Particular of the spectral centroid in time: from the top, mouthpieces #1, #2 and #4 Progress in Additive Manufacturing Fig. 15 Spectral centroids in time: from the top, mouthpieces #1, #2 and #4 Fig. 16 Particular of the spectral centroid in time: from the top, mouthpieces #1, #2 and #4 Fig. 16 Particular of the spectral centroid in time: from the top, mouthpieces #1, #2 and #4 1 3 3 Progress in Additive Manufacturing Fig. 17 Fundamental pitch distribution: from the top, mouthpieces #1, #2 and #4 Fig. 17 Fundamental pitch distribution: from the top, mouthpieces #1, #2 and #4 Fig. 18 Fundamental pitch distribution of the first 6 notes: from the top, mouthpieces #1, #2 and #4 Fig. 18 Fundamental pitch distribution of the first 6 notes: from the top, mouthpieces #1, #2 and #4 3 Progress in Additive Manufacturing value, the higher will be the brightness perception of the sound. parameters, such as loudness, sharpness, fluctuation strength and roughness, have been considered, according to definitions introduced in Sect. 2. The same result in terms of spectral centroid can be shown focusing on the first six notes played by the musi- cian at each track (Fig. 16). These notes are, respectively: B flat, F, D, B flat, F and D (of the lower octave). The overall time behaviour is similar, but mouthpiece #4 has 4 of the 6 notes with a spectral centroid peak value of sev- eral percentage points lower compared to the commercial and the SLA mouthpieces. It means a lower timbre when compared to the other brass and SLA items. i Figure 19 illustrates the behaviour of time-varying loudness measured in sones (one sone is arbitrarily set equal to the loudness of a 1000 Hz tone at a sound level of 40 dB above the standard reference level) for all the tested mouthpieces, computed according to the algo- rithms included in ISO 532-1 regulation [18]. Neglecting the effect of the time-shifting of the brass played track (yellow line), it is possible to see that the trombone with brass mouthpiece has higher loudness levels during all the played track compared to the SLA and FDM mouthpieces (respectively, in orange and cyan colour). The difference between metal commercial and experimental instruments increases during the audio signal. 3.2 Sound and psychoacoustic analysis Moreover, following all the previous analysis, the worst performances are obtained with the FDM component, while the SLA manufacturing guarantees a sufficient sound volume.l As the last conventional audio signal analysis, the fun- damental pitch is evaluated for all the versions under com- parison. As can be seen in Fig. 17, there are five regions, highlighted with green circles, in which there are some pitch discrepancies, where the brass version has a clearer pitch distribution without disturbances, even if in general a similar behaviour among the three mouthpieces behaviour can be noticed.i fi Moving to the fluctuation strength parameter, computed according to the approach described in [32], Fig. 19 shows almost comparable behaviour of brass and SLA mouthpieces during the played tracks, while the FDM version has lower performances, especially at 25 s played time, confirming the evaluation carried out with other indicators. Afterwards, when the time-varying sharpness—that is evaluated follow- ing standards DIN 45692 and ISO 532-1 [31]—is consid- ered, there are some peaks in correspondence of pauses However, if the analysis focuses on the first 6 notes played by the musician (Fig. 18), it can be noticed that almost the same values in terms of fundamental pitches can be found. This means that in general AM results in components that have sound characteristics close to parts produced with traditional manufacturing (mean errFDM = 0.81% , mean errSLA = 0.66%). Also, a psychoacoustical approach has been used to study the acoustic features of musical sound: several Fig. 19 From the top, time-varying loudness, fluctuation strength and time-varying sharpness distribution for the brass (in yellow), SLA (in orange) and the FDM (cyan) mouthpieces Fig. 19 From the top, time-varying loudness, fluctuation strength and time-varying sharpness distribution for the brass (in yellow), SLA (in orange) and the FDM (cyan) mouthpieces 3 Progress in Additive Manufacturing Table 2 Psychoacoustic average performances of tested mouthpieces N5 loudness (sones) Average fluctuation (vacil) Average sharpness (acum) Average roughness (asper) Brass 42.58 0.169 0.953 0.3879 SLA 39.76 (− 6.6%) 0.151 (− 10%) 0.927 (− 2.7%) 0.429 (+ 9%) FDM 35.70 (− 16%) 0.135 (− 20%) 0.941 (− 1.2%) 0.464 (+ 16%) Table 2 Psychoacoustic average performances of tested mouthpieces made by the player during the played tracks. These could be associated with environmental noise in the recording room which increases instantaneously the sharpness level. 4 Conclusion and future developments The aim of this paper is the development of a methodology that can be used to compare and evaluate the musical per- formances of musical instruments produced with traditional and new manufacturing technologies based on additive man- ufacturing techniques. The methodology relies on a Design of Experiment approach to understand which design factor is more relevant in terms of sound quality. In the following, digital sound processing is accomplished in terms of time and frequency domain analysis by applying several math- ematical tools, such as frequency spectrum, spectrogram, cross-correlation, spectral centroid and fundamental pitch function. In parallel to conventional audio signal analysis, psychoacoustic parameters are computed to compare the acoustic sound quality of tested components. Just to provide the reader with an easy and fast com- parison among psychoacoustic parameters for the tested mouthpieces, the N5 percentile of the loudness level in sones (a level below which is 95% of the reported loud- ness), the mean sharpness level (in acum), the mean fluc- tuation strength (in vacils) and the mean roughness level (in asper) are collected in Table 2 along with the percentage error compared to the brass mouthpiece which is taken as a benchmark. The meaning of these measurement units can be found in reference [18]. For what concerns the roughness estimation, this metric has not yet been standardised, which reflects on several proposed methods of calculation. This is due to the difficulty of accurately quantifying the perceived masking depth. In this research, the Daniel and Weber algo- rithm [34] is used to estimate the total roughness level of the audio signal. q y p Even if authors acknowledge that some variables in the complex relationship between musician and instru- ment where not taken under consideration, the conclusion stemming from this research is that AM processes, espe- cially SLA, are a good alternative for the production of the musical components from a merely design and manu- facturing perspective. Indeed, the sound characteristics are not overturned by the change in material and produc- tion process, even if plastic material or photo-sensible resins are used instead of metallic materials, particularly when SLA technology is used. Moreover, AM allows for extreme customization capability compared to traditional manufacturing techniques. When AM is adopted, sym- bols, writings or other distinctive symbols can be added to musical instruments, increasing end-user satisfaction. 3.2 Sound and psychoacoustic analysis Leaving out these peaks, all three mouthpieces have a com- parable amount of sharpness level during the audio signal and no important conclusion can be drawn from this kind of analysis. instruments manufactured in AM could be easily compared with commercial pieces. 4 Conclusion and future developments Rapid changes to the geometry of musical instruments can be made, by changing the 3D Computer-Aided Design (CAD) model before the production process. Finally, plas- tic material does not suffer from glueing of lips when playing wind instruments on cold winter days, as it hap- pens when brass or other metals are used, making AM very attractive. From the psychoacoustic analysis, it is possible to state that the brass mouthpiece has the highest level of acoustic sound quality. AM variants show worse performances in absolute value, but the SLA version has negligible perfor- mance difference (less than 10% of error) in terms of all the analysed psychoacoustic parameters (Table 2). The conclusions that can be drawn from the sound and psychoacoustic analysis are that on the one hand, FDM technology offers poorer sound performances compared to SLA, remarking and confirming the results coming from the statistical DOE analysis which are in agreement. On the other hand, the SLA mouthpiece has demonstrated sufficient sound quality performance after analysing several audio signal parameters. To sum up, the exploitation of SLA in future applications can be justified by the customization pos- sibility offered by AM technology and good sound qualities. SLA can be considered a good technology to design and produce extremes and innovative components for musical instruments without losing acoustic and sound quality per- formances. Moreover, thanks to the methodology described in this study, new and experimental designs of musical Further studies involving wave propagation in the air, perception by the human ear and neural stimuli should be made in future works. Other temporal, harmonic, 1 3 Progress in Additive Manufacturing 5. Cottrell S, Howell J (2019) Reproducing musical instrument components from manufacturers’ technical drawings using 3D printing: Boosey & Hawkes as a case study. J New Music Res 48(5):449–457. https://doi.org/10.1080/09298215.2019.1642362 perceptual and spectral audio descriptors will be investi- gated in future works to quantitatively evaluate the tim- bre of AM components. Moreover, other studies involv- ing different musical instruments should be carried out to confirm AM maturity about the production of musical components; in this scenario, this work can be considered a preliminary contribution towards that direction. 6. Holmström J, Holweg M, Khajavi SH, Partanen J (2016) The direct digital manufacturing (r)evolution: definition of a research agenda. Oper Manag Res 9(1–2):1–10. https://doi.org/10.1007/ s12063-016-0106-z 7. 4 Conclusion and future developments Aita-Holmes C, Liacouras P, Wilson WO, Grant GT (2015) Digi- tal capture, design, and manufacturing of an extraoral device for a clarinet player with Bell’s palsy. J Prosthet Dent 114(2):297–300. https://doi.org/10.1016/j.prosdent.2015.02.029 Acknowledgements Authors wish to thank the following students of Mechanical and Aerospace Engineering at the School of Engineering and Architecture of the University of Bologna for their support of the research: Michele Ascani, Luca Crudeli, Lisa Lucci, Sarah Merighi, Davide Neri and Giovanni Santucci. A special thanks to the expert musician and professional trombone player Mr Lorenzo Rocculi, who played the musical pieces and two Professors of Music. 8. Lorenzoni V, Doubrovski EL, Verlinden JC (2013) Embracing the digital in instrument making: Towards a musician-tailored mouthpiece by 3D printing. Proceedings of the stockholm music acoustics conference 2013, SMAC 2013, Stockholm (Sweden), 30 July-3 August 2013 9. Zoran A, Maes P (2008) Considering virtual and physical aspects in acoustic guitar designl Funding Open access funding provided by Alma Mater Studiorum - Università di Bologna within the CRUI-CARE Agreement. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 10. Zoran A (2011) The 3D printed flute: digital fabrication and design of musical instruments. J New Music Res 40(4):379–387. https://doi.org/10.1080/09298215.2011.621541 p g 11. de Gans B-J, Duineveld PC, Schubert US (2004) Inkjet printing of polymers: state of the art and future developments. Adv Mater 16(3):203–213. https://doi.org/10.1002/adma.200300385 Data availability Neither data, material, nor code is available due to future work in the same project. 12. Klapuri A, Davy M (eds) (2006) Signal processing methods for music transcription. Springer, New York Declarations 13. Savio G, Meneghello R, Concheri G (2019) Design of vari- able thickness triply periodic surfaces for additive manufactur- ing. Prog Addit Manuf 4(3):281–290. https://doi.org/10.1007/ s40964-019-00073-x Conflict of interest No potential conflict of interest was reported by the authors. 14. Celentano F, May N, Simoneau E, DiPasquale R, Shahbazi Z, Shahbazmohamadi S (2016) 3D printing for manufacturing antique and modern musical instrument parts. In: Proceedings of the ASME 2016 international mechanical engineering congress and exposition. Emerging technologies, vol 14. 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https://openalex.org/W2099633906	https://repositorio.aemet.es/bitstream/20.500.11765/485/1/18539-63638-1-PB.pdf	English	null	Development and evaluation of the BSC-DREAM8b dust regional model over Northern Africa, the Mediterranean and the Middle East	Tellus. Series B, Chemical and physical meteorology	2,012	cc-by	14,880	(Manuscript received 6 September 2011; in final form 30 March 2012) The BSC-DREAM8b model and its predecessor are analysed in terms of aerosol optical depth (AOD) for 2004 over Northern Africa, the Mediterranean and the Middle East. We discuss the model performance and we test and analyse its behaviour with new components. The results are evaluated using hourly data from 44 AERONET stations and seasonally averaged satellite observations. The operational versions strongly underestimate the winter AOD over the Sahel and overestimate the AOD over the Middle East and the Mediterranean achieving a low average annual correlation (0.35). The use of a more detailed size distribution and a corrected wash-out ratio, together with a new dry deposition scheme, improves the transport over the Mediterranean, although underestimations remain over the Sahel and overestimations over the Middle East. The inclusion of a ‘preferential source’ mask improves the localisation of the main North African sources and consequently the dust transport towards Europe and the Atlantic. The use of a more physically based dust emission scheme and a new soil texture database leads to significant improvements in the representation of emissions and the transport over the Sahel, achieving an average annual correlation of 0.53. In this case, the use of a preferential source mask does not introduce significant improvements. Keywords: mineral dust, model simulation, model comparison, aerosol optical depth, seasonal variability 2005; Grini et al., 2006; Pe´ rez et al., 2006a; Rodwell and Jung, 2008). Dust storms produce a variety of problems to inhabitants in and around desert areas such as deaths and damage caused by traffic accidents, road disruption, avia- tion operations and impacts on human health, such us allergies, respiratory diseases and eye infections (WHO, 2005). It is also thought to be a risk factor for epidemics of lethal meningitis in sub-Saharan Africa (Thomson et al., 2006) and increased incidence of paediatric asthma attacks in the Caribbean (Gyan et al., 2005). Tellus B 2012. # 2012 S. Basart et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Citation: Tellus B 2012, 64, 18539, http://dx.doi.org/10.3402/tellusb.v64i0.18539 Development and evaluation of the BSC-DREAM8b dust regional model over Northern Africa, the Mediterranean and the Middle East By SARA BASART1, CARLOS PE´ REZ2, SLODOBAN NICKOVIC3, EMILIO CUEVAS4 and JOSE´ MARI´ A BALDASANO1,5, 1Earth Sciences Department, Barcelona Supercomputing Center- Centro Nacional de Supercomputacio´n, Barcelona, Spain; 2NASA Goddard Institute for Space Studies and Department of Applied Physics and Applied Math, Columbia University, New York, USA; 3World Meteorological Organization, Geneva, Switzerland; 4Izan˜a Atmospheric Research Center, Meteorological State Agency of Spain (AEMET), Santa Cruz de Tenerife, Spain; 5Environmental Modelling Laboratory, Technical University of Catalonia, Barcelona, Spain Corresponding author. email: sara.basart@bsc.es PUBLISHED BY THE INTERNATIONAL METEOROLOGICAL INSTITUTE IN STOCKHOLM SERIES B CHEMICAL AND PHYSICAL METEOROLOGY PUBLISHED BY THE INTERNATIONAL METEOROLOGICAL INSTITUTE IN STOCKHOLM SERIES B CHEMICAL AND PHYSICAL METEOROLOGY AL INSTITUTE IN STOCKHOLM S C A M 1. Introduction Pe´ rez et al., 2006a, b; Papanastasiou et al., 2010) concerning both the horizontal and vertical extent of the dust plume in the Mediterranean Basin. Furthermore, daily evaluation of BSC-DREAM8b with near-real-time observations is conducted at BSC-CNS (www.bsc.es/ plantillaH.php?cat_id522). Currently, the daily opera- tional model evaluation includes satellites (MODIS and MSG) and AErosol RObotic NETwork (AERONET) sun photometers. DREAM and BSC-DREAM8b have also been validated and tested over longer time periods in the European region (e.g. Jime´ nez-Guerrero et al., 2008; Pay et al., 2010, 2012; Basart et al., 2012) and against measure- ments at source regions for the SAMUM I (Haustein et al., 2009) and BODEX campaigns (Todd et al., 2008). Substantial impacts of dust upon climate and environ- ment (IPCC, 2007) have increased the need to better understand and predict the atmospheric dust cycle. In this regard, a number of experimental and operational dust forecast systems have been developed in recent years, such as the global models Navy Aerosol Analysis and Prediction System (Westphal et al., 2009) and the aerosol model at the European Centre for Medium-range Weather Forecasts (Morcrette et al., 2008, 2009), and some regional models as BSC-DREAM8b (Nickovic et al., 2001; Pe´ rez et al., 2006a, b), SKIRON (Nickovic and Dobricic, 1996; Kallos et al., 1997; Nickovic et al., 1997), CHIMERE-DUST (Menut, 2008; Schmechtig et al., 2011), MOCAGE (Martet and Peuch, 2009), Chinese Unified Atmospheric Chemistry Environment Dust (Zhou et al., 2008) and NMMB/BSC- Dust (Pe´ rez et al., 2011). The updated BSC-DREAM8b model is operated and further developed at the Barcelona Supercomputing Center- Centro Nacional de Supercomputacio´ n (BSC-CNS; www. bsc.es/projects/earthscience/DREAM/). BSC-DREAM8b is one of the regional mineral dust models participating in the NA-ME-E Node of the SDS-WAS Programme. The history of the model starts with its predecessor, a single particle size dust model (Nickovic and Dobricic, 1996) initially developed in the World Laboratory Centre, Erice, Italy in the period 19911993. This model was implemented in the Tunisian Meteorological Service, where the first ever- successful operational regional dust forecast was performed during MarchMay 1995. The model was transferred to the University of Athens in 1995 where it was further improved and routine daily dust forecasts were established during 19961998 within the SKIRON project. 1. Introduction A large amount of mineral dust is mobilised over arid regions and injected into the atmosphere under specific weather conditions. In many regions, mineral dust repre- sents the major contribution to atmospheric optical thick- ness (Tegen et al., 1997), and evidence exists of increasing dust production in recent decades (Prospero and Lamb, 2003; Mahowald et al., 2010). Observations and simulations have demonstrated that in regions of high dust emission such as North Africa, dust aerosols strongly influence surface and tropospheric radiation budgets and conse- quently the atmospheric circulation from short term to climate scales (e.g. Miller and Tegen, 1998; Haywood et al., The seriousness of the problem inspired the develop- ment of the Sand and Dust Storm Warning Advisory and Assessment System (SDS-WAS) Programme under the umbrella of the World Meteorological Organization (http://www.wmo.int/sdswas). The SDS-WAS Programme intends to achieve comprehensive, coordinated and sustained observations and modelling capabilities of dust storms, in order to improve their monitoring state, (page number not for citation purpose) S. BASART ET AL. 2 2 increase the understanding of their formation processes and enhance the ability of countries to deliver timely and quality forecasts, observations, information and knowl- edge to users through an international partnership of research and operational communities. At present, the SDS-WAS Programme consists of two Regional Nodes that deal with both operational and scientific aspects related to atmospheric dust monitoring and forecasting in their respective regions, that is Northern Africa, Middle East and Europe (NA-ME-E) (http://sds-was.aemet.es/) and Asia (www.sds.cma.gov.cn). northernmost African sources, from 2006 the operational dust forecast at BSC-CNS was extended further covering a larger part of Europe to the North, sub-Saharan Africa to the South and the Arabian Peninsula to the East (Fig. 1). BSC-DREAM8b provides the operational dust forecast at BSC-CNS since May 2009. The main improved features of BSC-DREAM8b (Pe´ rez et al., 2006a, b) with respect to the original DREAM model version are a more detailed size bin distribution and the inclusion of dustradiation interactions. In the past years, both operational versions have been used for dust forecasting and as dust research tools in North Africa and the Mediterranean (e.g. Amiridis et al., 2009; Klein et al., 2010; Alonso-Pe´ rez et al., 2011). Several case studies have outlined the good skills of DREAM (e.g. Balis et al., 2006; Kishcha et al., 2007) and BSC-DREAM8b (e.g. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 3 where C is a constant tuning parameter, u* is the friction velocity, ut is the threshold friction velocity above which dust production starts, S represents the source term (which includes the desert mask), bk is the fraction of each texture class considered in the model and is the mass fraction of bin k in the soil and gk represents the ratio of the mass available for uplift and the total mass of the respective bin k. developments need to be evaluated in order to achieve better understanding and knowledge of dust source processes, size distributions and optical properties. The evaluation of regional mineral dust models is difficult. Measurements of aerosol properties from ground or space exist. However, all these remote-sensing measurements are highly integrated: not only over the atmospheric column but also over all aerosol components. Thus investigations for the treatment of a particular aerosol type may be limited to seasons and regions, when or where that aerosol type dominates the aerosol composition. The threshold friction velocity of the dry soil utkd depends on the particle size which is defined following Bagnold (1941): ut kd ¼ Ak ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2grk qk qa qa r (2) (2) In this contribution, we first evaluate the performance and discuss the limitations of DREAM and BSC- DREAM8b and we test and discuss updates in the emission and deposition schemes. The domain covers Northern Africa, the Mediterranean Basin and the Middle East. The evaluation is performed using hourly aerosol optical depth (AOD) data from the AERONET network (Holben et al., 1998) for year 2004. Satellite aerosol products are used to compare and analyse the modelled dust seasonal cycles. The paper is organised as follows. Section 2 describes the main characteristics of DREAM and BSC-DREAM8b as well as the new components that will be evaluated. Section 3 describes the observational data and introduces the evalua- tion strategy. In Section 4, we discuss the results of the evaluation of several model versions. Finally, conclusions are included in Section 5. where g is the gravity acceleration, rk is the particle radius size and rk and ra are the density of the particle and the ambient air, respectively. The parameter Ak is a function of the particle Reynolds number (Rr)rk(2rkutk)/n and n0.000015 m2 s1. For the considered particle sizes in the model Ak is specified by using available empirical data (White, 1979). DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL In this formulation, utkd decreases with decreasing particle size (rk), neglecting cohesive forces among small particles. In practice, this emission scheme directly entrains dust-sized particles into the atmosphere and includes the influence of soil structure and particle size distribution by means of S, bk and gk. Soil moisture effects on the threshold friction velocity are included following Fecan et al. (1999): utk ¼ utk ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ 1:21ðw w0Þ0:68 q (3) (3) 1. Introduction Further model developments continued at ICoD, University of Malta, and resulted in the DREAM version of the model (Nickovic et al., 2001), which was routinely run at ICoD during the period 19982005. In May 2005, the operational DREAM dust forecasting system was transferred to the Environmen- tal Modelling Laboratory of the Technical University of Catalonia (UPC) and in September 2006 to BSC-CNS where the model and its further developments are run on a routine basis. While the forecast model domain had traditionally covered a region comprising the Mediterranean and the The implementation of new model versions for opera- tional applications requires extensive verification. Ongoing Fig. 1. Mediterranean (in red) and North Africa-Europe- Mediterranean (in blue) forecast domains of the operational versions of BSC-DREAM. Fig. 1. Mediterranean (in red) and North Africa-Europe- Mediterranean (in blue) forecast domains of the operational versions of BSC-DREAM. 2. Model description, new components and experimental set-up where w is the ground wetness predicted by the model and w? is the amount of adsorbed water which is an increasing function of the clay fraction in the soil. The DREAM model solves the Euler-type partial differ- ential non-linear equation for dust mass continuity, and it is fully embedded as one of the governing prognostic equa- tions in the atmospheric NCEP/Eta atmospheric model (Janjic, 1977, 1979, 1984, 1990, 1994, 1996a, b; Mesinger et al., 1988; Zhao and Carr, 1997). In this section, we describe the original and the current operational versions of the model together with other new components that will be evaluated. Table 1 lists the main characteristics of the different versions evaluated in this contribution. In addition, DREAM includes a viscous sublayer between the surface and the lowest atmospheric model layer (Janjic, 1994), by assuming a physical similarity between mass/heat/momentum exchanges over surfaces such as ocean with that of mobilised dust particle over desert surfaces (Chamberlain, 1983; Segal, 1990). The model includes a simple wet-scavenging scheme which uses a basic precipitation model with a constant washout ratio (see Nickovic et al., 2001). Dry deposition scheme follows Giorgi (1986) which calculates dry deposi- tion velocity relative to the bottom transport model as a function of particle size and it is applied to any type of surface. This scheme includes processes of deposition by turbulent and Brownian diffusion, gravitational settling, interception and impaction on the surface roughness elements. Furthermore, the model considers desert dust aerosols as inert and particle growth under humid conditions is not taken account. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 5 Fig. 2. In the top and central panels, it is shown the source term based on the arid and semi-arid categories of the 10-min SSiB (a) and Olson World Ecosystem and the 1-km USGS land use datasets (b). In the panel c, regional distributions of the source function (G) from Ginoux et al. (2001) are shown in the grey scale. The red circles indicate dust emission source areas discussed in the text: (1) Bode´ le´ , (2) Mali, (3) Mauritania, (4) Western Sahara-Morocco, (5) Algeria-Adrar, (6) North of Algeria-Tunisia, (7) Lybia desert, (8) An-Nafud desert and (9) Rub’ Al Khali desert. remapping the arid and semi-arid categories of the Olson World Ecosystems database (EPA, 1992) at 10-min resolu- tion to the regional model domain (Fig. 2a). For soil textures, a combination of the Zobler near-surface global soil texture (Staub and Rosenzweig, 1987) at 18 resolution and the UNEP/GRID gridded FAO/UNESCO 4-km soil units is used. For each soil texture class the fractions of clay, small silt, large silt and sand are estimated from the soil texture triangle (Hillel, 1982). In this version of the model, the four different particle classes are assumed to have a particle radius of 0.73, 6.1, 18 and 38 mm, respectively. For long-range transport, only the first two dust classes are relevant for the analysis since their lifetime is larger than about 12 h. To obtain the AOD we use a constant-specific cross-section (r k): AODðkÞ ¼r kM (4) (4) where M is the column mass loading in the two smaller size classes (0.73 and 6.1 mm). The fine component of the AOD (AODfine) is calculated with the column mass of the first dust particle class, and the coarse component (AODcoarse) is calculated with the column mass of the second dust particle class. From 2003 onwards a research version of the model started to emerge. Following the idea of improving weather forecasts by including the dust radiative effect, an online interactive dustradiation scheme was under development in the period 20022005 (Nickovic, 2002, 2005) and was successfully finalised in Pe´ rez et al. (2006a) showing the positive impact of including dustradiation interactions in the short-term weather forecast. The main features of BSC-DREAM8b (hereafter referred as M8, see Table 1), described in Pe´ rez et al. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL (2006a), are a new source function S based on the arid and semi-arid categories of the 1-km USGS land use dataset (Fig. 2b), a more detailed particle size distribution which includes eight size bins within the 0.110 mm radius range according to Tegen and Lacis (1996) as listed in Table 2, a source size distribution derived from D’Almeida (1987), and dust radiative feedbacks (Pe´ rez et al., 2006a). Because important overestimations of the simulated dust concentration were observed during intense wet events in the Mediterranean Basin in M4, a corrected wash-out ratio was introduced in M8. Fig. 2. In the top and central panels, it is shown the source term based on the arid and semi-arid categories of the 10-min SSiB (a) and Olson World Ecosystem and the 1-km USGS land use datasets (b). In the panel c, regional distributions of the source function (G) from Ginoux et al. (2001) are shown in the grey scale. The red circles indicate dust emission source areas discussed in the text: (1) Bode´ le´ , (2) Mali, (3) Mauritania, (4) Western Sahara-Morocco, (5) Algeria-Adrar, (6) North of Algeria-Tunisia, (7) Lybia desert, (8) An-Nafud desert and (9) Rub’ Al Khali desert. AODfine and AODcoarse are calculated in this version with bins 14 and 58, respectively. For M8, the AOD is calculated with the following expression: AODðkÞ ¼ X 8 k¼1 AODkðkÞ ¼ X 8 k¼1 3 4qkrk MkQextðkÞk (5) 2.1. Operational versions In the operational versions of the model, the dust emission scheme parameterises the vertical dust flux Fk for each particle size bin k following Shao et al. (1993): Fk ¼ C S bk u3 1 utk u !2 2 4 3 5 for u utk (1) (1) In the original DREAM version (hereafter referred as M4, see Table 1), the source mask S is calculated from velocity, sition N used by 3: Shao e velocity, sition used by 3: Shao e alysis: model version, status, texture-type dataset, threshold friction v mber of bins and radiative feedbacks. l Vertical flux Source size distribution Preferential sources Wet deposition Dry depo S93 N01 No N01 G86 S93 D87 No Corrected G86 S93 D87N01 No Corrected Z01 S93 D87N01 G01 Corrected Z01 MB95 D87 No Corrected Z01 MB95 D87 G01 Corrected Z01 87); D87N01: D’Almeida (1987) modified with the correction factors : Marticorena and Bergametti (1995); N01: Nickovic et al. (2001); S9 ysis: mode ber of bins Vertical flux S93 S93 S93 S93 MB95 MB95 ); D87N01 Marticoren DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL S. BASART ET AL. S. BASART ET AL. 6 Table 2. Dust size bins introduced in the BSC-DREAM8b model. The bin intervals are taken from Tegen and Lacis (1996). Here rmin rmax are minimum and maximum radius and reff is effective radius for each size bin. N8 is based on M8 with the dust deposition scheme based on Zhang et al. (2001) and a more physically based dust emission scheme. In this case, a horizontal saltation flux H is simulated according to White (1979) as follows: Bin number rmin rmax (in mm) reff (in mm) 1 0.1 0.18 0.15 2 0.18 0.3 0.25 3 0.3 0.6 0.45 4 0.6 1 0.78 5 1 1.8 1.3 6 1.8 3 2.2 7 3 6 3.8 8 6 10 7.1 H ¼ cs qa g u3 X 4 k¼1 1 þ utk u ! 1 u2 tk u2 ! sk for u utk (6) (6) where cs is a dimensionless factor taken as 2.61 (White, 1979), ra is the air density, u* is the wind friction velocity, g is the gravitational constant, u*tk is the threshold wind friction velocity and sk is the relative surface area of each soil population k (i.e. clay, silt, fine-medium sand and coarse sand). In this case, the threshold friction velocity shows an optimum particle diameter range for uplifting between 60 and 80 mm and is parameterised using the semi-empirical relationship of Iversen and White (1982). For smaller and larger particles, the threshold friction velocity increases due to inter-particle cohesion forces and gravity, respectively. As in the previous model versions, soil moisture effects on the threshold friction velocity are included following Fecan et al. (1999). use of a topographic preferential source mask in combina- tion with the two different emission schemes (DG8 and NG8). The main features are described below and sum- marised in Table 1. 2.2.1. Source size distribution and dry deposition. D8 is based on M8 with a modified mass size distribution at sources and a new dry deposition scheme based on Zhang et al. (2001). The modified source size distribution intro- duces a correction in the proportion between emitted coarse and fine fractions to the D’Almeida’s (1987) mass size distribution. These correction factors are the same than those used in the original model version by Nickovic et al. (2001). S. BASART ET AL. The new deposition scheme based on Zhang et al. (2001) replaces the one of Giorgi (1986) and calculates particle dry deposition velocities as a function of particle size and density as well as relevant meteorological variables and land use. As a difference from earlier size-dependent particle dry deposition models (like the Giorgi’s scheme), this latter dry deposition scheme produces more realistic deposition velocities for submicron particles in better agreement with the experimental results (Zhang et al., 2001). This deposition scheme includes simplified empirical parameterisations for the deposition processes of Brownian diffusion, impaction, interception and gravitational settling based on the seminal work by Slinn (1982). The vertical flux F follows the empirical relationship of Marticorena and Bergametti (1995): F ¼ C S d H (7) (7) where C is the tuning constant, S represents the source term (which includes the desert mask) and d is the so-called horizontal to vertical flux ratio reflecting the availability of dust in the soil. To specify the soil size distribution, we use the soil textures of the hybrid STATSGO-FAO soil map. In this database, the FAO two-layer 5-min global soil texture is remapped into a global 30-s regular latitude longitude grid. We use four soil populations in the model distinguishing among fine-medium sand and coarse sand according to the criteria used in Tegen et al. (2002). 2.2.3. Preferential sources. Enclosed basins containing former lake beds or riverine sediment deposits provide an abundance of small clay-sized particles that are loosely bound, and dominate global dust emission according to the Total Ozone Mapping Spectrometer Aerosol Index (TOMS AI) satellite retrieval (Prospero et al., 2002). Modelling studies show that inclusion of these ‘preferred’ source regions improves the realism of the model dust load in the vicinity of the sources (Zender et al., 2003). To identify these regions, we test the source function G described in Ginoux et al. (2001) based upon topography which is described as follows: 2.2.2. Emission scheme. Wind tunnel studies have shown that for particles with radius B35 mm, the threshold friction velocity increases with decreasing radius (Iversen and White, 1982), and at sources the direct emission of small dust particles by wind is negligible. Sandblasting and disaggregation of clay and silt particles by large particles in saltation dominate the vertical flux of dust, which is strongly sensitive to the size distribution of saltating particles. 2.2. New model components (5) In addition to the evaluation of the operational versions of the model, we test and compare the behaviour of the model with several new components, including a new dry deposi- tion scheme and a modified source size distribution (D8), a more physically based dust emission scheme (N8), and the where for each size bin k, rk is the particle mass density, rk is the effective radius, Mk is the column mass loading and Qext(l)k is the extinction efficiency factor at wavelength l which was calculated using Mie scattering theory. 2.3. Model experimental set-up and tuning In the present work, quality-assured direct-sun data in the 440870 nm wavelength range is used, as these channels are highly accurate and they are available in all AERONET instruments. In order to achieve a good temporal and spatial coverage, the selection of the AERONET stations for the present model evaluation is based on two criteria: their localisation in the simulation domain (box at 0458N and 268W608E) and the amount of data during our study year. All operational stations in 2004 that collected data on at least 100 h, over a minimum period of 2 months have been selected. Fig. 3 shows the location of the 44 selected AERONET sites and Table 3 lists additional information including type of site, observation periods, percentage of cloud screened data and the availability of SDA products. We evaluate and discuss annual simulations over the extended operational domain, which includes Northern Africa, the Mediterranean and the Middle East (Fig. 1, blue grid). The simulated dust distributions consist of 366 daily runs for year 2004. The initial state of the dust concentration is defined by the 24-h forecast of the previous-day model run. Only in the ‘cold start’ of the model, concentration is set to zero. The cold start of the model is initiated on 23 December 2003. The Final Analyses of the National Centers of Environmental Prediction (at 1818) at 0 UTC are used as initial meteorological conditions and boundary conditions at intervals of 6 h. The resolution is set to 1/38 in the horizontal and to 24 layers extending up to approximately 15 km in the vertical. In addition to the AERONET comparison, we qualita- tively compare the modelled dust AOD to seasonal satellite aerosol distributions from TOMS, Multiangle Imaging SpectroRadiometer (MISR) and Moderate Resolution Imaging Spectroradiometer (MODIS) sensors (Fig. 7). In the visible and near IR, deserts are highly reflective, and accurate retrievals of AOD are difficult because single- view multispectral satellite instruments are generally unable to separate the atmospheric and surface contributions to the measured radiances. Among other causes, differences between the different aerosol satellite products are partly due to the use of different sensors that work in different spectral channels, orbits and spatial resolutions as well the algorithms used for the production of the corresponding aerosol product. In spite of these differences, there are some features in common. S. BASART ET AL. Gi ¼ zmax zi zmax zmin !5 (8) (8) 2.3. Model experimental set-up and tuning Because dust emission is a non-linear function of wind speed and depends on many other factors that are poorly known at the scale of the model grid, dust emission in models is constrained with observations (Cakmur et al., 2006). In this analysis, we optimise the tuning parameter C in eqs. (1) and (7) for all the experiments by minimising the weighted sum of the squared difference between the model and observed values of AOD from the measure- ments available during the study period presented in the next section. A posteriori model calibration is necessary to compare different model versions and understand regional differences among them. Simulated seasonal average AOD for the different model experiments are shown in Figs. 46 for winter (DJF) corresponding to December, January and February; spring (MAM) corresponding to March, April and May; summer (JJA) corresponding to June, July, August and autumn (SON) corresponding to September, October and November. TOMS AI (Torres et al., 1998; Prospero, 2002; Ginoux and Torres, 2003) is a semi-quantitative aerosol product that indicates the presence of absorbing aerosols like desert dust. The algorithm is based on the perturbation of the backscattered UV flux that originates below the aerosol layer and it could retrieve aerosol data in partially clouded pixels. The MISR is a unique blend of directional and spectral data that allows using aerosol retrieval algorithms that do not depend on explicit radiometric surface proper- ties. As such, MISR can retrieve aerosol properties like TOMS AI (Torres et al., 1998; Prospero, 2002; Ginoux and Torres, 2003) is a semi-quantitative aerosol product that indicates the presence of absorbing aerosols like desert dust. The algorithm is based on the perturbation of the backscattered UV flux that originates below the aerosol layer and it could retrieve aerosol data in partially clouded pixels. The MISR is a unique blend of directional and spectral data that allows using aerosol retrieval algorithms that do not depend on explicit radiometric surface proper- ties. As such, MISR can retrieve aerosol properties like DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 7 2000). These instruments rely on extinction measurements of the direct and scattered solar radiation at several nominal wavelengths (between 340 and 1020 nm). In addition, direct-sun AOD processing includes the Spectral Deconvolution Algorithm (SDA) described in O’Neill et al. (2003). This algorithm yields submicron and super-micron AOD (hereafter referred to as AODfine and AODcoarse, respectively) at a standard wavelength of 500 nm from which the fraction of fine mode (FMF) to total AOD can be computed. The algorithm fundamentally depends on the assumption that the coarse mode A˚ ngstro¨ m exponent and its derivative are close to zero. which is defined as the probability to have accumulated alluvium sediments in the grid cell i of altitude zi, and where zmax and zmin are the maximum and the minimum eleva- tions in the surrounding 108108 topography, respectively (Fig. 2c). We analyse the impact of the preferential source mask of Ginoux et al. (2001) in conjunction with the two emission schemes in D8 and N8. In these model simula- tions, the source term S is multiplied by G in D8 and N8 to which we refer as DG8 and NG8, respectively. 3.2. Evaluation strategy AOD over a variety of terrain, including highly reflective surfaces like deserts (Martonchik et al., 2004). The MISR observation repeat time is only three or four visits per month for those latitudes where Earth’s large desert areas occur. The MODIS aerosol algorithm is comprised of two independent algorithms, one for deriving aerosols over land and the second for aerosols over ocean (Levy et al., 2003; Remer et al., 2005). In the present analysis, we use the collection C005 AOD data (cloud free) at 550 nm from MODIS/Aqua sensor. However, the algorithm over land was developed only for low ground reflectance (i.e. over dark vegetation). For this reason, we also include the MODIS/Aqua Deep Blue product which employs radiances from blue channels for which the surface re- flectance is low enough that the presence of dust brightens the total reflectance and enhances the spectral contrast (Hsu et al., 2004). Thus, the MODIS/Aqua Deep Blue product basically provides information over arid and semi- arid areas. Fig. 7 includes a combination of the seasonal averages of MODIS/Aqua AOD Deep Blue and the collection C005 AOD data. AOD over a variety of terrain, including highly reflective surfaces like deserts (Martonchik et al., 2004). The MISR observation repeat time is only three or four visits per month for those latitudes where Earth’s large desert areas occur. The MODIS aerosol algorithm is comprised of two independent algorithms, one for deriving aerosols over land and the second for aerosols over ocean (Levy et al., 2003; Remer et al., 2005). In the present analysis, we use the collection C005 AOD data (cloud free) at 550 nm from MODIS/Aqua sensor. However, the algorithm over land was developed only for low ground reflectance (i.e. over dark vegetation). For this reason, we also include the MODIS/Aqua Deep Blue product which employs radiances from blue channels for which the surface re- flectance is low enough that the presence of dust brightens the total reflectance and enhances the spectral contrast (Hsu et al., 2004). Thus, the MODIS/Aqua Deep Blue product basically provides information over arid and semi- arid areas. Fig. 7 includes a combination of the seasonal averages of MODIS/Aqua AOD Deep Blue and the collection C005 AOD data. 3.1. Observational data For the quantitative model evaluation, we use column- integrated aerosol optical properties routinely observed within AERONET (Holben et al., 1998; Smirnov et al., S. BASART ET AL. 8 Fig. 3. Spatial distribution of 44 selected AERONET stations over the study domain. Acronyms are described in the Table 3. The different colours indicate the different regions which are deﬁned as: Sahel, Eastern Tropical Atlantic (E.Trop.Atl), Eastern subTropical Atlantic (E.sub-Trop.Atl), Northwestern Africa (NW.Afr), Western Iberian Peninsula (W.IP), Eastern Iberian Peninsula-Western Mediterranean (E.IP-W.Med), Central Mediterranean (C.Med), Eastern Mediterranean (E.Med) and Middle East. Fig. 3. Spatial distribution of 44 selected AERONET stations over the study domain. Acronyms are described in the Table 3. The different colours indicate the different regions which are deﬁned as: Sahel, Eastern Tropical Atlantic (E.Trop.Atl), Eastern subTropical Atlantic (E.sub-Trop.Atl), Northwestern Africa (NW.Afr), Western Iberian Peninsula (W.IP), Eastern Iberian Peninsula-Western Mediterranean (E.IP-W.Med), Central Mediterranean (C.Med), Eastern Mediterranean (E.Med) and Middle East. 3.2. Evaluation strategy In order to quantitatively compare the modelled optical data in the mid-visible spectrum with measurements at 550 nm, AOD at 550 nm from AERONET observations are obtained from data between 440 and 870 nm following the A˚ ngstro¨ m’s law. Because AERONET data are acquired at 15-min intervals on average, all AERONET measure- ments within 930 min of the model outputs have been extracted and used for the model comparison on an hourly basis. In order to quantitatively compare the modelled optical data in the mid-visible spectrum with measurements at 550 nm, AOD at 550 nm from AERONET observations are obtained from data between 440 and 870 nm following the A˚ ngstro¨ m’s law. Because AERONET data are acquired at 15-min intervals on average, all AERONET measure- ments within 930 min of the model outputs have been extracted and used for the model comparison on an hourly basis. The presence of different types of aerosols mixed with dust in the measurement points should introduce a negative bias in the comparison between dust model outputs and observations. In general, the model should underpredict the dust AOD for increasing A˚ ngstro¨ m exponents (a) because of the influence of anthropogenic pollution (Pe´ rez et al., 2006b). In order to evaluate mineral dust models, observa- tions have to be segmented into their different aerosol components, and the contribution of dust has to be extracted. In the aerosol characterisation for North Africa, Southern Europe and the Middle East presented in Basart el al. (2009), in addition to a, its spectral curvature rep- resented by daa(440, 675) a(675, 870) was used to discriminate mineral dust contributions. Desert dust aero- sols were observed mainly for aB0.75 for which the fine mode contributions were always less than 40%. ‘Pure desert dust’ conditions were associated to the highest As shown by the satellite data in Fig. 7, in general, MODIS features a higher AOD than MISR. This could be linked to the lower MISR observation frequency. While its temporal resolution (the repeat time is only three or four visits per month) is high enough to capture the major seasonal dust activity, it may be too low to reproduce some regional sources. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 9 Fig. 4. Seasonal averages of the dust AOD modelled by M4 (top panels) and M8 (bottom panels). Fig. 5. 3.2. Evaluation strategy Seasonal averages of the dust AOD modelled by D8 (top panels) and N8 (bottom panels). Fig. 6. Seasonal averages of the dust AOD modelled by DG8 (top panels) and NG8 (bottom panels). Fig. 4. Seasonal averages of the dust AOD modelled by M4 (top panels) and M8 (bottom panels). Fig. 4. Seasonal averages of the dust AOD modelled by M4 (top panels) and M8 (bottom panels). Fig. 5. Seasonal averages of the dust AOD modelled by D8 (top panels) and N8 (bottom panels). Fig. 5. Seasonal averages of the dust AOD modelled by D8 (top panels) and N8 (bottom panels). Fig. 6. Seasonal averages of the dust AOD modelled by DG8 (top panels) and NG8 (bottom panels). Fig. 6. Seasonal averages of the dust AOD modelled by DG8 (top panels) and NG8 (bottom panels). biomass burning aerosols were found associated to a1.5 and a mixture of different aerosol types (including desert dust) were found in 1 BaB1.5. extinctions, aB0.3 and da was negative or slightly positive. However, in the regions around the Mediterranean Basin and Persian Gulf where different classes of particles can be found, mixed dust was associated to 0.75 BaB1 and positive values of da. In addition, fine anthropogenic and Following these previous considerations, we implemen- ted a simple method to separate the contribution of dust (N), the y of the SDA X X X X X X X X X X X X X X X X X X X nd urban areas (R/U) and in littoral areas (C); coordinates, altitude, ﬁrst and last measurement date, number of measurements o.), the percentage of cloud screened data of the selected AERONET stations (F) in the observation period and the availabilit e Class Lon. (8/E) Lat. (8/N) Alt. (m) First date (dd/mm/yyyy) Last date (dd/mm/yyyy) N Hr. Dy. Mo. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 11 Station name Code Zone Class Lon. (8/E) Lat. (8/N) Alt. (m) First date (dd/mm/yyyy) Last date (dd/mm/yyyy) N Hr. Dy. Mo. F (%) SDA Rome_Tor_Vergata ROM C. Med R/U 12.6 41.8 130.0 01/01/2004 31/12/2004 2256 601 106 8 40.05 Saada SAA NW. Afr D 8.2 31.6 420.0 01/07/2004 31/12/2004 6053 1360 158 6 25.67 Saih_Salam SAI Middle East D 55.3 24.8 84.0 26/06/2004 16/10/2004 5537 1191 111 5 21.36 X SEDE_BOKER SED E. Med D 34.8 30.9 480.0 01/01/2004 02/12/2004 11 904 2915 310 12 24.54 X Sir_Bu_Nuair SIR Middle East R/UC 54.2 25.2 10.0 04/08/2004 04/10/2004 1512 445 60 3 39.11 X Solar_Village SVI Middle East D 46.4 24.9 764.0 13/01/2004 31/12/2004 10 086 2409 284 11 23.55 X Toulon TUL W. Med R/UC 6.0 43.1 50.0 15/11/2004 31/12/2004 430 128 24 2 42.09 Umm_Al_Quwain UMM Middle East R/UC 55.7 25.5 20.0 31/05/2004 12/10/2004 5336 1335 135 6 19.03 X Villefranche VIL W. Med R/UC 7.3 43.7 130.0 07/01/2004 10/11/2004 7120 1936 235 11 32.81 over our study area, based on the combination of AOD, a and da. For the quantitative model evaluation against AERONET data, we only take into account desert dust (i.e. aerosols associated with high extinctions and coarse-particle dominated fraction). Aerosol data with aB1 and daB0.1 have been considered as desert dust. All data with a1.5 is associated to fine anthropogenic aerosols and have been considered no-dust situations. Therefore, in the model comparison we have ascribed observed dust AOD of 0 for a1.5. Measurements out- side these ranges are associated with mixed aerosols and not included in the quantitative model evaluation. Furthermore, at those sites where the SDA products are available (mainly in the Middle East and Mediterranean regions as shown in Table 2) the AOD evaluation is complemented with AODcoarse which is fundamentally associated to maritime/oceanic aerosols and desert dust. Since sea-salt is related to low AOD (B0.03; Dubovik et al., 2002) and mainly affects coastal stations, high AODcoarse values are mostly related to mineral dust. Regionally averaged time series of the observed and simulated daily AOD are displayed for Sahel, and the Eastern Tropical North Atlantic, North-Western Africa and the Middle East in Fig. 8. Regionally averaged time series of the observed and simulated daily AODcoarse are displayed for the Mediterranean and the Middle East in Fig. 9. 3.2. Evaluation strategy F (%) R/UC 53.1 25.5 24.5 20/09/2004 28/12/2004 2021 580 83 4 33.22 D 1.5 15.3 305.0 01/01/2004 31/12/2004 10 967 2815 316 12 30.27 R/U 55.1 24.2 192.0 02/06/2004 20/09/2004 3959 1024 100 4 19.86 R/UC 53.0 24.1 5.0 23/06/2004 25/08/2004 2245 482 44 3 19.44 R/U 4.9 43.9 32.0 01/01/2004 31/12/2004 7131 1368 220 12 34.52 R/UC 50.6 26.2 25.0 08/10/2004 31/12/2004 2423 610 77 3 27.33 D 2.7 13.5 250.0 01/01/2004 31/12/2004 11000 2842 325 12 32.00 R/UC 2.9 36.5 230.0 01/01/2004 27/12/2004 4598 1998 264 12 33.55 R/UC 9.5 38.8 140.0 04/01/2004 14/10/2004 4179 1259 197 10 39.17 R/U 31.2 30.0 50.0 29/10/2004 31/12/2004 1061 338 52 3 58.56 Atl O 22.9 16.7 60.0 01/01/2004 31/12/2004 1583 477 70 4 37.23 ed R/U 5.1 44.1 100.0 02/01/2004 15/11/2004 6905 1979 242 11 37.12 Atl R/UC 17.0 14.4 0.0 01/01/2004 31/12/2004 9742 2520 301 12 28.69 R/UC 52.3 24.5 0.0 11/07/2004 21/12/2004 4422 1435 161 6 32.38 R/UC 54.4 24.5 15.0 01/01/2004 31/08/2004 7128 1813 203 8 22.05 R/UC 56.3 25.5 81.0 28/06/2004 31/12/2004 5057 1252 161 7 21.55 R/U 1.6 9.8 400.0 24/02/2004 31/12/2004 4517 1166 136 7 31.39 R/UC 6.7 37.1 0.0 01/01/2004 31/12/2004 9235 2437 302 12 23.7 R/UC 15.0 37.6 736.0 01/01/2004 15/04/2004 871 226 40 3 39.25 R/U 7.9 38.6 293.0 02/01/2004 31/12/2004 7860 2205 266 12 29.75 R/UC 25.3 35.3 20.0 01/01/2004 31/12/2004 9634 2352 272 12 24.9 D 54.3 23.0 209.0 22/06/2004 22/12/2004 7899 1715 183 7 23.39 D 5.9 13.3 285.0 01/06/2004 31/12/2004 6488 1659 195 7 29.6 R/U 4.3 8.3 350.0 22/10/2004 31/12/2004 840 390 66 3 36.06 R/UC 34.3 36.6 3.0 20/01/2004 27/12/2004 7223 1832 235 11 19.83 N. Atl HO 16.5 28.3 2391.0 26/06/2004 31/12/2004 5358 1260 145 7 29.79 R/U 55.8 24.1 1059.0 27/06/2004 13/10/2004 2971 719 83 5 35.20 R/UC 12.6 35.5 45.0 01/01/2004 30/12/2004 2561 848 139 8 30.63 R/U 18.1 40.3 30.0 01/01/2004 07/10/2004 5489 1671 219 10 34.28 R/UC 54.7 24.7 10.0 08/08/2004 02/10/2004 2298 559 56 3 25.53 D 53.8 23.1 204.0 07/06/2004 11/10/2004 6001 124 1166 5 19.14 R/UC 54.5 24.4 10.0 04/10/2004 31/12/2004 2912 590 75 3 28.57 R/U 34.8 31.9 40.0 01/01/2004 05/12/2004 9441 2592 299 12 33.09 D 1.4 12.2 290.0 01/01/2004 31/12/2004 7133 1911 271 12 34.51 R/U 4.5 42.0 750.0 21/01/2004 30/12/2004 7389 2010 265 12 32.69 DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL In order to evaluate the performance of the different model configurations, we use a set of statistics. Discrete statistics such as correlation coefficient (r), mean bias (MB) and root mean square error (RMSE), measure the skill of the model when performing diagnostic analyses of dust AOD at specific points where AERO- NET sites are located. Comparisons are made at annual and monthly levels as well as the four seasonal periods, DJF, MAM, JJA and SON. Regionally averaged time series of the monthly MB by regions are shown in Fig. 10. The Taylor’s diagram (Taylor, 2001) by regions shown in Fig. 11 concisely summarises the degree of correspon- dence between simulated and observed fields. On this diagram the correlation coefficient (r), the centred RMSE and the standard deviation of the simulated values are shown. Together these statistics provide a quick summary of the degree of pattern correspondence, allow- ing one to gauge how accurately a model simulates the observations. 4.1. Operational versions Mali-Mauritania border source, presenting maximum aerosol concentrations in spring and summer. The winter dust activity is greatest in low latitudes; as the year progresses, dust activity shifts to higher latitudes (Prospero et al., 2002; Klose et al., 2010). The transport is driven by the latitudinal shift of the Intertropical Front which corresponds to the convergence zone between the dry northern winds, called the Harmattan, and the humid monsoon winds coming from the South. From late February to early May the Harmattan is at its maximum (Sultan et al., 2005) with strong winds in the Sahel. During summertime, the dust activity is at maximum (Prospero et al., 2002; Engelstaedter et al., 2006; Schepanski et al., 2007) and the dust transport moves to northern latitudes. Although much of the dust is raised locally over the Middle East, substantial amounts of dust come from the Sahara (Middelton and Goudie, 2001). Over much of the Arabian Peninsula, dust is active all year long, but is relatively low in winter months. Dust activity in the Middle East grows strong in spring and summer, and weakens in autumn (Prospero et al., 2002). Overall, the simulated dust AOD at sources is generally weaker in M8 than in M4 (Fig. 4) with the exception of certain areas around of the Red Sea and the Middle East. This is partly explained by the increased transport bin resolution in M8 making long-range transport more efficient. Overall, the simulated dust AOD at sources is generally weaker in M8 than in M4 (Fig. 4) with the exception of certain areas around of the Red Sea and the Middle East. This is partly explained by the increased transport bin resolution in M8 making long-range transport more efficient. In winter and spring, the satellites highlight a strong aerosol signal over the Sahel and the Gulf of Guinea (Fig. 7). Dust is carried southwestward from the Bode´ le´ in Western Chad and adjacent areas at low levels by the northeasterly Harmattan winds (Chiapello et al., 1995; Cavalieri et al., 2010). During these seasons, both M4 and M8 strongly underestimate the AOD in the Sahelian sites (Figs. 8 and 10) with a winter bias between 0.5 and 1.0, and achieve very low annual correlations (0.24 for M4 and 0.18 for M8, see Fig. 11). 4. Results and discussion Mineral dust is the most important aerosol constituent in Northern Africa and the Middle East. As shown in the satellite observations (Fig. 7), in Northern Africa there are two major dust sources, the Bode´ le´ Basin and the 12 12 S. BASART ET AL. Fig. 7. Seasonal average of the observed aerosols for 2004 for TOMS AI (left panels), MISR AOD (central panels) as well as a combination of MODIS Aqua AOD products (Collection 005 and Deep Blue; right panels). Fig. 7. Seasonal average of the observed aerosols for 2004 for TOMS AI (left panels), MISR AOD (central panels) as well as a combination of MODIS Aqua AOD products (Collection 005 and Deep Blue; right panels). 4.1. Operational versions The interaction of desert dust and biomass burning aerosols in the region partly hampers the dust model evaluation but does not explain the significant mismatch of the model. Note that in M4 and M8, AOD DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 13 13 ral series of the modelled (M4, M8, D8, DG8, N8 and NG8) versus direct-sun AERONET measurem ent, AE) averaged by regions in daily basis. From the top to the bottom: Sahel, Eastern Tropical Nor ons. The legend includes the annual values (calculated in hourly basis) of the correlation coefﬁcient (r) quare error (RMSE) of each model version averaged for each region represented. Fig. 8. Temporal series of the modelled (M4, M8, D8, DG8, N8 and NG8) versus direct-sun AERONET measurements (AOD a A˚ ngstro¨ m exponent, AE) averaged by regions in daily basis. From the top to the bottom: Sahel, Eastern Tropical North Atlantic Middle East regions. The legend includes the annual values (calculated in hourly basis) of the correlation coefﬁcient (r), mean bias and root mean square error (RMSE) of each model version averaged for each region represented. Fig. 8. Temporal series of the modelled (M4, M8, D8, DG8, N8 and NG8) versus direct-sun AERONET measurements (AOD and A˚ ngstro¨ m exponent, AE) averaged by regions in daily basis. From the top to the bottom: Sahel, Eastern Tropical North Atlantic and Middle East regions. The legend includes the annual values (calculated in hourly basis) of the correlation coefﬁcient (r), mean bias (MB) and root mean square error (RMSE) of each model version averaged for each region represented. 14 oral series of the modelled AODcoarse (M4, M8, D8, DG8, N8 and NG8) versus SDA AERONET retrie nd Middle East regions in daily basis. The legend includes the annual values (calculated in hourly basis) o mean bias (MB) and root mean square error (RMSE) of each model version averaged for each region re S. BASART ET AL. S. BASART ET AL. Fig. 9. Temporal series of the modelled AODcoarse (M4, M8, D8, DG8, N8 and NG8) versus SDA AERONET retrievals aver Mediterranean and Middle East regions in daily basis. The legend includes the annual values (calculated in hourly basis) of the co coefﬁcient (r), mean bias (MB) and root mean square error (RMSE) of each model version averaged for each region represente Fig. 9. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 15 15 Fig. 10. Temporal series of the monthly evolution of mean bias (MB) obtained from the comparison between modelled (M4, M8, D8, DG8, N8 and NG8) versus direct-sun AERONET measurements averaged by regions. Regions are deﬁned in Fig. 3. Fig. 10. Temporal series of the monthly evolution of mean bias (MB) obtained from the comparison between modelled (M4, M8, D8, DG8, N8 and NG8) versus direct-sun AERONET measurements averaged by regions. Regions are deﬁned in Fig. 3. other dust source areas, including the zone of the Chotts in Algeria and Tunisia, in summer, as well as the region of Libya, Egypt and Sudan, in spring (see Fig. 7). From winter to summer, M4 and M8 (see Fig. 4) show a clear overestimation of the dust activity in Algeria when com- pared to satellite estimates. Maximum simulated dust transport from the North of Algeria and Tunisia is observed in spring over the Central-Eastern Mediterranean shifting towards the Western Mediterranean in summer (Fig. 4). In spring, M4 clearly tends to overestimate the AOD over the Central-Western Mediterranean AERONET sites (Figs. 8 and 10). Dust events in the Western-Central Mediterra- nean are usually driven by low-pressure systems with the presence of rain (Avila et al., 1998; Moulin et al., 1998). The AOD is better represented in M8 than M4 (Fig. 11) due to a lower dust emission in Algeria and a more efficient dust wet removal in this version. On the contrary, the strong dust emissions in Northern Mali during summer indicated by the observations are not reproduced by both model versions. In summer, M8 simulates a lower AOD than M4 at sources in Chad, Niger, Mali and Southern Algeria showing a better match with satellite observations (Fig. 7). The overestima- tion of M4 is also observed at AERONET sites along the values are below 0.1 south of 128N in winter (Fig. 4). This result suggested a severe underestimation in the emission from South Saharan sources, a problem in the low-level dust transport over the region or a combination thereof. Under the influence of the Harmattan, dust storms in the Sahara are quite frequent, for example, on the alluvial plain of Bilma (Niger) and Faya Largeau (Chad). Dust is transported from these regions to the Gulf of Guinea (e.g. Middleton and Goudie, 2001). 4.1. Operational versions Temporal series of the modelled AODcoarse (M4, M8, D8, DG8, N8 and NG8) versus SDA AERONET retrievals averaged by Mediterranean and Middle East regions in daily basis. The legend includes the annual values (calculated in hourly basis) of the correlation coefﬁcient (r), mean bias (MB) and root mean square error (RMSE) of each model version averaged for each region represented. Fig. 9. Temporal series of the modelled AODcoarse (M4, M8, D8, DG8, N8 and NG8) versus SDA AERONET retrievals averaged by Mediterranean and Middle East regions in daily basis. The legend includes the annual values (calculated in hourly basis) of the correlation coefﬁcient (r), mean bias (MB) and root mean square error (RMSE) of each model version averaged for each region represented. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL With respect to the latter, a detailed analysis of the simulated dust transport in M4 and M8 reveals a very efficient dry deposition along the southwestward dust transport (not shown). Also some important sources over Eastern Niger and the Mali-Mauritania border appear to be misrepresented by M4 and M8 throughout the year (particularly in summer) in comparison with satellite data. Figures 8 and 10 show that the model tends to overestimate the summer AOD in the Eastern Tropical North Atlantic AERONET sites where it achieves annual correlations of 0.24 for M4 and 0.34 for M8 that may reflect deficiencies in the transport from the Mali-Mauritania border source. In contrast to TOMS AI retrievals, MISR and MODIS AOD indicate significant aerosol content over several S. BASART ET AL. 16 Fig. 11. Taylor’s diagrams for M4, M8, D8, DG8, N8 and NG8 model version against the ﬁltered AOD direct-sun AERONET observations by regions. The radial distance from the origin is proportional to the standard deviation of a pattern. The centred root mean square (RMS) difference between the test and reference ﬁeld is proportional to their distance apart (in the same units as the standard deviation). The correlation between the two ﬁelds is given by the azimuthal position of the test ﬁeld. Regions are deﬁned in Fig. 3. Fig. 11. Taylor’s diagrams for M4, M8, D8, DG8, N8 and NG8 model version against the ﬁltered AOD direct-sun AERONET observations by regions. The radial distance from the origin is proportional to the standard deviation of a pattern. The centred root mean square (RMS) difference between the test and reference ﬁeld is proportional to their distance apart (in the same units as the standard deviation). The correlation between the two ﬁelds is given by the azimuthal position of the test ﬁeld. Regions are deﬁned in Fig. 3. The different satellite estimates do not show a clear seasonal pattern in Arabia (see Fig. 7), where the MODIS Deep Blue AOD product presents low extinction values in comparison with the relatively strong signal in TOMS AI or MISR AOD particularly in summer and autumn. In the Middle East, the maximum local desert dust activity is detected in spring (Smirnov et al., 2002; Kim et al., 2007). In contrast to M8, M4 simulates a maximum AOD in winter and spring over the An-Nafud desert in the northern part of the Arabian Peninsula (Fig. 4). DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 17 particularly in summer with an annual MB of 0.20 and 0.33, respectively. In summer, the presence of sea-breeze circula- tions cause regional dust transport from inland regions to the Persian Gulf (Eck et al., 2008) favouring a mixing of desert dust and fine pollution aerosols (Basart et al., 2009). The daily variability is rather poorly captured (correlations of 0.29 and 0.14 in M8 and M4, respectively). During this season, the Middle East is affected by long-range transport from Iraq and Southern Iraq deserts (Liu et al., 2000), and the dust transport is more efficient in M8. As shown in Fig. 9, the AODcoarse is reasonably well reproduced by M4 and M8 indicating that the observed AOD overestimations are linked to overestimations in the fine fraction of the dust AOD. observe an increase in the Atlantic dust transport (see Fig. 5) in better agreement with satellite observations (Fig. 7), and significant changes in dust mass distribution in the simulation domain. In contrast to D8, the simulated dust AOD by N8 decreases in the Maghreb, South of Algeria and Mauritania, and increases in the Bode´ le´ Depression, South of Niger, Nigeria, Benin and Chad through the year in better agreement with satellite observa- tions. In comparison with AERONET observations, the improvement is clearly visible in the Sahel (Fig. 11) where the model achieves a winter correlation of 0.60 and winter MB of 0.18. Also better results are obtained in the Eastern Tropical North Atlantic AERONET sites (Capo Verde and Dakar) with an annual correlations of 0.58 (see Fig. 11). Maximum differences between D8 and N8 are detected in the Maghreb, Algeria and Mauritania in spring and summer (Fig. 5). In contrast to D8, the emissions in N8 in the North of Mauritania are strongly inhibited in spring as well as in the North of Algeria and North of Libya in summer. Dust outbreaks with origin in the area of Mauritania are linked to Atlantic trajectories that are capable to reach the Iberian Peninsula as shown in the MODIS observation during spring and summer (Fig. 7). The comparison with AERONET shows that D8 presents better seasonal correlation than N8 (0.59 for D8 and 0.47 for N8, Fig. 11) in the Western Iberian Peninsula during this period. If we take a closer look at the coarse component of the AOD (see Fig. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 9), in areas affected by long-range transport as the Mediterranean Basin, AODcoarse is reasonably well reproduced by D8 and N8 particularly in the Western-Central Mediterranean AERO- NET sites (with annual correlations of 0.58 for D8 and 0.56 for N8 for Western Mediterranean sites; and 0.52 for D8 and 0.45 for N8 for Central Mediterranean sites). As a summary, the evaluation of the operational versions has highlighted significant limitations of the modelling system. The model strongly underestimates the AOD in the Sahel and the Eastern Tropical North Atlantic regions in winter and spring. It also overestimates the AOD in Northern Algeria and the dust transport towards the Western and Central Mediterranean mainly in spring. The MaliMauritania source is not represented in the model. The model places a hot spot in Southern Algeria not highlighted by the satellite. Finally the AOD overestima- tion in the Middle East is, at least partly, related to the overestimation of the fine dust fraction. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL In spring, the comparison with AERONET (Fig. 8) shows that M4 presents better seasonal correlations than M8 (0.59 for M4 and 0.53 for M8). This is partly linked to the different source mask used in each model version (see Table 1 and Fig. 2a and b). In both model versions, maximum summer activity shifts towards southern latitudes over the Rub’ Al Khali desert (in the southwestern part of the Arabian Peninsula, see Fig. 4). M4 and M8 suffer strong overestimations (Fig. 11) dust transport in the Eastern Tropical and sub-Tropical North Atlantic (Fig. 10). While there are important differences between M4 and M8, both model versions strongly overestimate the satellite (Fig. 7) and AERONET (Fig. 10) AOD, especially M4. The overestimated dust emissions in Algeria are always related to the lee of the Atlas Mountains and are a common pattern in M4 and M8. Several causes could induce the overestimation of dust emission in this area. For example, the misrepresentation of a small-scale atmospheric convec- tion processes in this complex region by the meteorological driver could overestimate the surface winds and conse- quently the dust emissions. Otherwise, threshold friction velocity is essentially a property of the soil surface, rather than that of a soil particle. In the model, the soil humidity is climatologically described, and this could introduce errors in the calculation of the threshold friction velocity. 4.2.2. Emission scheme. When we evaluate the physically based dust emission scheme model version (N8), we 4.2. New model components It is not straightforward to attribute the discrepancies to specific aspects of the model since the emission scheme depends on multiple surface, soil and meteorological features and includes threshold pro- cesses and non-linear relationships. One of the main responsible of the regional differences between the different model versions is how dust sources are defined in each model version. This introduces differences in the mass available for uplift of the respective size class, and consequently the modelled dust concentrations. Otherwise, the physically based emission scheme better reproduces the balance between fine and coarse fractions despite worse results observed in the total AOD with respect to the original emission scheme of the model. These results highlight the complexity of the problem since model improvements in some regions (or dust parameter- isations) might be accompanied by deterioration in some others. and summer and decrease the AOD seasonal concentra- tions in Sudan in better agreement with the MODIS and MISR aerosol products (Fig. 7). The annual correlation increases from 0.46 with D8 to 0.49 with DG8 and decreases from 0.45 with N8 to 0.42 with NG8 in the North-Western African sites (Fig. 11). Both DG8 and NG8 show slightly worse results than D8 and N8 in the Mediterranean AERONET sites (see Fig. 11). Also, the Northern Mauritania border source is further omitted by the topographic preferential source approach. As a result, on average, in the AOD comparison with the AERONET sites located in North-Western Africa region the annual correlation of DG8 increases 6% with respect D8 and NG8 decreases 7% with respect N8. The preferential source mask restricts the dust produc- tion in North-Eastern Saudi Arabia and Iraq (see Fig. 6). On the contrary, in summer it shows an increase of the dust production in Ash Sharqiyah and in the South-Eastern Saudi Arabia which is associated to the overestimations observed in June (Fig. 8) in the AERONET sites located in areas around the Persian Gulf. On average, the annual correlation in the Middle East decreases from 0.39 with D8 to 0.30 with DG8 and from 0.39 with N8 to 0.26 with NG8 (see Fig. 11). On the other hand, DG8 and NG8 are capable of reproducing some summer episodes in the Persian Gulf associated to regional sea-breeze circulations as the dust event on mid-July (Fig. 8). 4.2.3. Preferential source approach. The inclusion of the topographic preferential source mask G from Ginoux et al. 4.2. New model components 4.2.1. Source size distribution and dry deposition. One of the possible reasons for the strong mismatch in the Sahel is a too efficient dry deposition scheme in the model. With the inclusion of a new dry deposition scheme, D8 considerably improves the transport over the Sahel in winter and autumn. On average, the winter correlation in the Sahelian sites increases from 0.16 with M8 to 0.43 with D8. However, severe underestimation still persists in D8 with an MB of 0.43 (Fig. 10). Besides the improvement in the Sahel, over the rest of the domain and seasons the total AOD behaviour in D8 is similar to that of M8. In the Middle East, as in the case of the operational versions, D8 and N8 overestimate the AERONET obser- vations (Fig. 11), particularly in summer (Figs. 8 and 10). In contrast to D8, maximum seasonal AOD values in N8 are confined to the South-Eastern Arabian Peninsula. High AOD values are simulated by D8 in Iraq and the An-Nafud desert, in the north part of the Arabian Peninsula. This is, at least partly, linked to the slightly better results obtained by D8 in the Eastern Mediterranean sites during summer, which are affected by long-range dust transport from the Middle East (seasonal correlations are 0.30 for D8 and 0.25 for N8, see Fig. 11). As shown in Fig. 9, the AODcoarse is reasonably well reproduced by N8 indicating that the observed AOD overestimations are linked to overestima- tions in the fine fraction of the dust AOD (not shown here) as in the case of M8. In D8, the overestimation (particularly in summertime) of the total AOD in the Middle East is related to the coarse fraction as shown in Fig. 9. On the contrary, the modified source size distribution of the model included in D8 improves the simulation of the fine fraction in this region (not shown here). These results emphasise the necessity to conduct specific sensitivity tests of the mass size distribu- tion at sources and its relation to coarse and fine AOD. As the results show, in some regions the physically based emission scheme presents worse results than the model versions that include the original emission scheme of the 4.2.2. Emission scheme. When we evaluate the physically based dust emission scheme model version (N8), we S. BASART ET AL. 18 model in the comparison against the total AOD from AERONET measurements. 4.2. New model components (2001) (see Fig. 2c) in both emission schemes (DG8 and NG8) enhances the dust emission in the Bode´ le´ during winter and autumn in better agreement with satellite observations (Fig. 6). Consequently, an increase of Atlantic dust transport during winter and autumn is observed as well as a better correspondence with AERONET observations in the Sahelian and Eastern Tropical North Atlantic sites during wintertime (Figs. 8 and 10). The annual correlation in DG8 and NG8 increases by 12 and 10% with respect to D8 and N8 on average for the Sahelian AERONET sites (Fig. 11). In spite of these improvements, underestimations persist in this region. These underestimations are common features observed in the dust global models which affect the Atlantic transport to America (Huneeus et al., 2011) and it could be linked to missing or underestimated sources in the model, for example in Bilma (west of Bode´ le´ Depression in Eastern Niger) as indicated in the MODIS AOD observations (Fig. 7). Furthermore, during the northward migration of the monsoon trough at the end of the Harmattan season, unstable atmospheric conditions develop as warm and humid air flows over the heated land surface, generating intense meso-scale convective systems. These systems have the potential to entrain large quantities of dust into the atmosphere. These meteorological processes are largely underrepresented by the numerical models (e.g. Knippertz et al., 2009) and they could explain underestimations of the simulated dust concentrations in this region. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL 19 and Algeria) are in better agreement with the satellites. Therefore, dust concentrations are increased in the Sahel during wintertime and consequently, the Atlantic dust transport towards America. The annual correlation is increased about 12% in the Sahelian sites. The long-range dust transport towards Europe is better reproduced parti- cularly in North-Western Africa and the Western Iberian Peninsula. In the South-Eastern part of the Arabian Peninsula, the preferential source mask increases the dust emission causing overestimations of the model and a reduction of the annual correlation of about 9%. available, we performed an evaluation of the simulated fine and coarse AOD (i.e. AODcoarse and AODfine). Satellite aerosol products from TOMS, MISR and MODIS sensors are also included in the analysis to check the ability of the model to qualitatively reproduce the observed seasonal spatial desert dust distributions. As a result, the weaknesses and strengths of the model in the prediction of dust concentrations are discussed in terms of inaccuracies of the emission, transport and deposition of desert dust. The comparison with satellite-derived data highlights that the versions based on the original emission scheme misrepresent dust sources over Eastern Nigeria and at the Mali-Mauritania border and overestimate the AOD in Northern Algeria. In contrast, N8 shows its ability to reproduce the dust fields in the Bode´ le´ Depression, South of Niger, Nigeria, Benin and Chad. On the other hand, emissions from the Maghreb region are reduced in N8. In the Middle East, the seasonal satellite products do not show a clear pattern. In all the model versions maximum summer activity are found in southern latitudes over the Rub’ Al Khali desert. As a difference to the versions based on the original emission scheme, N8 inhibits dust produc- tion in Iraq and the An-Nafud desert during winter and spring. For NG8, the inclusion of the preferential source mask does not introduce an improvement in the modelled AOD dust fields. In fact, the Mauritania border and the region of Libya, Egypt and Sudan sources are mostly omitted by the topographic preferential source approach. Moreover, over- estimations observed during summer in the Middle East in NG8 are also found in any other model version and there is a decrease of the annual correlation of about 13% in this region. In this latter case, only the modelled dust fields in the Sahel region present improvements with respect to the observations. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL Overall, the implementation of a new deposition scheme after Zhang et al. (2001) shows an improvement of the results of the operational model versions. By regions, NG8 is the version better reproducing the localisation and magnitude of the main dust sources in North Africa (i.e. Bode´ le´ ), and D8 shows the best match with the observa- tions in the Middle East. On average for the Mediterra- nean, Iberian Peninsula and North-Western Africa regions, DG8 is the version that better captures the observed background dust AOD and the timing of the observed dust peaks. Finally, considering the entire domain of simulation, N8 is the version with most accurate results not only in source regions but also areas affected by long- range dust transport. Regional differences between the different model versions are mainly associated to how dust sources are defined in each model version. However, the inclusion of a physically based emission schemes highlight the improvement that represents the refinement of soil textures and dust emission processes. The results of the quantitative comparison with the AERONET measurements have shown that M4 and M8 strongly underestimate the AOD in the Sahel during the winter Harmattan season and overestimate the AOD in spring over the Mediterranean and in summer over the Middle East achieving rather low annual correlations (0.35). The implementation of a new deposition scheme after Zhang et al. (2001) in D8 considerably improves the transport over the Sahel in winter and autumn. However, significant underestimations over the Sahelian AERONET sites and overestimations in the Middle East still persist. In the overall domain an annual correlation of 0.42 is obtained for D8. For N8, an increase in the Atlantic dust transport and significant changes in the spatial dust mass distribution in Northern Africa are in better agreement with the observations. However, in the Middle East, overestimations persist during summertime. As a result, the dust fields of N8 show reasonably good correspondence with the AOD AERONET data obtaining an annual correlation of 0.53. Inaccuracies observed in the compar- ison with observed AODcoarse and AODfine from AERO- NET retrievals emphasise the necessity to conduct more detailed sensitivity tests of the mass size distribution of the model at sources and its relation to coarse and fine AOD. DEVELOPMENT AND EVALUATION OF THE BSC-DREAM8B DUST REGIONAL MODEL The results presented in this study will be used to improve the operational dust predictions of BSC- DREAM8b and for selecting the most appropriated model components in long-term dust reanalysis. In the near future, it is planned to implement some of these updates (tested and analysed in the present work) in a new operational version at BSC-CNS. For DG8, the preferential source mask constitutes a useful approach particularly in North African sources com- pared with D8. In this case, the placement and magni- tude of the main dust sources (i.e. Bode´ le´ , Mauritania 5. Conclusions In this contribution, the original version of the DREAM model (M4) and the updated BSC-DREAM8b (M8) together with four research model versions have been analysed for a complete annual cycle over a domain covering Northern Africa, the Mediterranean and the Middle East. In addition to a more detailed size bin resolution and an corrected wash-out ratio in the wet deposition, the analysed research model versions include a new dry deposition scheme based on Zhang et al. (2001) and a modified source size distribution (D8), a more physically based dust emission scheme which includes a new soil texture (N8), and the inclusion of a topographic preferential source mask in their respective emission schemes (DG8 and NG8). The preferential source mask is based upon approach from Ginoux et al. (2001). 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https://openalex.org/W2934396678	https://www.mdpi.com/1422-0067/20/7/1669/pdf?version=1554290826	English	null	Overexpression of PtDXS Enhances Stress Resistance in Poplars	International journal of molecular sciences	2,019	cc-by	14,696	Received: 21 February 2019; Accepted: 2 April 2019; Published: 3 April 2019 Abstract: 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the rate-limiting enzyme in the plastidial methylerythritol phosphate pathway (MEP). In this study, PtDXS (XM_024607716.1) was isolated from Populus trichocarpa. A bioinformatics analysis revealed that PtDXS had high homology with the DXSs of other plant species. PtDXS expression differed among plant tissues and was highest in young leaves and lowest in roots. The recombinant protein was produced in Escherichia coli BL21 (DE3), puriﬁed, and its activity evaluated. The puriﬁed protein was capable of catalyzing the formation of 1-deoxy-D-xylulose-5-phosphate (DXP) from glyceraldehyde-3-phosphate and pyruvate. A functional color assay in E. coli harboring pAC-BETA indicated that PtDXS encodes a functional protein involved in the biosynthesis of isoprenoid precursors. The treatment of P. trichocarpa seedlings with 200 µM abscisic acid (ABA), 200 mM NaCl, 10% polyethylene glycol6000, and 2 mM H2O2 resulted in increased expression of PtDXS. The ABA and gibberellic acid contents of the transgenic lines (Poplar Nanlin 895) were higher than wild types, suggesting that DXS is important in terpenoid biosynthesis. Overexpression of PtDXS enhanced resistance to S. populiperda infection. Furthermore, the transgenic lines showed decreased feeding by Micromelalopha troglodyta, supporting the notion that PtDXS is a key enzyme in terpenoid biosynthesis. Keywords: Populus trichocarpa; DXS; elicitor treatments; ABA; GA Overexpression of PtDXS Enhances Stress Resistance in Poplars Hui Wei 1,†, Ali Movahedi 1,†, Chen Xu 1,2,†, Weibo Sun 1,†, Amir Almasi Zadeh Yaghuti 1, Pu Wang 1, Dawei Li 1 and Qiang Zhuge 1,* 1 Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, College of Biology and the Environment, Nanjing Forestry Universit Nanjing 210037, China; 15850682752@163.com (H.W.); ali_movahedi@njfu.edu.cn (A.M.); xuchenidea@hotmail.com (C.X.); czswb@njfu.edu.cn (W.S.); Amir_20364@yahoo.com (A.A.Z.Y.); 18705155218@163.com (P.W.); dwli@njfu.edu.cn (D.L.) 1 Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China; 15850682752@163.com (H.W.); ali_movahedi@njfu.edu.cn (A.M.); xuchenidea@hotmail.com (C.X.); czswb@njfu.edu.cn (W.S.); Amir_20364@yahoo.com (A.A.Z.Y.); 18705155218@163.com (P.W.); dwli@njfu.edu.cn (D.L.) 2 Jiangsu Provincial Key Construction Laboratory of Special Biomass Resource Utilization, Nanjing Xiaozhuang University, Nanjing 211171, China g y j g * Correspondence: qzhuge@njfu.edu.cn; Fax: +86-25-8542-8701 † These authors are contributed equally as the ﬁrst author. International Journal of Molecular Sciences International Journal of Molecular Sciences www.mdpi.com/journal/ijms 1. Introduction Higher plants have two distinct isoprenoid biosynthesis pathways, the chloroplastic methylerythritolphosphate (MEP) pathway, which is responsible for producing monoterpenoids and diterpenoids, and the cytosolic mevalonic acid (MVA) pathway, which is responsible for synthesizing sesquiterpenoids and triterpenes [1–3]. Both the MEP and MVA pathways provide isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) for isoprenoid biosynthesis [4,5]. All known isoprenoids are necessary for life [6]. For example, the isoprenoid vitamin A plays a role in human growth and development as well as in immune system maintenance. Isoprenoids are also implicated in defense against biotic and abiotic stresses, intracellular signal transduction and vesicular transport, and formation of cellular and organelle membranes [7–11]. In the first important step in the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) catalyzes the condensation of pyruvate and glyceraldehyde-3-phosphate (G-3-P) to form Int. J. Mol. Sci. 2019, 20, 1669; doi:10.3390/ijms20071669 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 1669 2 of 21 1-deoxy-D-xylulose 5-phosphate (DXP) [12]. DXP synthesis requires Mg2+ or Mn2+ and thiamine pyrophosphate (TPP) as a cofactor [13,14]. Next, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol-4-phosphate (MEP), which requires NADPH as a cofactor as well as Mg2+ or Mn2+ [15]. DXS genes from Ginkgo biloba, Aquilaria sinensis, Arabidopsis thaliana, conifers, maize, and soybean have been cloned and characterized [16–19]. Transformation of tomato plants with bacterial DXSs resulted in signiﬁcant increases in carotenoid content [20]. In DXS-overexpressing A. thaliana, the contents of terpenoids–tocopherols, chlorophyll, carotenoids, and abscisic acid (ABA) were signiﬁcantly higher than wild types (WT) [21]. In addition, the contents of these terpenoids in DXS-underexpressing A. thaliana were signiﬁcantly lower than WT plants. The chlorophyll content of DXS–over- and under-expressing A. thaliana was 130% to 142% and 65% to 84% that of the WT, respectively. In addition, the hypocotyls of DXS–over- and under-expressing A. thaliana were signiﬁcantly shorter and longer than WT plants, respectively. AtDXS from A. thaliana was transferred into lavender, resulting in a signiﬁcantly higher essential oil content in T0 and T1 transgenic seedlings than in WT plants, but the carotenoid and chlorophyll contents were unaffected [22]. Transfer of a bacterial DXS gene into potato resulted in tuber lengthening and premature ﬂowering, indicating DXS is involved in phenotypic regulation. In addition, the carotene content increased two-fold and that of lycopene by six- to seven-fold compared to the control [21]. 2.1. Molecular Cloning and Sequence Analysis of PtDXS A 2900 bp full-length cDNA, named PtDXS (GenBank accession no. XM_024607716.1) was isolated from leaves of P. trichocarpa. It contained a 2166 bp ORF encoding a peptide of 721 amino acids, a 351 bp 5′-untranslated region, and a 383 bp 3′-untranslated region (Figure S1). The PtDXS protein had a predicted molecular mass of 77.72 kDa and an isoelectric point of 6.62 (http://web.expasy.org/ cgi-bin/protparam/protparam). The deduced amino acid sequence of PtDXS was similar to those of DXSs from other species; e.g., Alpinia ofﬁcinarum (AEK69518.1, 80.72% identity), Hevea brasiliensis (ABD92702.1 86.78% identity), Ricinus communis (XP_015573388.1, 86.17% identity), and Theobroma cacao (XP_017975597.1, 86.56% identity) (Figure S2). Plant DXS proteins contain a chloroplast transit peptide, which is consistent with the subcellular localization of the MEP pathway [23]. A predicted chloroplast transit peptide with a conserved VXA cleavage site [24] was present at the N-terminus of PtDXS and was rich in hydroxylated serine and threonine residues and lacked acidic amino acids such as aspartic acid and glutamic acid (Figure S2). Thiamin pyrophosphate (TPP) is a cofactor of DXS and is indispensable for its activity [25]. PtDXS also contains an N-terminal TPP-binding domain, which begins and concludes with the highly conserved sequences GDG and LNDN, respectively (Figure S2). A glutamic acid residue, which is thought to be related to transketolase activity, was found in the middle of PtDXS. Moreover, DRAG and PSD domains, which are considered to be related to pyridine binding, were present at the C-terminus, similar to the DXSs of other plant species (Figure S2). 1. Introduction Thus, DXS is the rate-limiting enzyme in isoprenoid synthesis and a variety of physiological processes. In this work, we report the cloning and characterization of DXS from Populus trichocarpa. We also evaluated PtDXS expression in different plant tissues and in the presence of abiotic stresses. Overexpression of PtDXS enhanced the tolerance of poplar to biotic stresses. 2.2. Structural and Phylogenetic Analyses of PtDXS The predicted three-dimensional structures of PtDXS and AtDXS were determined using the SWISS-MODEL server (http://www.expasy.org/swissmod/SWISS-MODEL.html) (Figure 1). PtDXS and AtDXS (NP_193291.1) comprised coils, helices, and strands. These two proteins also contain a TPP-binding domain [26] in the N-terminal region and an NADH-binding domain, which plays an 3 of 21 ntain a Int. J. Mol. Sci. 2019, 20, 1669 and AtDXS (NP_193291 important functional role. Therefore, we speculated PtDXS might have a biological function similar to AtDXS. important functional role. Therefore, we speculated PtDXS might have a biological function similar to AtDXS. Figure 1. Predicted tertiary structures of AtDXS and PtDXS Green, cyan, and yellow, α-helices, β-strands, and random coils, respectively. Red and magenta, TPP and DRAG domain motifs, respectively. (A) Tertiary structure of AtDXS; (B) Tertiary structure of PtDXS. Figure 1. Predicted tertiary structures of AtDXS and PtDXS Green, cyan, and yellow, α-helices, β-strands, and random coils, respectively. Red and magenta, TPP and DRAG domain motifs, respectively. (A) Tertiary structure of AtDXS; (B) Tertiary structure of PtDXS. Figure 1. Predicted tertiary structures of AtDXS and PtDXS Green, cyan, and yellow, α-helices, β-strands, and random coils, respectively. Red and magenta, TPP and DRAG domain motifs, respectively. (A) Tertiary structure of AtDXS; (B) Tertiary structure of PtDXS. Figure 1. Predicted tertiary structures of AtDXS and PtDXS Green, cyan, and yellow, α-helices, β-strands, and random coils, respectively. Red and magenta, TPP and DRAG domain motifs, respectively. (A) Tertiary structure of AtDXS; (B) Tertiary structure of PtDXS. A phylogenetic tree was constructed using the full-length amino acid sequence of DXS proteins (Figure 2). Type-I DXS genes are constitutively expressed, mainly in plant green tissues, and produce carotenoid and chlorophyll precursors. Type-II DXS genes are present in certain plant tissues [27]. The existence of type-III DXS genes has been suggested [28]. Type-I DXSs may play an important role in plant primary metabolism, and type-II DXSs in plant secondary metabolism [29]. Thus, based on the analysis of phylogenetic tree and the transcript profile of PtDXS, we speculate that PtDXS (GenBank accession no. XM_024607716.1) may be a type-I DXS, and that it plays an important role in the basic life processes of plants. A phylogenetic tree was constructed using the full-length amino acid sequence of DXS proteins (Figure 2). Type-I DXS genes are constitutively expressed, mainly in plant green tissues, and produce carotenoid and chlorophyll precursors. Type-II DXS genes are present in certain plant tissues [27]. 2.2. Structural and Phylogenetic Analyses of PtDXS The existence of type-III DXS genes has been suggested [28]. Type-I DXSs may play an important role in plant primary metabolism, and type-II DXSs in plant secondary metabolism [29]. Thus, based on the analysis of phylogenetic tree and the transcript proﬁle of PtDXS, we speculate that PtDXS (GenBank accession no. XM_024607716.1) may be a type-I DXS, and that it plays an important role in the basic life processes of plants. 2.3. Protein Expression, Purification, and Western Blot Analysis 2.3. Protein Expression, Puriﬁcation, and Western Blot Analysis p f y To study the function of PtDXS protein, we firstly needed to obtain PtDXS protein. By using prokaryotic expression and protein purification technology, we successfully expressed and purified PtDXS protein (Figure 3). To identify whether the prokaryotic expression system could express the target protein, the SDS-PAGE analysis was performed. IPTG-induced bacteria showed a specific band of the expected size, 78.6 kDa (including the 6×His tag, Figure 3A). This was consistent with the predicted protein (77.72 kDa). In addition, SDS-PAGE analysis has been carried out to identify whether the expressed PtDXS protein exists in the supernatant or in the precipitation. The result showed that the target protein was detected in the precipitate, suggesting its sequestration in inclusion bodies (Figure 3B). The processes of denaturation and renaturation were used to release inclusion bodies. Inclusion bodies were denatured in 2, 4, 6, and 8 M urea (Figure 3C). Continuously, we obtained the pure target protein PtDXS through the combined techniques of dilution, renaturation phase changes in dialysis, re-dissolution and finally, Ni-IDA resin. To isolate PtDXS fusion protein, we captured His-tagged protein using Ni-IDA resin, washed frequently in buffer containing 20 mM imidazole and eluted in buffer containing 250 mM imidazole (Figure 3D E) To study the function of PtDXS protein, we ﬁrstly needed to obtain PtDXS protein. By using prokaryotic expression and protein puriﬁcation technology, we successfully expressed and puriﬁed PtDXS protein (Figure 3). To identify whether the prokaryotic expression system could express the target protein, the SDS-PAGE analysis was performed. IPTG-induced bacteria showed a speciﬁc band of the expected size, 78.6 kDa (including the 6×His tag, Figure 3A). This was consistent with the predicted protein (77.72 kDa). In addition, SDS-PAGE analysis has been carried out to identify whether the expressed PtDXS protein exists in the supernatant or in the precipitation. The result showed that the target protein was detected in the precipitate, suggesting its sequestration in inclusion bodies (Figure 3B). The processes of denaturation and renaturation were used to release inclusion bodies. Inclusion bodies were denatured in 2, 4, 6, and 8 M urea (Figure 3C). Continuously, we obtained the pure target protein PtDXS through the combined techniques of dilution, renaturation phase changes in dialysis, re-dissolution and ﬁnally, Ni-IDA resin. To isolate PtDXS fusion protein, we captured His-tagged protein using Ni-IDA resin, washed frequently in buffer containing 20 mM imidazole, and eluted in buffer containing 250 mM imidazole (Figure 3D,E). 2.3. Protein Expression, Purification, and Western Blot Analysis 2.3. Protein Expression, Puriﬁcation, and Western Blot Analysis containing 20 mM imidazole, and eluted in buffer containing 250 mM imidazole (Figure 3D,E). Western blotting of PtDXS showed the presence of His-DXS for confirming that the produced peptide in E. coli BL21 (DE3) was DXS (Figure 3E). g g Western blotting of PtDXS showed the presence of His-DXS for conﬁrming that the produced peptide in E. coli BL21 (DE3) was DXS (Figure 3E). 4 of 21 4 of 21 Int. J. Mol. Sci. 2019, 20, 1669 Int J Mol Sci 2019 20 x FOR Figure 2. Phylogenetic tree of Populus trichocarpa. Amino acid sequences of PtDXS (XP_024463484.1) and other DXS proteins. The tree was constructed using the neighbor-joining method in MEGA 5.1 and was bootstrapped 1000 times. Bootstrap percentages are indicated at the branch points. In all cases, the tree topologies obtained using the neighbor-joining, minimum-evolution, and maximum-parsimony methods were identical. The GenBank accession numbers of the DXS sequences are as follows: Alpinia officinarum (AEK69518.1), Medicago truncatula (CAD22530.1), Pinus taeda (ACJ67021.1), Catharanthus roseus (ABI35993.1), Jatropha curcas (XP_012076628.1), Fragaria vesca subsp. Vesca (XP_011459218.1), Colwellia psychrerythraea (KGJ90592.1), Hevea brasiliensis (ABD92702.1), Bixa orellana (AMJ39459.1), Elaeis guineensis (XP_019710182.1), Helianthus annuus (OTG31837.1), Phoenix dactylifera (XP_008795130.1), Prunus persica (XP_007225144.1), Ricinus communis (XP_015573388.1), Taraxacum kok-saghyz (AMB19705.1), Theobroma cacao (XP_017975597.1), Aquilaria sinensis (AHI62962.1), Astragalus membranaceus (AID51428.1), Bixa orellana (AMJ39460.1), Catharanthus roseus (CAA09804.2), Crataegus pinnatifida var. Major (ALL29183.1), Eucommia ulmoides (AFU93069.1), Hevea brasiliensis (BAF98289.1), Morus alba (ALD62471.1), Tripterygium wilfordii (AKP20998.1), Arabidopsis thaliana (OAP04569.1), Bixa orellana (AMJ39461.1), Camellia sinensis (ANB66336.1), Herrania umbratica (XP_021276650.1), Jatropha curcas (XP_012065282.1), Lavandula angustifolia (AGQ04154.1), Magnolia champaca (ART66976.1), Manihot esculenta (XP_021634514.1), Ricinus communis (XP_002533688.1), Salvia miltiorrhiza (ACQ66107.1), Stevia rebaudiana (ALJ30087.1), Theobroma cacao (XP_017981933.1), Aquilaria sinensis (AFU75320.1), Arabidopsis thaliana (NP_196699.1), Bixa orellana (AMJ39462.1), Gossypium raimondii (XP_012474408.1), Jatropha curcas (XP_012076626.1), Magnolia champaca (ART66977.1), Manihot esculenta (XP_021597449.1), Medicago truncatula (XP_003603440.1), Nelumbo nucifera (XP_010254310.1), Ricinus communis (XP_002514364.1), Theobroma cacao (EOY31423 1) and Ziziphus jujuba (XP 015885917 1) Figure 2. Phylogenetic tree of Populus trichocarpa. Amino acid sequences of PtDXS (XP_024463484.1) and other DXS proteins. The tree was constructed using the neighbor-joining method in MEGA 5.1 and was bootstrapped 1000 times. Bootstrap percentages are indicated at the branch points. In all cases, the tree topologies obtained using the neighbor-joining, minimum-evolution, and maximum-parsimony methods were identical. The GenBank accession numbers of the DXS sequences are as follows: Alpinia ofﬁcinarum (AEK69518.1), Medicago truncatula (CAD22530.1), Pinus taeda (ACJ67021.1), Catharanthus roseus (ABI35993.1), Jatropha curcas (XP_012076628.1), Fragaria vesca subsp. 2.3. Protein Expression, Purification, and Western Blot Analysis 2.3. Protein Expression, Puriﬁcation, and Western Blot Analysis Vesca (XP_011459218.1), Colwellia psychrerythraea (KGJ90592.1), Hevea brasiliensis (ABD92702.1), Bixa orellana (AMJ39459.1), Elaeis guineensis (XP_019710182.1), Helianthus annuus (OTG31837.1), Phoenix dactylifera (XP_008795130.1), Prunus persica (XP_007225144.1), Ricinus communis (XP_015573388.1), Taraxacum kok-saghyz (AMB19705.1), Theobroma cacao (XP_017975597.1), Aquilaria sinensis (AHI62962.1), Astragalus membranaceus (AID51428.1), Bixa orellana (AMJ39460.1), Catharanthus roseus (CAA09804.2), Crataegus pinnatiﬁda var. Major (ALL29183.1), Eucommia ulmoides (AFU93069.1), Hevea brasiliensis (BAF98289.1), Morus alba (ALD62471.1), Tripterygium wilfordii (AKP20998.1), Arabidopsis thaliana (OAP04569.1), Bixa orellana (AMJ39461.1), Camellia sinensis (ANB66336.1), Herrania umbratica (XP_021276650.1), Jatropha curcas (XP_012065282.1), Lavandula angustifolia (AGQ04154.1), Magnolia champaca (ART66976.1), Manihot esculenta (XP_021634514.1), Ricinus communis (XP_002533688.1), Salvia miltiorrhiza (ACQ66107.1), Stevia rebaudiana (ALJ30087.1), Theobroma cacao (XP_017981933.1), Aquilaria sinensis (AFU75320.1), Arabidopsis thaliana (NP_196699.1), Bixa orellana (AMJ39462.1), Gossypium raimondii (XP_012474408.1), Jatropha curcas (XP_012076626.1), Magnolia champaca (ART66977.1), Manihot esculenta (XP_021597449.1), Medicago truncatula (XP_003603440.1), Nelumbo nucifera (XP_010254310.1), Ricinus communis (XP_002514364.1), Theobroma cacao (EOY31423.1), and Ziziphus jujuba (XP_015885917.1). 5 of 21 5 f 21 Int. J. Mol. Sci. 2019, 20, 1669 Figure 3. (A) Analysis of the fusion protein by 12% SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lanes 2–4: colonies 1–3 (induced with 1 mM IPTG). The molecular weight of the target protein was approximately 78.6 kDa (black arrow). (B) Analysis of the supernatant and precipitate by SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lane 2: colonies induced by 1 mM IPTG; lane 3: precipitate; lane 4: supernatant. (C) Denaturation of PtDXS. Lane M: molecular mass marker; lane 1: 2 M urea; lane 2: 4 M urea; lane 3: 6 M urea; and lane 4: 8 M urea. (D) Ni-IDA affinity chromatography of the fusion protein. Marks 1 and 2: flow-through; mark 3: wash; and mark 4: elution. (E) Purification of PtDXS. Lane M: molecular weight marker; lanes 1, 2: flow-through; lane 3: wash; lane 4: elution; lane 5: western blot analysis of purified PtDXS using a l l tib d i t th 6 Hi t Figure 3. (A) Analysis of the fusion protein by 12% SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lanes 2–4: colonies 1–3 (induced with 1 mM IPTG). The molecular weight of the target protein was approximately 78.6 kDa (black arrow). (B) Analysis of the supernatant and precipitate by SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lane 2: colonies induced by 1 mM IPTG; lane 3: precipitate; lane 4: supernatant. (C) Denaturation of PtDXS. 2.3. Protein Expression, Purification, and Western Blot Analysis 2.3. Protein Expression, Puriﬁcation, and Western Blot Analysis Lane M: molecular weight marker; lanes 1, 2: ﬂow-through; lane 3: wash; lane 4: elution; lane 5: western blot analysis of puriﬁed PtDXS using a monoclonal antibody against the 6×His tag. 2.3. Protein Expression, Purification, and Western Blot Analysis 2.3. Protein Expression, Puriﬁcation, and Western Blot Analysis Lane M: molecular mass marker; lane 1: 2 M urea; lane 2: 4 M urea; lane 3: 6 M urea; and lane 4: 8 M urea. (D) Ni-IDA afﬁnity chromatography of the fusion protein. Marks 1 and 2: ﬂow-through; mark 3: wash; and mark 4: elution. (E) Puriﬁcation of PtDXS. Lane M: molecular weight marker; lanes 1, 2: ﬂow-through; lane 3: wash; lane 4: elution; lane 5: western blot analysis of puriﬁed PtDXS using a monoclonal antibody against the 6×His tag. A l f h f b % A E l Figure 3. (A) Analysis of the fusion protein by 12% SDS-PAGE. Lane M: molec Figure 3. (A) Analysis of the fusion protein by 12% SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lanes 2–4: colonies 1–3 (induced with 1 mM IPTG). The molecular weight of the target protein was approximately 78.6 kDa (black arrow). (B) Analysis of the supernatant and precipitate by SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lane 2: colonies induced by 1 mM IPTG; lane 3: precipitate; lane 4: supernatant. (C) Denaturation of PtDXS. Lane M: molecular mass marker; lane 1: 2 M urea; lane 2: 4 M urea; lane 3: 6 M urea; and lane 4: 8 M urea. (D) Ni-IDA affinity chromatography of the fusion protein. Marks 1 and 2: flow-through; mark 3: wash; and mark 4: elution. (E) Purification of PtDXS. Lane M: molecular weight marker; lanes 1, 2: flow-through; lane 3: wash; lane 4: elution; lane 5: western blot analysis of purified PtDXS using a l l tib d i t th 6 Hi t Figure 3. (A) Analysis of the fusion protein by 12% SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lanes 2–4: colonies 1–3 (induced with 1 mM IPTG). The molecular weight of the target protein was approximately 78.6 kDa (black arrow). (B) Analysis of the supernatant and precipitate by SDS-PAGE. Lane M: molecular mass marker; lane 1: negative control; lane 2: colonies induced by 1 mM IPTG; lane 3: precipitate; lane 4: supernatant. (C) Denaturation of PtDXS. Lane M: molecular mass marker; lane 1: 2 M urea; lane 2: 4 M urea; lane 3: 6 M urea; and lane 4: 8 M urea. (D) Ni-IDA afﬁnity chromatography of the fusion protein. Marks 1 and 2: ﬂow-through; mark 3: wash; and mark 4: elution. (E) Puriﬁcation of PtDXS. 2.5. Tissue Speciﬁcity of PtDXS Expression Mature and young leaves, upper and lower stems, petioles, and roots from 2-month-old seedlings of P. trichocarpa, which were grown on sterile half-strength Murashige and Skoog medium (pH 5.8), were chosen to extract total RNA. QRT-PCR was used to determine the transcript level of the PtDXS gene in different tissues (mature and young leaves, upper and lower stems, petioles, and roots). PtDXS was expressed in all tissues examined, and was highest in young leaves and mature leaves, followed by the lower region of the stem, upper region of the stem, petiole, and root. PtDXS expression in young leaves was almost 4.6-fold higher than roots (Figure 4A). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 21 (pH 5.8), were chosen to extract total RNA. QRT-PCR was used to determine the transcript level of the PtDXS gene in different tissues (mature and young leaves, upper and lower stems, petioles, and roots). PtDXS was expressed in all tissues examined, and was highest in young leaves and mature leaves, followed by the lower region of the stem, upper region of the stem, petiole, and root. PtDXS expression in young leaves was almost 4.6-fold higher than roots (Figure 4A). Figure 4. Comparison of PtDXS expression in various tissues and in response to stresses. (A) Comparison of the PtDXS expression in the indicated tissues. Comparison of the PtDXS expression level in young leaves followed by different treatments and times (B) 200 μM ABA, (C) 2 mM H2O2, (D) 200 mM NaCl, and (E) 10% PEG6000. All experiments have been normalized by βactin as an internal reference; bars represent means ± standard deviation (SD; n = 3); three independent biological experiments were performed with three repeats; asterisks represent significant differences in comparing with 0 h treatment as the control (Student’s t-test; , , and *, p < 0.05, p < 0.01, and p < Figure 4. Comparison of PtDXS expression in various tissues and in response to stresses. (A) Comparison of the PtDXS expression in the indicated tissues. Comparison of the PtDXS expression level in young leaves followed by different treatments and times (B) 200 µM ABA, (C) 2 mM H2O2, (D) 200 mM NaCl, and (E) 10% PEG6000. monoclonal antibody against th 2.4. Functional Analysis of PtDXS 2.4. Functional Analysis of PtDXS To assess the function of PtDXS, preliminary enzymatic assays were conducted using HPLC-MS/MS. A comparison of the retention times and mass fragmentation patterns of samples with those of the DXP standard confirmed that recombinant PtDXS catalyzed formation of DXP from G-3-P and pyruvate. By contrast, no peak or mass fragmentation pattern was detected in the To assess the function of PtDXS, preliminary enzymatic assays were conducted using HPLC-MS/MS. A comparison of the retention times and mass fragmentation patterns of samples with those of the DXP standard conﬁrmed that recombinant PtDXS catalyzed formation of DXP from G-3-P and pyruvate. By contrast, no peak or mass fragmentation pattern was detected in the negative control (Figure S3A). py y , p g p negative control (Figure S3A). To further analyze the biological activity of PtDXS, the color complementation method was used. E. coli lacks genes related to β-carotene synthesis. However, transgenic E. coli reportedly produces carotene [30]. To confirm the function of PtDXS, single vector pTrc was transformed into E. coli as the negative control and two pTrc and pAC-BETA vectors were transformed into E. coli as the positive control. In addition, pTrc-PtDXS and pAC-BETA vectors were transformed into E. coli. Colonies of E. coli containing pTrc-PtDXS and pAC-BETA (Figure S3Bc) were darker than E. coli containing the empty vectors pTrc and pAC-BETA (Figure S3Bb). In addition, the co-transformations demonstrated that PtDXS encodes the target functional protein. Therefore, To further analyze the biological activity of PtDXS, the color complementation method was used. E. coli lacks genes related to β-carotene synthesis. However, transgenic E. coli reportedly produces carotene [30]. To conﬁrm the function of PtDXS, single vector pTrc was transformed into E. coli as the negative control and two pTrc and pAC-BETA vectors were transformed into E. coli as the positive control. In addition, pTrc-PtDXS and pAC-BETA vectors were transformed into E. coli. Colonies of E. coli containing pTrc-PtDXS and pAC-BETA (Figure S3Bc) were darker than E. coli containing the empty vectors pTrc and pAC-BETA (Figure S3Bb). In addition, the co-transformations demonstrated that PtDXS encodes the target functional protein. Therefore, PtDXS plays an important role in the synthesis of carotene. Int. J. Mol. Sci. 2019, 20, 1669 6 of 21 2.5. Tissue Speciﬁcity of PtDXS Expression All experiments have been normalized by βactin as an internal reference; bars represent means ± standard deviation (SD; n = 3); three independent biological experiments were performed with three repeats; asterisks represent significant differences in comparing with 0 h treatment as the control (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < 0.001, respectively). Figure 4. Comparison of PtDXS expression in various tissues and in response to stresses. (A) Comparison of the PtDXS expression in the indicated tissues. Comparison of the PtDXS expression level in young leaves followed by different treatments and times (B) 200 μM ABA, (C) 2 mM H2O2, (D) 200 mM NaCl, and (E) 10% PEG6000. All experiments have been normalized by βactin as an internal reference; bars represent means ± standard deviation (SD; n = 3); three independent biological experiments were performed with three repeats; asterisks represent significant differences in comparing with 0 h treatment as the control (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < Figure 4. Comparison of PtDXS expression in various tissues and in response to stresses. (A) Comparison of the PtDXS expression in the indicated tissues. Comparison of the PtDXS expression level in young leaves followed by different treatments and times (B) 200 µM ABA, (C) 2 mM H2O2, (D) 200 mM NaCl, and (E) 10% PEG6000. All experiments have been normalized by βactin as an internal reference; bars represent means ± standard deviation (SD; n = 3); three independent biological experiments were performed with three repeats; asterisks represent significant differences in comparing with 0 h treatment as the control (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < 0.001, respectively). 0.001, respectively). 2.6. Expression Level of PtDXS under Abiotic Stresses 2019, 20, 1669 signiﬁcantly revealed higher expression in responding to H2O2 stress along certain time points 6, 24 and 48 h in comparing with the other stresses and times (Figure S4). signiﬁcantly revealed higher expression in responding to H2O2 stress along certain time points 6, 24 and 48 h in comparing with the other stresses and times (Figure S4). 2.7. Overexpression of PtDXS in Nanlin895 Poplar and Response of Transgenic Poplars to 100 mM NaCl and 3% PEG6000 Stresses To analyze the PtDXS functional roles in transgenic poplars, we used the Agrobacterium-mediated transformation to overexpress PtDXS gene into WT poplars. Twelve independent transgenic poplars were achieved through screening of regenerated kanamycin-resistant poplars (Figure S5). PCR was performed to determine whether T-DNA was inserted into the genome of Nanlin895 poplars, and qRT-PCR and western blot were used to conﬁrm expression of the target gene (Figure S6). When we used the 35S primer as the upstream primer and PtDXS-R primer as the downstream prime, the genomic PCR analyses illustrated that the twelve transgenic lines had the expected bands, but the wild-type poplars had no bands according to the analysis of 1% agarose gel electrophoresis (Figure S6A). In PCR reactions, the PtDXS-F and PtDXS-R primers isolated one longer fragment > 2166 bp in WT poplars, because of introns, whereas expected targeted bands were appeared in the transgenic lines (Figure S6B). In addition, the result of PCR for nptII (kanamycin resistance) gene showed that the twelve transgenic lines exhibited expected bands, but the wild-type poplars exhibited no bands (Figure S6C). The result of southern blot revealed that 2–4 copies of PtDXS gene were overexpressed in transgenic lines (Figure S6D). The twelve transgenic lines also analyzed by qRT-PCR to show the PtDXS transcript levels. qRT-PCR result showed that PtDXS gene were expressed in Nanlin895 poplars (Figure S6E). Total protein from WT and transgenic plants was extracted and analyzed by 12% SDS-PAGE (Figure S6F). Furthermore, the results of western blot revealed the presence of PtDXS expressed peptides using His-DXS. Western blotting also revealed that the PtDXS gene was stably integrated into the genomes of the transgenic lines and led to expression of the DXS protein using the expression system of the poplars (Figure S6G). y p p g The growth of wild-type poplars was severely inhibited under the 100 mM NaCl and 3% PEG6000 stress treatments, and the leaves were etiolated and browned (Figure S7A). 0.001, respectively). 2.6. Expression Level of PtDXS under Abiotic Stresses 2.6. Expression Level of PtDXS under Abiotic Stresses First, 200 mM NaCl, 200 μM abscisic acid (ABA), 10% PEG6000, and 2 mM hydrogen peroxide (H2O2) were chosen for introducing to 2-month-old seedlings of P. trichocarpa grown on sterile half-strength MS medium (pH 5.8). QRT-PCR showed that the expression of PtDXS was upregulated by 200 mM NaCl, 200 μM ABA, 2 mM H2O2, and 10% PEG6000. Treatment with 200 μM ABA significantly upregulated the expression level of PtDXS from 3 to 48 h, with a peak at 12 h (Figure 4B). Treatment with 2 mM H2O2 caused to upregulate the PtDXS expression significantly after 3 h, which persisted for 48 h with a peak at 8 h (Figure 4C). Treatment with 200 mM NaCl downregulated the expression of PtDXS at 1 and 3 h, followed by an increase from 6–48 h with a peak at 48 h (Figure 4D). Treatment with 10% PEG6000 downregulated the expression of PtDXS at1 to 2 days and the expression of PtDXS increased subsequently and reached the maximum on the First, 200 mM NaCl, 200 µM abscisic acid (ABA), 10% PEG6000, and 2 mM hydrogen peroxide (H2O2) were chosen for introducing to 2-month-old seedlings of P. trichocarpa grown on sterile half-strength MS medium (pH 5.8). QRT-PCR showed that the expression of PtDXS was upregulated by 200 mM NaCl, 200 µM ABA, 2 mM H2O2, and 10% PEG6000. Treatment with 200 µM ABA signiﬁcantly upregulated the expression level of PtDXS from 3 to 48 h, with a peak at 12 h (Figure 4B). Treatment with 2 mM H2O2 caused to upregulate the PtDXS expression signiﬁcantly after 3 h, which persisted for 48 h with a peak at 8 h (Figure 4C). Treatment with 200 mM NaCl downregulated the expression of PtDXS at 1 and 3 h, followed by an increase from 6–48 h with a peak at 48 h (Figure 4D). Treatment with 10% PEG6000 downregulated the expression of PtDXS at1 to 2 days, and the expression of PtDXS increased subsequently, and reached the maximum on the 7th day. (Figure 4E). These data suggested that PtDXS protein might play a role in the response to abiotic stresses. In addition, PtDXS gene 7 of 21 Int. J. Mol. Sci. 0.001, respectively). 2.6. Expression Level of PtDXS under Abiotic Stresses The transgenic poplar seedlings remained green and alive under 100 mM NaCl and 3% PEG6000 stress treatments, whereas the most WT poplar seedlings revealed yellow and could not be alive (Figure S7B), and most transgenic poplars can survive. The result showed that the survival rates of wild-type poplars were lower than transgenic poplars under 100 mM NaCl and 3% PEG6000 treatments (Figure S7C). Therefore, we resulted that the PtDXS overexpression may be able to improve the osmotic stress. 2.8. Expression Levels of MVA-, MEP-, ABA-, and GA-Related Genes in PtDXS-Overexpressing Plants .8. Expression Levels of MVA-, MEP-, ABA-, and GA-Related Genes in PtDXS-Overexpressing Plants Expression Levels of MVA-, MEP-, ABA-, and GA-Related Genes in PtDXS-Overexpressing Plants Expression levels of the MEP-related genes DXR, HDS, HDR, MCT, and CMK were increased signiﬁcantly in the transgenic plants (Figure 5A). Expression levels of the MVA-related genes HMGS, HMGR, and MVD revealed signiﬁcant diferences between the transgenic and WT poplars (Figure 5B). In addition, the expression levels of DXS, DXR, HDS, HDR, MVK, and MVD were increased signiﬁcantly by the overexpression of PtHMGR (Figure S8). The expression levels of MVA- and MEP-related genes revealed signiﬁcant differences between the PtDXS- and PtHMGR-overexpressing plants. Therefore, we speculated that the MEP pathway might play a leading role in the biosynthesis of terpenoids, and the MVA pathway an auxiliary role. Furthermore, transgenic lines exhibited higher expression levels of the downstream genes IDI, GPS, GPPS, and GGPPS in comparing with WT poplars (Figure 5C). Changes in the expression levels of MVA- and MEP-related genes may affect those of ABA- and GA-related genes. The expression levels of NCED1, NCED5, NCED6, ZEP1, ZEP2, and ZEP3 were signiﬁcantly higher in the transgenic plants, but that of NCED3 was lower (Figure 5D). The expression levels of GA20OX-1, GA20OX-2, GA20OX-3, and GA20OX-4 were signiﬁcantly higher in the transgenic plants (Figure 5F). In addition, while IAA1 gene revealed signiﬁcant lower expression in transgenic 8 of 21 Int. J. Mol. Sci. 2019, 20, 1669 poplars, IAA2, IAA6, IAA18, and IAA19 genes revealed no signiﬁcant differences between transgenic and WT poplars (Figure 5E). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 21 Figure 5. Expression levels of MEP- MVA-, ABA-, IAA-, and GA-related and downstream genes in the transgenic and WT poplar leaves. (A) Expression levels of DXR, MCT, CMK, HDS, and HDR. (B) Expression levels of ACL, HMGS, HMGR, MVK, and MVD. (C) Transcript levels of IDI, GPS, GPPS, and GGPPS. (D) Expression levels of NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3. (E) Expression levels of IAA1, IAA2, IAA6, IAA18, and IAA19. (F) Expression levels of GA20OX-1, GA20OX-2, GA20OX-3, and GA20OX-4. All experiments have been normalized using β-actin as an internal reference; bars represent means ± SD (n = 3). Three independent experiments were performed. The asterisks represent significant differences relative to WT plants (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < 0.001, respectively). 2.9. ABA, GA3, and GA4 Contents of PtDXS-Overexpressing Plants 2.9. ABA, GA3, and GA4 Contents of PtDXS-Overexpressing Plants HPLC-MS/MS was used to quantify the ABA, GA3, and GA4 contents of the transgenic and WT plants (Figure 6A,D). The ABA content of the transgenic plants (2 ± 0.2 ng/g) was approximately 1.6–2-fold that of the control plants. In addition, there was a significant difference in ABA content between the transgenic and WT plants. The GA3 content of the transgenic plants was approximately 4–14-fold that of the control plants. The highest GA3 content was in the T3 plants (1.51 ng/g). The GA4 content of the T1 plants was 0.65 ± 0.05 ng/g. By contrast, GA4 was not detected in the T3 and WT plants. HPLC-MS/MS was used to quantify the ABA, GA3, and GA4 contents of the transgenic and WT plants (Figure 6A,D). The ABA content of the transgenic plants (2 ± 0.2 ng/g) was approximately 1.6–2-fold that of the control plants. In addition, there was a signiﬁcant difference in ABA content between the transgenic and WT plants. The GA3 content of the transgenic plants was approximately 4–14-fold that of the control plants. The highest GA3 content was in the T3 plants (1.51 ng/g). The GA4 content of the T1 plants was 0.65 ± 0.05 ng/g. By contrast, GA4 was not detected in the T3 and WT plants. 2.8. Expression Levels of MVA-, MEP-, ABA-, and GA-Related Genes in PtDXS-Overexpressing Plants Expression levels of MEP- MVA-, ABA-, IAA-, and GA-related and downstream genes in Expression levels of MEP- MVA-, ABA-, IAA-, and GA-related and downstream genes in 2.8. Expression Levels of MVA-, MEP-, ABA-, and GA-Related Genes in PtDXS-Overexpressing Plants Figure 5. Expression levels of MEP- MVA-, ABA-, IAA-, and GA-related and downstream genes in the transgenic and WT poplar leaves. (A) Expression levels of DXR, MCT, CMK, HDS, and HDR. (B) Expression levels of ACL, HMGS, HMGR, MVK, and MVD. (C) Transcript levels of IDI, GPS, GPPS, and GGPPS. (D) Expression levels of NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3. (E) Expression levels of IAA1, IAA2, IAA6, IAA18, and IAA19. (F) Expression levels of GA20OX-1, GA20OX-2, GA20OX-3, and GA20OX-4. All experiments have been normalized using β-actin as an internal reference; bars represent means ± SD (n = 3). Three independent experiments were performed. The asterisks represent signiﬁcant differences relative to WT plants (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < 0.001, respectively). Figure 5. Expression levels of MEP- MVA-, ABA-, IAA-, and GA-related and downstream genes in the transgenic and WT poplar leaves. (A) Expression levels of DXR, MCT, CMK, HDS, and HDR. (B) Expression levels of ACL, HMGS, HMGR, MVK, and MVD. (C) Transcript levels of IDI, GPS, GPPS, and GGPPS. (D) Expression levels of NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3. (E) Expression levels of IAA1, IAA2, IAA6, IAA18, and IAA19. (F) Expression levels of GA20OX-1, GA20OX-2, GA20OX-3, and GA20OX-4. All experiments have been normalized using β-actin as an internal reference; bars represent means ± SD (n = 3). Three independent experiments were performed. The asterisks represent significant differences relative to WT plants (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < 0.001, respectively). Figure 5. Expression levels of MEP- MVA-, ABA-, IAA-, and GA-related and downstream genes in the transgenic and WT poplar leaves. (A) Expression levels of DXR, MCT, CMK, HDS, and HDR. (B) Expression levels of ACL, HMGS, HMGR, MVK, and MVD. (C) Transcript levels of IDI, GPS, GPPS, and GGPPS. (D) Expression levels of NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3. (E) Expression levels of IAA1, IAA2, IAA6, IAA18, and IAA19. (F) Expression levels of GA20OX-1, GA20OX-2, GA20OX-3, and GA20OX-4. All experiments have been normalized using β-actin as an internal reference; bars represent means ± SD (n = 3). Three independent experiments were performed. The asterisks represent signiﬁcant differences relative to WT plants (Student’s t-test; , , and , p < 0.05, p < 0.01, and p < 0.001, respectively). 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS Overexpressing Plants 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS-Overexpressing Plants (D) Comparison of the ABA, GA3, and GA4 contents of transgenic and WT plants. Asterisks represent significant differences relative to WT poplars (Student’s t-test; , and , p < 0.01, and p < 0.001, respectively). Figure 6. ABA, GA3, and GA4 contents in the transgenic and WT plants by HPLC-MS/MS. (A) Raw data and ABA, GA3, and GA4 contents of WT plants. (B) Raw data and ABA, GA3, and GA4 contents of T1 plants. (C) Raw data and ABA, GA3, and GA4 contents of T3 plants. (D) Comparison of the ABA, GA3, and GA4 contents of transgenic and WT plants. Asterisks represent signiﬁcant differences relative to WT poplars (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). Figure 6. ABA, GA3, and GA4 contents in the transgenic and WT plants by HPLC-MS/MS. (A) Raw data and ABA, GA3, and GA4 contents of WT plants. (B) Raw data and ABA, GA3, and GA4 contents of T1 plants. (C) Raw data and ABA, GA3, and GA4 contents of T3 plants. (D) Comparison of the ABA, GA3, and GA4 contents of transgenic and WT plants. Asterisks represent significant differences relative to WT poplars (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). Figure 6. ABA, GA3, and GA4 contents in the transgenic and WT plants by HPLC-MS/MS. (A) Raw data and ABA, GA3, and GA4 contents of WT plants. (B) Raw data and ABA, GA3, and GA4 contents of T1 plants. (C) Raw data and ABA, GA3, and GA4 contents of T3 plants. (D) Comparison of the ABA, GA3, and GA4 contents of transgenic and WT plants. Asterisks represent signiﬁcant differences relative to WT poplars (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). Figure 6. ABA, GA3, and GA4 contents in the transgenic and WT plants by HPLC-MS/MS. (A) Raw data and ABA, GA3, and GA4 contents of WT plants. (B) Raw data and ABA, GA3, and GA4 contents of T1 plants. (C) Raw data and ABA, GA3, and GA4 contents of T3 plants. (D) Comparison of the ABA, GA3, and GA4 contents of transgenic and WT plants. Asterisks represent significant differences relative to WT poplars (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). Figure 6. ABA, GA3, and GA4 contents in the transgenic and WT plants by HPLC-MS/MS. 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS Overexpressing Plants 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS-Overexpressing Plants Micromelalopha troglodyta on WT and PtDXS Overexpressing Plants Most terpenoids have bacteriostatic and bactericidal effects and can enhance plant disease resistance. In addition, terpenoids can resist natural enemies. For example, terpenoids as food inhibitors and toxic substances have a direct effect on insect feeding, as well as a toxic influence on insects during feeding [31]. Septotis populiperda causes large-spot disease of poplar. Conidia appeared in WT plants 2 days after inoculation, compared to 4 days in the transgenic plants (Figure 7A). Moreover, the spread of pathogens was greater in the WT than in the transgenic plants (Figure 7B). Micromelalopha troglodyta larvae nibble on poplar leaves, which markedly reduces the quality of poplar. Feeding of Micromelalopha troglodyta was reduced on the leaves of the transgenic plants (Figure 8A,C). In addition, Micromelalopha troglodyta preferred leaves of the WT to those of the transgenic plants (Figure 8A,C). Finally, feeding by Micromelalopha troglodyta on WT poplar leaves t th th t t i l t (Fi 8D) Most terpenoids have bacteriostatic and bactericidal effects and can enhance plant disease resistance. In addition, terpenoids can resist natural enemies. For example, terpenoids as food inhibitors and toxic substances have a direct effect on insect feeding, as well as a toxic inﬂuence on insects during feeding [31]. Septotis populiperda causes large-spot disease of poplar. Conidia appeared in WT plants 2 days after inoculation, compared to 4 days in the transgenic plants (Figure 7A). Moreover, the spread of pathogens was greater in the WT than in the transgenic plants (Figure 7B). Micromelalopha troglodyta larvae nibble on poplar leaves, which markedly reduces the quality of poplar. Feeding of Micromelalopha troglodyta was reduced on the leaves of the transgenic plants (Figure 8A,C). In addition, Micromelalopha troglodyta preferred leaves of the WT to those of the transgenic plants (Figure 8A,C). Finally, feeding by Micromelalopha troglodyta on WT poplar leaves was greater than that on transgenic plants (Figure 8D). Int. J. Mol. Sci. 2019, 20, 1669 9 of 21 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 9 of 21 Figure 6. ABA, GA3, and GA4 contents in the transgenic and WT plants by HPLC-MS/MS. (A) Raw data and ABA, GA3, and GA4 contents of WT plants. (B) Raw data and ABA, GA3, and GA4 contents of T1 plants. (C) Raw data and ABA, GA3, and GA4 contents of T3 plants. 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS Overexpressing Plants 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS-Overexpressing Plants (A) Raw data and ABA, GA3, and GA4 contents of WT plants. (B) Raw data and ABA, GA3, and GA4 contents of T1 plants. (C) Raw data and ABA, GA3, and GA4 contents of T3 plants. (D) Comparison of the ABA, GA3, and GA4 contents of transgenic and WT plants. Asterisks represent signiﬁcant differences relative to WT poplars (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). 10 of 21 10 of 21 Int. J. Mol. Sci. 2019, 20, 1669 Int. J. Mol. Sci. 2019, 20, x FOR Figure 7. Necrotic symptoms of WT and transgenic plants infected with S. populiperda. (A) Leaves of WT and transgenic plants after inoculation. (B) Plaques on WT and transgenic plants. Three independent experiments were performed; three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and *, p < 0.01, Figure 7. Necrotic symptoms of WT and transgenic plants infected with S. populiperda. (A) Leaves of WT and transgenic plants after inoculation. (B) Plaques on WT and transgenic plants. Three independent experiments were performed; three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). Figure 7. Necrotic symptoms of WT and transgenic plants infected with S. populiperda. (A) Leaves of WT and transgenic plants after inoculation. (B) Plaques on WT and transgenic plants. Three independent experiments were performed; three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). Figure 7. Necrotic symptoms of WT and tran ansgenic plants infected with S. populiperda. (A) Leaves of c symp opulipe Figure 7. Necrotic symptoms of WT and transgenic plants infected with S. populiperda. (A) Leaves of WT and transgenic plants after inoculation. (B) Plaques on WT and transgenic plants. Three independent experiments were performed; three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and *, p < 0.01, Figure 7. Necrotic symptoms of WT and transgenic plants infected with S. populiperda. (A) Leaves of WT and transgenic plants after inoculation. (B) Plaques on WT and transgenic plants. 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS Overexpressing Plants 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS-Overexpressing Plants Three independent experiments were performed; three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). WT and transgenic plants after inoculation. (B) Plaques on WT and transgenic plants. Three independent experiments were performed; three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and *, p < 0.01, and p < 0.001, respectively). and p < 0.001, respectively). and p < 0.001, respectively). Figure 8. M. troglodyte feeding assay. (A) Leaves of WT and PtDXS-transgenic plants after initiating to be eaten by first instar larvae. (B) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by third instar larvae. (C) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by fifth instar larvae. (D) Quantitative data for leaves of WT and PtDXS-transgenic plants. Three independent experiments were performed; asterisks represent significant differences relative Figure 8. M. troglodyte feeding assay. (A) Leaves of WT and PtDXS-transgenic plants after initiating to be eaten by first instar larvae. (B) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by third instar larvae. (C) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by fifth instar larvae. (D) Quantitative data for leaves of WT and PtDXS-transgenic plants. Three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; , and p < 0.001, respectively). Figure 8. M. troglodyte feeding assay. (A) Leaves of WT and PtDXS-transgenic plants after initiating to be eaten by ﬁrst instar larvae. (B) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by third instar larvae. (C) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by ﬁfth instar larvae. (D) Quantitative data for leaves of WT and PtDXS-transgenic plants. Three independent experiments were performed; asterisks represent signiﬁcant differences relative to WT plants (Student’s t-test; , and p < 0.001, respectively). and p < 0.001, respectively). and Figure 8. M. troglodyte feeding assay. (A) Leaves of WT and PtDXS-transgenic plants after initiating to be eaten by first instar larvae. (B) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by third instar larvae. (C) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by fifth instar larvae. 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS Overexpressing Plants 2.10. Resistance to Septotis Populiperda Infection of PtDXS-Overexpressing Plants and Feeding of Micromelalopha troglodyta on WT and PtDXS-Overexpressing Plants (D) Quantitative data for leaves of WT and PtDXS-transgenic plants. Three independent experiments ere performed asterisks represent significant differences relati e Figure 8. M. troglodyte feeding assay. (A) Leaves of WT and PtDXS-transgenic plants after initiating to be eaten by first instar larvae. (B) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by third instar larvae. (C) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by fifth instar larvae. (D) Quantitative data for leaves of WT and PtDXS-transgenic plants. Three independent experiments were performed; asterisks represent significant differences relative to WT plants (Student’s t-test; * and p < 0 001 respectively) Figure 8. M. troglodyte feeding assay. (A) Leaves of WT and PtDXS-transgenic plants after initiating to be eaten by ﬁrst instar larvae. (B) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by third instar larvae. (C) Leaves of WT and PtDXS-transgenic plants after after initiating to be eaten by ﬁfth instar larvae. (D) Quantitative data for leaves of WT and PtDXS-transgenic plants. Three independent experiments were performed; asterisks represent signiﬁcant differences relative to WT plants (Student’s t-test; ***, and p < 0.001, respectively). to WT plan 3 Discussion 3. Discussion DXS expression is highest at the early stage of leaf development [37] in plants whose leaves are the major organs of terpenoid synthesis. The expression of DXSs in different types of the same species may be the same or different. The expression of DXS in Aquilaria sinensis (Lour.) Spreng was high in stems, leaves and roots [16]. DXS1 from Salvia miltiorrhiza was expressed in leaves, stems, and roots, and was highest in leaves and lowest in root. However, expression of DXS2 from S. miltiorrhiza was highest in root [38]. In this study, PtDXS was constitutively expressed in mature and young leaves, upper and lower regions of stems, petioles, and roots of poplar. PtDXS expression was highest in young leaves, followed by mature leaves, and was lowest in root; this is in agreement with a previous report [39]. Type-I DXSs are expressed in all tissues, except root, of M. truncatula, Lycopersicon esculentum, and Nicotiana tabacum [40], which is consistent with our results. PtDXS plays an important role in primary metabolism and growth and development. Hormones, light, circadian rhythm, developmental degree, and sucrose concentration regulate the expression of DXSgenes [41]. Treatment of Dendrobium ofﬁcinale with ABA, salicylic acid (SA), jasmonic acid (JA), and brassinosteroids resulted in increased DXS expression in the protocorm [35]. In Aquilaria sinensis, the expression of AsDXS1 is regulated by physical and chemical factors, but that of AsDXS2 and AsDXS3 is regulated only by physical and chemical factors. These three genes were regulated by methyl jasmonate, but at different times [16]. Treatment with 200 mM NaCl resulted in PtDXS expression 18-fold higher than that in untreated plants. Treatment with 200 µM ABA signiﬁcantly upregulated the expression of PtDXS from 3 to 48 h, with a peak at 12 h. Treatment with 2 mM H2O2 resulted in peak expression of PtDXS at 48 h, when it was 18-fold that of the control. The expression of PtDXS decreased during the ﬁrst 2 days of treatment with 10% PEG6000, and increased to a peak at 7 days. Thus, we speculate that PtDXS may facilitate development and improvement of poplar resistance to salt and drought stresses. Recombinant PtDXS was capable of catalyzing the formation of DXP from G-3-P and pyruvate. The color complementation method has been used to assess the function of DXS in Amomum villosum and Camptotheca acuminata [42]. As reported by Cunningham [43], E. to WT plan 3 Discussion 3. Discussion 3. Discussion The enzymes of the MEP pathway produce precursor compounds DXS is the rate limiting 3. Discussion The enzymes of the MEP pathway produce precursor compounds. DXS is the rate-limiting enzyme in the MEP pathway We characterized and evaluated the expression of DXS from The enzymes of the MEP pathway produce precursor compounds. DXS is the rate-limiting enzyme in the MEP pathway. We characterized and evaluated the expression of DXS from P. 3. Discussion The enzymes of the MEP pathway produce precursor compounds. DXS is the rate-limiting 3. Discussion The enzymes of the MEP pathway produce precursor compounds. DXS is the rate-limiting enzyme in the MEP pathway. We characterized and evaluated the expression of DXS from P t i h PtDXS d d t i ith TPP bi di d DRAG d i Th i id The enzymes of the MEP pathway produce precursor compounds. DXS is the rate-limiting enzyme in the MEP pathway. We characterized and evaluated the expression of DXS from P. 11 of 21 Int. J. Mol. Sci. 2019, 20, 1669 trichocarpa. PtDXS encoded a protein with TPP-binding and DRAG domains. The amino acid sequence of PtDXS (XP_024463484) showed a high level of similarity with those of Alpinia ofﬁcinarum (AEK69518.1), Hevea brasiliensis (ABD92702.1), Ricinus communis (XP_015573388.1), and Theobroma cacao (XP_017975597.1). Type-I and -II DXSs may play an important role in plant primary and secondary metabolism, respectively, and type-III DXSs are commonly found in plant genomes, but the functions of their encoded enzymes are unknown [32]. PtDXS is a type-I DXS and plays an important role in basic life processes. p DXS expression is related to ﬂower and fruit development, and circadian rhythm. In addition, the expression patterns of DXS genes in different families (such as Type-I, -II, and -III DXSs) may vary in the same species. The expression level of DXS in rose petals increased from the bud to blooming stages. Subsequently, the expression level of DXS decreased as ﬂowers decayed. Thus, DXS is related to ﬂower development [33]. The circadian clock regulates the expression level of DXS in jasmine petal. Tomato SlDXS2 is highly expressed in ﬂower [34] and DXS from Dendrobium candidum in the protocorm [35]. In addition, the expression level of DXS differs according to the stage of fruit development [36]. to WT plan 3 Discussion 3. Discussion coli strains lacking genes related to carotenoid synthesis do not produce lycopenes. The result of a colorimetric assay indicated that PtDXS encodes a functional protein, which increased the accumulation of β-carotene via the MEP pathway. Therefore, PtDXS could help to demonstrate an increasing metabolic ﬂux to the synthesis of isoprene compounds according to the color complementation. Genes may be co-expressed with other upstream or downstream genes in their metabolic pathways. Upregulation of the expression of potato DXS in A. thaliana increased the expression of downstream GGPPS genes, increasing the carotenoid content. In addition, upregulation of phytoene synthase (PSY) expression increased the β-carotene content of transgenic plants [44]. In this study, the expression levels of the MEP-related genes DXR, HDS, HDR, MCT, and CMK as well as downstream genes were increased signiﬁcantly in the transgenic plants. By contrast, the expression levels of Int. J. Mol. Sci. 2019, 20, 1669 12 of 21 the MVA-related genes HMGS, HMGR, and MVD were decreased. These data suggest that the MEP pathway may play a leading role in terpenoid biosynthesis, while the MVA pathway plays an auxiliary role. y DXS is related to terpenoid synthesis and regulates the generation of downstream products. As the ﬁrst key enzyme in terpenoid biosynthesis, overexpression or inhibition of DXS can cause changes in downstream metabolites. The carotenoid, chlorophyll, and ABA contents of Arabidopsis harboring exogenous DXS increased [44,45]. However, the ABA and GA contents of Arabidopsis decreased following transfer of DXS1 from Solanum tuberosum [44]. The expression of DXS from Nicotiana tabacum was positively related to the carotenoid content of fruit, and silencing of SIDXS2 reduced the β-carotene content [34]. In this study, the transgenic plants had signiﬁcantly greater ABA and GA contents than the WT. Thus, the GA and ABA contents are correlated with the expression level of DXS, and thus DXS plays an important role in their biosynthesis. Infection of the root of wheat, maize, and rice by mycorrhizal fungi alters the expression levels of DXSs [46]. Mycorrhizal fungi infect the roots of Medicago truncatula, which increases the concentration of MtDXS2 [23]. Potato late blight is related to the expression level of DXS, and a decrease in DXS expression results in decreased levels of terpenoids, which are related to disease resistance [44]. In this study, the transgenic plants showed enhanced resistance to S. populiperda infection as well as reduced feeding by M. troglodyta. to WT plan 3 Discussion 3. Discussion We cloned full-length PtDXS, which is related to isoprenoid biosynthesis, from P. trichocarpa. We also assessed the expression pattern of PtDXS in various tissues and under abiotic stresses and analyzed the expression levels of MEP- and MVA-related genes in the transgenic and WT plants. Overexpression of PtDXS increased the ABA and GA contents, and PtDXS expression was positively related to resistance to S. populiperda and negatively related to feeding by M. troglodyta. Our ﬁndings will facilitate further studies of the functions of PtDXS gene in P. trichocarpa. 4.1. Plant Materials and Treatments P. trichocarpa was sterilized and grown in Murashige and Skoog (MS) medium at 23 ◦C for 2 months. P. trichocarpa was also grown in MS medium supplemented with 200 mM NaCl, 200 µM ABA, or 2 mM H2O2. Leaves were collected after 1, 3, 6, 8, 12, 24, and 48 h. As drought stress, P. trichocarpa was treated with 10% polyethylene glycol (PEG) 6000 and leaves were collected after 1, 2, 3, 4, 5, 6, and 7 days. Total RNA was extracted from the young and mature leaves, upper and lower regions of stems, roots, and petioles using an RNAprep Pure Plant Kit (Biomiga Company, San Diego, CA, USA) according to the manufacturer’s instructions. The MMLV reverse transcriptase (TaKaRa, Japan) was used to synthesize cDNA according to the manufacturer’s instructions. 4.2. Cloning of Full-Length PtDXS and Rapid Ampliﬁcation of cDNA Ends PtDXS was ampliﬁed by PCR using speciﬁc primers (Table 1); the PCR system included 2 µL forward and reverse primers, 2.0 µL cDNA as template, 5.0 µL 10× PCR buffer (Mg2+ plus), 1 µL 10 mM dNTPs, 0.5 µL rTaq DNA polymerase (Takara, Japan) and the ddH2O was to a constant volume up to 50 µL. Also, the PCR reaction was performed as follows: 95 ◦C for 10 min, 35 cycles of 95 ◦C for 1.5 min, 58 ◦C for 1.5 min, and 72 ◦C for 2 min and, ﬁnally, 72 ◦C for 10 min. In addition, the PCR product was puriﬁed according to the manufacturer’s instructions (AXYGEN, Suzhou, China), and the puriﬁed product was cloned into the PEASY-T3 vector (TransGen Biotech, Beijing, China) based on the complementary cohesive end. The vector was transformed into Escherichia coli TransTI. Positive clones were selected by blue and white spot induced by X-Gal and IPTG and the reconstructed plasmids were sequenced by GenScript Company (Nanjing, China). 13 of 21 Int. J. Mol. Sci. 2019, 20, 1669 Table 1. The primers used in this study. Primer Direction Nucleotide Sequence(5′–3′) Primer Direction Nucleotide Sequence(5′–3′) PtDXS Forward ATGGCTCTCTCTGCATTTTCT q-IDI Forward CGGGTTGTTGGTCATGACTC PtDXS Reverse TTATGATGACATAATCTCCAGAGAGTTT q-IDI Reverse CTCTGTGCAGCATTCCTCAC 3′GSP-1-DXS Forward TTCCTGATAGGTACATTGACCATG q-GPS Forward AAGCTCACTCTGATGGGGAC 3′Outer Reverse TACCGTCGTTCCACTAGTGATTT q-GPS Reverse TGATAGCCTCCTTGTGTGGG 3′GSP-2-DXS Forward CTGGTCTCACACCATCTCAC q-GPPS Forward TGAAACGGAAACTGGCAGTG 3′Inter Reverse ACACCAATTTTGCATTCTTACAAC q-GPPS Reverse TACTGCCACCTTATTGCCCA UPM Forward CTAATACGACTCACTATAGGGCAAG CAAGCAGTGGTATCAAACGCAGAGT(long) q-GGPPS Forward CTGACCTTGTACCAGAGCCA CTAATACGACTCACTATAGGGC(short) q-GGPPS Reverse GAGCACTCACCCATTTCACC 5′GSP-1 Reverse CTGATTGATCTTTTTAAAAGATTGGC q-NCED1 Forward TAGAAACGGAGCCAACCCAT NUP Forward AAGCAGTGGTATCAACGCAGAGT q-NCED1 Reverse GCTATGCCCGAATGACCATG 5′GSP-2 Reverse GAGAGAGACAGAGACAGAGAGGTCT q-NCED3 Forward GGCTCCTCGTCATTTTCGTC q-PtDXS Forward GAATGCAAAGCCGTTGAAGC q-NCED3 Reverse GGCGTGTTTTGTTTTGGGTG q-PtDXS Reverse GTTTCAATTTTATCAGTGCCAAAA q-NCED5 Forward TCCACCGGACTCCATTTTCA q-Actin Forward GCCATCTCTCATCGGAATGGAA q-NCED5 Reverse TGTCTGTCTTCCAAGCCGAT q-Actin Reverse AGGGCAGTGATTTCCTTGCTCA q-NCED6 Forward GAGACTGACGAGGTGACCAA PET-PtDXS Forward CGGGATCCATGGCTCTCTCTGCATTT q-NCED6 Reverse CACCAATTCTGACCTCCCCT PET-PtDXS Reverse CCGCTCGAGTTATGATGACATAATCT q-ZEP1 Forward ACAGTCTCTTTCCCATGCCA pTrc-PtDXS Forward GAATGGCTCTCTCTGCATTT q-ZEP1 Reverse CCAGAAGCATGTACAGCACC pTrc-PtDXS Reverse AAGGAAAAAATTATGATGACATAATCT q-ZEP2 Forward TGACCTTGCTTGGGGATTCT pGWB9-PtDXS Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCTCTCTCTGCATTT q-ZEP2 Reverse TTGCTGCCATTCTTGCCATT pGWB9-PtDXS Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTTTATGATGACATAATCT q-ZEP3 Forward GGGATGAGAAGAGGAGGCTC q-DXR Forward TTGAAAAGGGTAGCAGAGTC q-ZEP3 Reverse TAACACAGCCAGCGTCCATA q-DXR Reverse TTGTTCTCCCTCTTGCTCAC q-GA20OX-1 Forward AGATCCTTTGGCGGTCTCAA q-HDS Forward TCTGTTGCGTTGCGAGTATC q-GA20OX-1 Reverse TCCGAGAGCTGCTTACCAAA q-HDS Reverse AAGACTGCCATGGTTTGTCC q-GA20OX-2 Forward TAACCACCCCTCATCACCAC q-HDR Forward TAACACCTCCCACCTCCAAG q-GA20OX-2 Reverse AGTGGGACTTGGAGTTCAGG q-HDR Reverse TAAGGGCATCTTCGACAACC q-GA20OX-3 Forward TACCCTCACTCTTGGCACTG q-MCT Forward ACTGCCAGGAAAGGAGAGAC q-GA20OX-3 Reverse CCCTGTTCACCACTGCTCTA q-MCT Reverse ACCAAGTACAGCAGCTCCAT q-GA20OX-4 Forward TTCTCCTGTCTCCTCCTCCA q-CMK Forward TTCTCATAAAGCCCCCACAG q-GA20OX-4 Reverse GCAGTCCTAACAAGCTCAGC q-CMK Reverse AGCAGGGGGCTCTAAATCAT q-IAA1 Forward CTCCTTCGAACTCCCAACCT 14 of 21 Int. J. Mol. Sci. 2019, 20, 1669 Table 1. Cont. 4.3. Construction of the Expression Vector According to the PtDXS sequence and the restriction enzyme sites in the PET-28a vector, forward and reverse primers were designed and synthesized (Table 1). The target gene was ampliﬁed by PCR, and the PCR product and vector were cut with BamHI and XhoI. Subsequently, the fusion plasmid was generated using the T4 ligation technique. Subsequently, the recombinant plasmid PET-28a-PtDXS was transformed into E. coli Top10, we can achieve the positive clones according to screening of solid Luria-Bertani broth (LB) medium containing 50 µg/mL kanamycin at 37 ◦C for 12 h. Moreover, the recombinant plasmid PET-28a-PtDXS was extracted by the plasmid extraction kit (AXYGEN) based on the manufacturer’s instructions. 4.2. Cloning of Full-Length PtDXS and Rapid Ampliﬁcation of cDNA Ends Primer Direction Nucleotide Sequence(5′–3′) Primer Direction Nucleotide Sequence(5′–3′) q-ACL Forward CCTCCACAAATCCCTGAAGA q-IAA1 Reverse AATTCGGCGAAACACTGGTC q-ACL Reverse CCCACACCATAACCCTGTTC q-IAA2 Forward GCTGAATTGGACCGGTTGTT q-HMGS Forward GTCTGCAATAGCTGGGAAGC q-IAA2 Reverse CACGCCCGATGGATTTTCAT q-HMGS Reverse GTGTTCATCGGTAGGCGTTT q-IAA6 Forward GTGAACAGGTTGCTGCTTCA q-HMGR Forward CTGGGCCATTGTTGCTTAAT q-IAA6 Reverse CGTCCTGACTTAATGGCTGC q-HMGR Reverse TCAACTCAGCAGCCCTTTTT q-IAA18 Forward TGTAGGTTGCCGGTTGTTTG q-MVD Forward ATGGGTGAGGATGGTGACTG q-IAA18 Reverse GCTTTGTGACTCCTTTGCCA q-MVD Reverse ATTGAGCCACATCCGATCCT q-IAA19 Forward AAGACATTCCCGCCTCTTGA q-MVK Forward GCAAACCCTATGGGGAAAAT q-IAA19 Reverse TTAGCCCTTCTGATGCCCAA q-MVK Reverse TGCATCAAAACATGGAAGGA Int. J. Mol. Sci. 2019, 20, 1669 15 of 21 Rapid ampliﬁcation of cDNA ends (RACE) was used to amplify the 3′ and 5′ untranslated regions of PtDXS. Next, the 5′ and 3′ fragments were sequenced using speciﬁc primers (Table 1). The 5′- and 3′-RACE ampliﬁed fragments were inserted into the PEASY-T3 vector and sequenced. The full-length sequence of PtDXS was obtained by aligning the obtained sequences. 4.4. Production and Puriﬁcation of the Target Protein We used electroporation to transform the recombinant plasmid PET-28a-PtDXS (with no mutation) into E. coli BL21 (DE3) cells. Next, recombinant E. coli BL21 (DE3) was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 220 rpm for 4 h at 37 ◦C. Non-induced culture medium, induced culture medium, supernatant, and sediment were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). p y y g p The target protein was present in inclusion bodies according to analysis of the supernatant and sediment. Inclusion body denaturation and renaturation were performed as follows: The precipitate was suspended in 20 mL of lysis buffer (20 mM Tris-HCl containing 1 mM phenylmethylsulfonyl ﬂuoride and protease inhibitor cocktail; pH 8.0), sonicated (400 W for 4 s, followed by at 8 s intervals for 20 min), centrifuged at 10,000 r/min for 20 min, and the precipitate collected. The inclusion bodies were washed three times with inclusion body detergent (20 mM Tris, 1 mM ethylenediaminetetraacetic acid, 2 M urea, 1 M NaCl, 1% Triton X-100; pH 8.0). The inclusion bodies were dissolved in solution buffer (20 mM Tris, 5 mM dithiothreitol, 8 M urea; pH 8.0), placed overnight at 4 ◦C, and centrifuged at room temperature for 15 min at 10,000 r/min. The resulting solution was dropped into buffer (20 mM Tris-HCl, 0.15 M NaCl; pH 8.0), and stirred slowly, and transferred to dialysis bags for dialysis overnight. Using a low-pressure chromatography system, the solution was loaded at a ﬂow rate of 0.5 mL/min onto a Ni-IDA-Sepharose CL-6B afﬁnity chromatography column. The column was rinsed with binding buffer (20 mM Tris-HCl, 10 mM imidazole, 0.15 M NaCl; pH 8.0) and washing buffer (20 mM Tris-HCl, 50 mM imidazole, 0.15 M NaCl; pH 8.0), and elution buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) was used to elute the target protein at a ﬂow rate of 1 mL/min. The collected solution was transferred to a dialysis bag and dialyzed overnight against 20 mM Tris-HCl, 0.15 M NaCl (pH 8.0). Puriﬁcation was veriﬁed by 12% SDS-PAGE and western blotting for the 6×His tag. 4.7. Determination of PtDXS Expression Movahedi et al. [47] proved that βactin (accession number: XM-006370951.1) could be stably expressed in leaves, stems, and roots. Thus, we decided to use βactin as an internal reference gene to normalize qPCR for assessing PtDXS expression. In addition, we performed RT-PCR to exhibit whether the βactin gene could be stably expressed in all tissues and experimental conditions (Figure S9). The PCR reaction was carried out following 95 ◦C for 7min, (95 ◦C for 30 s, 58 ◦C for 30 s, and 72 ◦C for 30 s)×26, and ﬁnally, 72 ◦C for 10 min. The expression level of PtDXS was measured by real-time quantitative polymerase chain reaction (qPCR) using total RNA from mature and young leaves, upper and lower regions of stems, petioles, and roots. The primers q-Actin-F and q-Actin-R were used to amplify a 152 bp fragment of P. trichocarpa actin (Table 1). The StepOnePlus™Real-Time PCR System (Applied Biosystems, ThermoFisher Company, Waltham, MA, USA) and SYBR Green Master reagents (Roche, F. Hoffmann-La Roche AG Company, Basel, Switzerland) were chosen to perform the quantitative PCR analysis. The real-time quantitative polymerase chain reaction system contained 1 µL each primer, 2 µL cDNA, 10 µL SYBR Green mixture, and 6 µL ddH2O. The real-time quantitative polymerase chain reaction was performed as follows: 95 ◦C for 5 min, 40 cycles at 95 ◦C for 15 s and 60 ◦C for 60 s. Triplicate measurements were performed for each parameter, and each data point represents a mean value. P. trichocarpa seedlings were subjected to the following stress treatments: 200 mM NaCl, 200 µM ABA, and 2 mM H2O2 for 0, 1, 3, 6, 8, 12, 24, or 48 h, and 10% PEG6000 for 0, 1, 2, 3, 4, 5, 6, or 7 days. The leaves were collected, and RNA was extracted using a RNeasy Plant Mini Kit (Biomiga Company, San Diego, CA, USA). Subsequently, real-time quantitative polymerase chain reaction (qPCR) was chosen to identify the transcription level of PtDXS gene, the PtActin gene was used as internal reference. The StepOnePlus™Real-Time PCR System (Applied Biosystems) and SYBR Green Master reagents (Roche) were chosen to perform the quantitative PCR analysis. The real-time quantitative polymerase chain reaction was performed as above description. The treatments were repeated three times for analyses of PtDXS expression in response to abiotic stress treatment. 4.6. Functional Analysis of PtDXS in Escherichia coli The plasmids pAC-BETA and pTrc-AtIPI provided by Francis X. Cunningham Lab. (Addgene Company, Watertown, MA, USA) were used to investigate the biological function of PtDXS. GGPP synthase (crtE), octahydrolycopene synthase (crtB), and octahydrolycopene desaturase (crtI), all of which are necessary for the synthesis of β-carotene, are present in pAC-BETA. Based on the PtDXS sequence and the restriction enzyme sites in the pTrc-AtIPI vector, forward and reverse primers were designed and synthesized (Table 1). The PtDXS gene was ampliﬁed by PCR, and PtDXS was cloned into pTrc-AtIPI by digestion with BglII and NotI and T4 ligation. The vectors pTrc-PtDXS and pAC-BETA were co-transformed into E. coli DH5α, and the co-transformants were screened on solid LB mediums containing 100 µg/mL ampicillin and 50 µg/mL chloramphenicol at 37 ◦C for 48 h. The two control groups were E. coli DH5α containing pTrc and pAC-BETA and E. coli DH5α containing pTrc. 4.5. Detection of 1-Deoxy-D-Xylulose-5-Phosphate In Vitro PtDXS activity was assayed by detecting DXP in vitro. One milliliter of a reaction mixture containing 110 mM fructose-1,6-diphosphate (pH 7.5), 10 mM MgCl2, 120 mM Tris-HCl (pH 7.5), 60 U aldolase, 60 U triose phosphate isomerase, and 5 mM β-mercaptoethanol was heated in a water bath at 25 ◦C for 1 h. Next, 110 mM sodium pyruvate, 2 mM thiamin pyrophosphate (TPP), and 100 µg of DXS were added to the reaction mixture. The solution was heated in a water bath at 37 ◦C for 16 h, and concentrated using a 10 kDa filter at 11,000 rpm for 2 min. The solution was finally freeze-dried to powder. E. coli BL21 (DE3) carrying an empty vector was included as the negative control. In addition, quantitative analysis of PtDXS reaction product was performed by high-performance liquid chromatography (HPLC)/mass spectrometry (MS) using the Agilent poroshell 120 SB-C18 reversed-phase column. The column temperature was set Int. J. Mol. Sci. 2019, 20, 1669 16 of 21 to 30 ◦C. The mobile phase contained 80% methanol and 20% water (0.1% formic acid) and was eluted using a gradient of 200 µL/min. The MS conditions were as follows: spray voltage of 4000 V (+)/3500 V (−), air curtain of 15 psi, atomizing gas pressure of 45 psi, auxiliary pressure of 60 psi, and atomization temperature of 340 ◦C. 4.8. Overexpression Plasmid Construction and Transformation The open reading frame (ORF) of PtDXS was cloned into pGWB9 using gateway technology. First, the ORF of PtDXS was cloned into the pDONRTM/Zeo entry vector using the BP clonase according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The method of electroporation was used to transform the recombinant plasmid pDONRTM/Zeo-PtDXS into E. coli Top10 cells and the positive clones were selected by solid LB medium containing 30 µg/mL zeocin. Int. J. Mol. Sci. 2019, 20, 1669 17 of 21 Second, using the LR clonase (Invitrogen), recombinant pDONRTM/Zeo-PtDXS was recombined with the pGWB9 vector, and the electroporation was used to transform the recombinant plasmid pGWB9-PtDXS into E. coli DH5α cells and the positive clones were selected by solid LB medium containing 50 µg/mL Kanamycin. pGWB9-PtDXS was introduced into Agrobacterium EHA105, which was used to infect poplar leaves. Transgenic operations are carried out in the following steps: Resistant shoots were obtained on selective differentiation medium containing 200 mg/L cefotaxime and 50 mg/L kanamycin. Next, putative resistant shoots were cultured on selective shooting MS medium containing 200 mg/L cefotaxime and 25 mg/L kanamycin. Subsequently, the putative resistant shoots were cultured on half-strength MS medium containing 200 mg/L cefotaxime and 15 mg/L kanamycin. Finally, the plants were transferred to the greenhouse. g To detect whether the PtDXS gene was inserted into the poplar genome, PCR reaction was performed using 35S primer and PtDXS-R primer as the upstream primer and the downstream primer as well as the genomic DNA of wild-type and potential transgenic lines as templates, respectively. Also, the PtDXS-F primer and PtDXS-R primer as well as the genomic DNA of wild-type and potential transgenic lines as templates were used to carry out PCR reaction. The kanamycin gene was indetiﬁed by PCR in twelve transgenic lines and the wild-type poplars. The copy number of transgenic lines was investigated by Southern blotting. The CTAB method was used to achieve 10 µg of genomic DNA, then the genomic DNA was digested with EcoRI at 37 ◦C for 4 h. Subsequently, the digested genomic DNA was separated on a 0.8% agarose gel at 15 V, and was transferred to a Hybond N+nylon membrane. Finally, based on the manufacturer’s instructions, blotting was performed (Roche, Basel, Switzerland). We perform the PCR to synthesize a digoxygenin (dig)-labeled Kan-tagged cDNA fragment (665 bp), and choose Kan-tagged cDNA fragment as a probe for Southern blotting. 4.8. Overexpression Plasmid Construction and Transformation In addition, in order to identify whether the PtDXS gene was expressed in the poplar genome, twelve transgenic lines were selected for real-time quantitative polymerase chain reaction (qPCR) analysis. Moreover, to identify whether the PtDXS gene was translated in poplar, total proteins from both WT and transgenic poplars were extracted and analyzed by 12% SDS-PAGE. Furthermore, the PtDXS gene linked to the PGWB9 vector by homologous recombination will be expressed in fusion with the 6×His tag located on the PGWB9 vector. So, western blotting was used to determine whether PtDXS could be speciﬁcally recognized by rabbit antiserum against His-DXS expressed in transgenic poplars. g p p After 1 month of subculture, shoot tips (3 cm high) were cut from wild-type and transgenic poplars and placed on medium containing 3% (w/v) PEG6000 or 100 mM NaCl under a 16 h light/8 h dark cycle at 23 ◦C. The survival rate was investigated after 1 week. 4.9. Expression Levels of MVA-, MEP-, ABA-, GA-, and IAA-Related Genes 4.9. Expression Levels of MVA-, MEP-, ABA-, GA-, and IAA-Related Genes Total RNA was isolated from the leaves of WT and transgenic poplars and the expression levels of MVA-, MEP-, ABA-, and GA-related genes were assessed by real-time quantitative polymerase chain reaction (qPCR) under the conditions. Furthermore, the expression levels of IAA-related genes in the transgenic and WT plants were determined. We choose PtActin as the reference gene, and the primers of MVA-, MEP-, ABA-, GA-, and IAA-related genes were shown in Table 1. The StepOnePlus™ Real-Time PCR System (Applied Biosystems) and SYBR Green Master reagents (Roche) were chosen to perform the quantitative PCR analysis. The real-time quantitative polymerase chain reaction was performed as above description. Triplicate measurements were performed for each parameter, and each data point represents a mean value. 4.10. Determination of ABA, GA3, and GA4 Contents We used the AB Qtrap6500 mass spectrometer and Agilent 1290 high-performance liquid chromatograph with electrospray ionization (ESI-HPLC) to determine the ABA, GA3, and GA4 contents of the leaves of transgenic and WT plants. Hormones were extracted according to the Int. J. Mol. Sci. 2019, 20, 1669 18 of 21 manufacturer’s instructions (Jiancheng Biotechnique, Nanjing, China). Subsequently, we used nitrogen to dry the collected hormone solutions and use 400 µL methanol containing 0.1% formic acid to dissolved samples processed in the previous step. Finally, the collected solution was ﬁltered through a 0.22-µm membrane and detected using HPLC-MS/MS. We used methanol containing 0.1% formic acid as the solvent to prepare standard solutions, and the concentrations of ABA, GA3, and GA4 were 0.1, 0.2, 0.5, 2, 5, 20, 50, and 200 ng/mL, respectively. The liquid phase conditions were as follows: a Poroshell 120 SB-C18 column (2.1 × 150, 2.7 m) was used in this study at a column temperature of 30 ◦C. The mobile phase included A:B = (methanol/0.1% formic acid):(water/0.1% formic acid). Elution gradient: 0–1 min, A = 20%; 1–9 min, A = 80%; 9–10 min, A = 80%; 10–10.1 min, A = 20%; 10.1–15 min, A = 20%. The injection volume was 2 µL. MS conditions were as follows: air curtain gas, 15 psi; spray voltage, 4500 v; atomization pressure, 65 psi; auxiliary pressure, 70 psi; atomization temperature, 400 ◦C. Supplementary Materials: Supplementarymaterialscanbefoundathttp://www.mdpi.com/1422-0067/20/7/166 Supplementary Materials: Supplementarymaterialscanbefoundathttp://www.mdpi.com/1422-0067/20/7/1669/s1. Author Contributions: H.W. and A.M. have designed, carried out and written the manuscript. H.W., A.M., C.X., W.S., A.A.Z.Y., P.W. and D.L. have modiﬁed the manuscript. A.M. and Q.Z. have supervised this project. Q.Z. has funded this project. Funding: This work was supported by the National Key Program on Transgenic Research (2018ZX08020002), the National Science Foundation of China (no. 31570650) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. Acknowledgments: We thank Tsuyoshi Nakagawa (Research Institute of Molecular Genetics Shimane University) for providing the pGWB9 plasmid. We gratefully thank Lihua Zhu (Nanjing Forestry University) for providing Septotis populiperda. We also thank Kees Venema (EEZ, CSIC, and Granada, Spain) for suggestion on 3D structure analysis. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.11. Disease-Response and Antifeedant Assays S. populiperda was grown at 23 ◦C on potato dextrose agar (PDA) for 1 week [48]. The same positions of 2-months-old transgenic and WT poplar leaves, grown in a greenhouse, were punctured using a 5 mL syringe needle and inoculated with a small amount of PDA containing the pathogen or the same amount of PDA only (negative control). Infected leaves were counted, and soft-rot symptoms were evaluated periodically. Each experiment was repeated at least three times. For the M. troglodyta feeding assay, we dipped the eggs of M. troglodyta in a brush and attached them to the back of the clean poplar leaves, then placed them in ﬂask followed by incubation for cultivation. 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https://openalex.org/W1972538112	https://zenodo.org/records/1517506/files/article.pdf	German	null	Zur Kasuistik der akuten tuberkulösen Peritonitis	Lung	1,914	public-domain	2,282	1) Die Tuberkulose. II. 1907. S. 1204. 3) Spez. Path. u. Ther. 1896. Bd. 17. I. 2. S. 765. 3) Spez. path. Anat. 1904. S. 494. ~) Lehrb. d. Chir. yon W~llstein u. Wilms. Bd. 2. 1910. S. 15. Aus der Chirurgischen Klinik I des K. Seraphimerlazaretts in Stockholm. (Prof. Dr. J. Berg.) Aus der Chirurgischen Klinik I des K. Seraphimerlazaretts in Stockholm. (Prof. Dr. J. Berg.) Aus der Chirurgischen Klinik I des K. Seraphimerlazaretts in Stockholm. (Prof. Dr. J. Berg.) ~) Lehrb. d. Chir. yon W~llstein u. Wilms. Bd. 2. 1910. S. 15. Zur Kasuistik der akuten tuberkul6sen Peritonitis. Von Dr. Abraham Troell, Privatdozent f'dr Chirurgie. Die Darstellung, die unsere Lehr- und Handbiicher yon den verschiedenen anatomischen Formen der Peritonealtuberkulose bringen, kann in Kiirze am besten dureh folgendes aus der grossen Tuberkulosemonographie C o r n e t s 1) entnommene Zitat wieder- gegeben werden : ,,Ausser der Ausstreuung yon Miliarkn6tehen ohne wesentliche entztindliche Erscheinungen bei akuter allgemeiner Miliartuberkulose (akute Tuberkulose des Peritoneums) tritt uns die tuberkulSse Peri- tonitis besonders in zwei Formen, als troekene adh~isive oder als exsudative Form entgegen, daneben zahlreich,e Dbergangs- und Misch- formen. Bei der t r o e k e n e n Form linden wir zahlreiche Darm- schlingen locker verklebt,... Am Perit.oneum... miliare... Knoten. Bei der e x s u d a ti v e n Form trcffen wir einen ser6sen, sulzig-fibr6sen, k~ig-eiterigen oder auch l~m.orrhagischen Erguss in weehselnder Menge .... Das Exsudat kann frei beweglich sein, Niufiger ist es durch . . . Abkapselung . . . fixiert .... " Ungef~hr dieselbe Besehreibung linden wir bei N o t h n a g e 1 ~), Kaufmann3), Poppert4) u. a. m. In dieselbe l~isst sich sehr wohl die Einteilung in drei Formen einfiigen, die unter klinischem Gesichtspunkt praktischer ist, n~imlieh 1. eine exsudative, ser6se, Abraham Troell. [2 136 2. eine adhiisive, fibrinSse und 3. eine ulzerSse, eiterige (abgekapselte, k~isig-eiterige, zuweilen recht grosse Exsudate)l). 2. eine adhiisive, fibrinSse und 3. eine ulzerSse, eiterige (abgekapselte, k~isig-eiterige, zuweilen recht grosse Exsudate)l). g g g Was die exsudative diffuse tuberkulOse Peritonitis anbelangt, so dfirfte die landl~iufige Erfahrung am meisten darauf hinaus- gehen, dasses sich der Regel nach um ein kl~res oder nahezu Mares Exsudat in reichlicher Menge ohne gleichzeitige Verwach- sungen, aber mit zahlreichen disseminierten mili~ren Tub,erkeln handelt (K.a. ufm~nn, Hiirtel u. a. m.). Na.ch Ziegler 2) deutet eine eiterige Beschaffenheit des Exsudats auf das Vorhanden- sein einer Mischinfektion. Mit Riicksicht auf diese Umst~nde glaubte ich, dass eine Mit- teilung der folgenden zwei Krankengeschichten yon gewissem Inter- esse sein wtirde, zumal sie mir ffir sowohl die Differentialdi~gnoso als die J~tiologie der Peritoniten belehrend zu sein scheinen. Fall 1. A. V., Konditorlehrling, 18 Jahre. Nr. 85, 1913. Aufgen. am 20. I., entlassen, gebessert, am 29. I, D i a g n o s e: Peritonitis tbc. exsudativa. Fall 1. A. V., Konditorlehrling, 18 Jahre. Nr. 85, 1913. Aufgen. am 20. I., entlassen, gebessert, am 29. I, D i a g n o s e: Peritonitis tbc. exsudativa. 1) K6rte (Handb. d. prakt. Chir. yon Bergmann, Bruns u. Micu- l icz. Bd. 3. 1903. S. 87), H~irtel (Ergebn. d. Chit. n. Orthop. yon Payr u. Kiittner. Bd. 6. 1913. S. 379) u. a. m 2) Spez. path. Anat. 1902. S. 600. 2) Spez. path. Anat. 1902. S. 600. Zur Kasuistik der akuten tuberkul6sen Peritonitis. Frtiher immer gesund (abgesehen yon in tier Kindheit durchgemachten Masern), hat niemals Beschwerden yon seiten der Verdauungsorgmm gehabt. Der Pat. erkrankte nun am Tage der Aufnahme friih 4 Uhr mit Kneipen im Epi- gastrium, welches Kneipen sich nach und nach nach der rechten Seite hiniiber- zog ~nd lokalisierte. Kopfschmerzen und mehrmals im Laufe des Tages sich wiederholende SchiittelfrOste, gefolgt yon Schweissen. Die Schmerzen in der rechten Seite nahmen zu. Kein Stuhl oder Flatus. Keine Neigung zum Erbrechen oder Erbrechen; auch nicht Harnbeschwerden. Nachmittags wurde ein Arzt konsultiert, der den Pat. in das Lazarett befSrdern liess. S t at. pr. bei der Aufnahme nachmittags 7 Uhr den 20. I.: Das Allge- meinbefinden ist etwas beeinflusst. Gelindes-Kopfweh. MRssige Schmerzen in der rechten Seite. Temp. 37,70; Puls 104, klein, Herz und Lungen o. B. p , ; , , g Der Bauch ist weich, zeigt keine Auftreibung oder Schmerzhaftigkeit, aus- genommen auf einem kleinen Gebiet medial yon Mc. Burneys Punkt, wo Pat. deutlich durckempfindlich ist. Eine Resistenz l~tsst sich nicht nachweisen. Es ist keine D~impfung vorhanden, auch keine deutliche Ddfense nmsculaire. Anf die Wahrscheinlichkeitsdiagnose Appendizitis hin wurde unmittelbar Operation vorgenommen (Troell): Der Bauch wurde mit einem Mc. Burneys Schnitt geOffnet. Beim Einschneiden in alas Peritoneum drang tells eine reichliche Menge freien, sehmigen, diinnen, graugelben Eiters, tells Oment yon sukkulenlem, gelatin6sem Aussehen bervor. Hier und da an. der Darmser.~sa fanden sich stecknadelspitzengrosse, gelatin5se KnStchen; dazu war die Serosa fleckweise mehr oder weniger gerStet. Die D~trme iiberall frei und ohne Ver- wachsungen. Der Appendix, der medial lag, war gleichfalls gerOtet und nahezu kleinfingerdick. Er wurde ligiert und mittels Paquelin abgebrannt. Ein kleines Stiickchen Oment wurde fiir histologische Untersuehung exzidiert. Nachdem Zur Kasuistik der akuten tubsrl/ulfisen Peritonitis. 3] 137 das Exsudat ausgetupft war, v,alrde die Bauchwuude in Etagen geschlossen. -- Der Appendix war ca. 7 cm lang. Im proximalen Dritteil der Schleimhaut waren zahlreiche, zum Tell zusammenfliessende, puuklfSrmige Blutungen zu sehen, der distale Tell desselben war geschwollen. , g Glatte Heilung folgte, und der Pat. wurde am 29. I. bet gutem Wohlsein entlassen. Die histologische Untersuchung ergab das Vorhandensein yon Tub e r k ul o s e sowohl in dem Omentsttick als auch in dem Appendix; keine auf akutc Appendizitis deutende SchleimhautverSnderungen. Fall 2. P. A., Schneider, 18 Jahre. Nr. 1204, 1913. Aufgen. am 3. XII., entlassen, gebessert, am 19. XII. Diagnose: Peritonitis exsudati~-a dif- fusa (tbc.?). Fall 2. P. A., Schneider, 18 Jahre. Nr. Zur Kasuistik der akuten tuberkul6sen Peritonitis. 1204, 1913. Aufgen. am 3. XII., entlassen, gebessert, am 19. XII. Diagnose: Peritonitis exsudati~-a dif- fusa (tbc.?). Kein Auftreten yon Appendizitis oder Tuberkulose in der Verwandtschaft. Vor 7--8 Jahren halle der Pat. langwierige DiarrhSen. Vor einigen Jahren wurde er wegen ,,Halsiibel" in einem Krankenbause g epflegt und halle damals hohes Fieber. Im iibrigen ist er gesund gewesen, bis er am vergangenen 30. No- vember beim Gehen gelinde Schmerzen im unteren Tell des Bauehes zu ftihlen anfing. Die Schmerzen nahmen stetig zu. Am Tags der Aufnahme (am 3. XII.) stellten sich nachmittags heftige Schmerzen innfitten des Epigastriums ein, abw~irts nach dem rechten Teil des Bauches bin ausstrahlend. Abends 8 Uhr ei,~ beftiger Schtittelfrost. Keine Neigung zum Erbrechen oder Erbrechen. Stuhl normal. S tat. pr. bei der Aufnahme am 3. XII. nachts 12 Uhr: Das Allgemein- befinden wenig beeinflusst. Der Pat. ist jedoch sehr bleich und sieht etwas bedrfick~ aus. Temp. 39,10, Puls 120. Keine R6tung im Rachen. In tier linkea Fossa supraclav, ist sin etwas unreines Atmungsger~.usch zu hSren, im tibrigen nichts yon Lungen nnd Herz. Der Ham enth/ilt etwas Albumin. l:ber der ganzen rechten H~lfte des Bauches be:kundet tier Pat. Druek- schmerzhaftigkeit, am s54rksten tiber Me. Burneys Punkt, wo doch keine dent- licbe D6fense konstatiert werden kann. Keine ausgesprocbene Empfindlichkeit per rectum. Diagnose: (Wurmfortsatz-??) Peritonitis. Operation unmittelbar (Troell): Mc. Burneys Schnitt. Die Diinn- d~irme lebbaft injiziert, ohne Verwachsungen oder andere Ver~indernngen. Aus dem Bauch quillt reichlich freier, sehmiger, ziemlich d(inner Eiter hervor, tier vorwiegend im kleinen Becken angesammelt zu sein scheint. Das C6kum wird mit dem medial liegenden, frei beweglicben Appendix, der kaum verSndert aus- sieht, vorgezogen. Er wird jedoeh exstirpiert (mittels Paquelin). Weder auf- warts nach der Gallenblasengegend, noeh abwarts im kleinen Becken kann etwas Abnormes palpiert werden. Die Bauchwunde wird volls~ndig zusammen- gen~ht. -- Der Appendix ist ca. 10 cm lang, die Schleimhaut geschwollen, aber nicht auffallend rot. Der Heilungsverlauf ging normal vonstatten. Der Pat. war bei der Ent- lassung symptomfrei und befand sich volls~ndig wohl. Zuvor wurde, am 11. XII., Pirquets Probe gemacht, die stark positiv ausfiel, und am 15. XII. eine RSntgenuntersuchung der Bauchh6hle vorgenommen; verkalkte Tbc.-Lymphome oder sonstige Veranderungen waren doch hierdurch nicht nachzuweisen (Dr. G. Forssell). Eine mikroskopische Unter- s u c h u n g des herausgenommenen Appendix liess erkennen, dass derselbe vSllig normal war. Eine bei der Operation entnommene Probe des Peritonealexsudats gab kein Wachstum in Bouillon. [4 138 Abraham Troell. Zur Kasuistik der akuten tuberkul6sen Peritonitis. Im e rsten Falle handelt es sieh um einen zuvor gesunden jungen Mann yon 18 Jahren, bei dem sich, nachdem er gut 12 Stunden einer akuten Appendizitis am n~chsten gleichende Symptome -- auch Sch~ittelfrSste -- dargeboten, bei der Operation herausstellt, dass er an einer tuberkulSsen Peritonitis mit typischen miliaren KnStchen in der Serosa und einem freien, graugelben, sehmigen Eiter lift. Der zweite Patient ist ein Mann yon gleichem Alter, der vor l~ngerer Zeit Symptome (langwierige Diar- rhSen, ,,Halsfibel") gehabt hat,. deren tuberkulSse Natur wenigstens einen gewissen Grad yon Wahrscheinlichkeit ffir sich hat. Auch er ist nun mit akuten Bauchsymptomen erkrankt. Nachdem sieh w~hrend der n~ichstvorhergehenden Tage gelinde aber zunehmende Beschwerden im unteren Tell des Bauches bemerkbax gemacht, haben sich nun den Tag'vor der Aufnahme in das Krankenhuus heftige Leibschmerzen eingestellt und, einige Stunden nach Beginn derselben, ist ein heftiger Sehiittelfrost aufgetreten. Kaum 12 Stunden nach dieser Verschlimmerung wird durch Laparotomie festgestellt, dass eine nicht begrenzte -- nicht yore Appendix, welcher normal ist, ausgehende -- Peritonitis vorliegt. Die Serosa der Diinnd~rme ist lebhaft injiziert, aber .ohne Kn6tchen oder Fibrinbel~ge. Eine grosse Menge freien, ziemlich dtinnen Eiters ist vor- handen. Uber die Art der trier vorliegenden Pro~esse ergab sowohl die makro- als die. mikrosl~opische Untersuchung im ersten Falle die sichere Diagnose Tuberkulose. Betreffs des zweiten Falles muss freilich einige Ungewissheit hier~iber obwalten. Aber setbst wenn dem" starken positiven Ausschlag, den Pir q u e t s Kutanreaktion ergab, keine entscheidende diagnostische Bedeutung beigemesse~l werden kann (vgl. H~rtel u. a. m.) und auch die frtihere Ana- mnese nieht fibersch~tzt werden darf, so l~sst andererseits da.s Nieht- vorhandensein gewShnlicher Eiter-.oder Darmbakterien in dem yon der Operation gewonnenen Eiter die Annahme yon Tuberkulose sehr berechtigt erseheinen. Eine gonorrh.oische Infektion lag nicht vor, und flit die Diagnose Pneum.o~rokkenperitonitis oder dergleichen kann kein Anhaltspunkt nachgewiesen werden. Ich halte nfich demnaeh ftir berechtigt, in diesen beiden F~llen das Vorhandensein einer a k u t auftretenden, f r e i e n, e i t e r i g e n Peritonitis auf tuberkulSser Basis anzunehmen, und zwar bei dem einen Patienten yon so frischer Beschaff, enheit, dass noch keine miliare Tuberkeln sich hatten bilden kSnnen. Das akute Ein- setzen, mit SchiittelfrSsten, kann auf eine plStzliche Aussaat in die PeritonealhShle yon einer Henge tuberkulSsen ~Iaterials hin- Zur Kasuistik der akuten tuberkul(isen Peritonitis. 5] 139 deuten, der Art, wie es z. B. Ausser der bereits angegebenen Literatur verweise ich auf K5 ni g, der im Zentralbl. f. Chir. (1884, S. 81) unter anderem zwei F~ille yon diffuser Peri- tonealtuberkulose mit grossen, abgekapselten Exsudaten (triibes Serum in grossen, begrenzten Hohlr~iumen) erwlihnt, und auf einen ahnliehen Fall yon L i n d f o r s (Hygiea, 1886, S. 38). Siehe auch u. a. B r o ca (Zentralbl. f. Chir. 1898, S. 1198), Lejars und Routier (Wien. reed. Woehenschr. 1898, S. 2043), T r ol le y (De la pbriton, tub. d. 1. Reg. I16acoec. Paris 1900) und II o u s s e a u (La pdrit, tb. aigue simul, l'appendie. Th~se, Paris 1901) und Arsber~ittelse fran Allm. o. Sahlgrenska sjukhuset i GSteborg f6r ar 1912 (S. 78 und 82), wie aueh schliesslieh P a llin: Einige F~ille yon almter Peritonitis, wahrseheinlieh yon den weiblichen Genitalien ausgegangen (Zentralbl. f. Gyn. 1910, Nr. 30) und E d 6 n: Eft egendomligt fall af tuberkul6s peritonit (Upsala lfik-s f6rb. 1913, N. F., Bd. 18, S. 157). Fber Enteritperitonitis siehe Lennander und NystrSm (Ups. llik-s fSrh. 1906, Bd. 12, N. F., S. 57',. Zur Kasuistik der akuten tuberkul6sen Peritonitis. bei dem Bersfen einer tub.erkul6sen Lymphdr{ise in Frage kommen kann. Die n~here Pathogenese muss indessen dahingestellt bleiben, da t~ts~ehliehe Anhaltspunkte ffir Annahmen in dieser oder jener Riehtung fehlen. Vom diagnostisehen Oesiehtspunkte aus dtirften die P~lle ver- dienen, in Erinnerung behalten zu werden, angesiehts der Ta.tsaehe, dass wir ja. bei der Opera.tion akuter Bauehfitlle nieht allzu selten Peritoniten antreffen, deren Entstehung wit nieht yon einer Appen- dizitis herleiten kSnnen, ffir deren Genes,e und bakteriologischen Ursprung wir aber zwisehen der Dia.gno~e Enteritis-Peritonitis, In- fluenza-, Pneumokokken- oder -- wenigstens bei Frauen -- Gono- kokken-Peritonitis sehwanken. Man sollte sieh, wie mir seheint, in derartigen F~illen mehr a.ls bisher gegenwiirtig, halten, dass die tuberkut6se Peritonealinfektion aueh in Formen auftritt, die grosse )[hnliehkeit mit den aufgezahlten eiterigen Peritoniten haben. Nieht nur die begrenzten, sondern aueh die freien tuberkulSsen Bauehfell- ergiisse kSnnen einen makroskopiseh rein eiterigen Charakter haben. Ausser der bereits angegebenen Literatur verweise ich auf K5 ni g, der im Zentralbl. f. Chir. (1884, S. 81) unter anderem zwei F~ille yon diffuser Peri- tonealtuberkulose mit grossen, abgekapselten Exsudaten (triibes Serum in grossen, begrenzten Hohlr~iumen) erwlihnt, und auf einen ahnliehen Fall yon L i n d f o r s (Hygiea, 1886, S. 38). Siehe auch u. a. B r o ca (Zentralbl. f. Chir. 1898, S. 1198), Lejars und Routier (Wien. reed. Woehenschr. 1898, S. 2043), T r ol le y (De la pbriton, tub. d. 1. Reg. I16acoec. Paris 1900) und II o u s s e a u (La pdrit, tb. aigue simul, l'appendie. Th~se, Paris 1901) und Arsber~ittelse fran Allm. o. Sahlgrenska sjukhuset i GSteborg f6r ar 1912 (S. 78 und 82), wie aueh schliesslieh P a llin: Einige F~ille yon almter Peritonitis, wahrseheinlieh yon den weiblichen Genitalien ausgegangen (Zentralbl. f. Gyn. 1910, Nr. 30) und E d 6 n: Eft egendomligt fall af tuberkul6s peritonit (Upsala lfik-s f6rb. 1913, N. F., Bd. 18, S. 157). Fber Enteritperitonitis siehe Lennander und NystrSm (Ups. llik-s fSrh. 1906, Bd. 12, N. F., S. 57',.
https://openalex.org/W2284239065	https://jukuri.luke.fi/bitstream/10024/532073/1/Pausch.pdf	English	null	A frameshift mutation in ARMC3 is associated with a tail stump sperm defect in Swedish Red (Bos taurus) cattle	BMC genomic data	2,016	cc-by	8,022	© 2016 Pausch et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pausch et al. BMC Genetics (2016) 17:49 DOI 10.1186/s12863-016-0356-7 Pausch et al. BMC Genetics (2016) 17:49 DOI 10.1186/s12863-016-0356-7 Open Access Abstract Background: Artificial insemination is widely used in many cattle breeding programs. Semen samples of breeding bulls are collected and closely examined immediately after collection at artificial insemination centers. Only ejaculates without anomalous findings are retained for artificial insemination. Although morphological aberrations of the spermatozoa are a frequent reason for discarding ejaculates, the genetic determinants underlying poor semen quality are scarcely understood. Results: A tail stump sperm defect was observed in three bulls of the Swedish Red cattle breed. The spermatozoa of affected bulls were immotile because of severely disorganized tails indicating disturbed spermatogenesis. We genotyped three affected bulls and 18 unaffected male half-sibs at 46,035 SNPs and performed homozygosity mapping to map the fertility disorder to an 8.42 Mb interval on bovine chromosome 13. The analysis of whole-genome re-sequencing data of an affected bull and 300 unaffected animals from eleven cattle breeds other than Swedish Red revealed a 1 bp deletion (Chr13: 24,301,425 bp, ss1815612719) in the eleventh exon of the armadillo repeat containing 3-encoding gene (ARMC3) that was compatible with the supposed recessive mode of inheritance. The deletion is expected to alter the reading frame and to induce premature translation termination (p.A451fs26). The mutated protein is shortened by 401 amino acids (46 %) and lacks domains that are likely essential for normal protein function. Conclusions: We report the phenotypic and genetic characterization of a sterilizing tail stump sperm defect in the Swedish Red cattle breed. Exploiting high-density genotypes and massive re-sequencing data enabled us to identify the most likely causal mutation for the fertility disorder in bovine ARMC3. Our results provide the basis for monitoring the mutated variant in the Swedish Red cattle population and for the early identification of infertile animals. Keywords: ARMC3, Tail stump sperm defect, Swedish Red cattle, MMAF, Flagellum, Male infertility, Spermatogenesis Keywords: ARMC3, Tail stump sperm defect, Swedish Red cattle, MMAF, Flagellum, Male inferti A frameshift mutation in ARMC3 is associated with a tail stump sperm defect in Swedish Red (Bos taurus) cattle Hubert Pausch1, Heli Venhoranta2, Christine Wurmser1, Kalle Hakala2, Terhi Iso-Touru3, Anu Sironen3, Rikke K. Vingborg4, Hannes Lohi5, Lennart Söderquist6, Ruedi Fries1 and Magnus Andersson2 Correspondence: Hubert.pausch@tierzucht.tum.de 1Lehrstuhl fuer Tierzucht, Technische Universitaet Muenchen, 85354 Freising, Germany Full list of author information is available at the end of the article Background f l Diagnoses of insufficient semen quality involve the ab- sence of spermatozoa, low sperm concentration, reduced motility or viability and morphological aberrations of spermatozoa [2]. Artificial insemination (AI) is widely used instead of nat- ural mating in many cattle breeding populations. Ejacu- lates of breeding bulls are collected once or twice a week and closely examined immediately after semen col- lection at highly specialized AI centers. Only ejaculates without apparent abnormalities are retained for AI. Up to 20 % of all collected ejaculates are rejected because they do not comply with current standards for AI [1]. A motile sperm flagellum is essential for the fertilization in vivo. Morphological aberrations of the sperm tail com- promise sperm motility and impair fertilization. Such aberrations are collectively referred to as multiple mor- phological abnormalities of the flagella (MMAF, [3]). Diag- noses of MMAF involve stump and short tail spermatozoa and dysplasia of the fibrous sheath. Sequence variants causing MMAF have been identified in, e.g., humans [3– 5], pigs [6, 7] and mice [8–10]. However, sequence Pausch et al. BMC Genetics (2016) 17:49 Page 2 of 9 Page 2 of 9 variants causing MMAF have not been identified in cattle so far. variants causing MMAF have not been identified in cattle so far. (~4 ml). The sperm count was only 10–20 % of the aver- age sperm count of control bulls. All spermatozoa were immotile because of multiple flagellar abnormalities such as rudimentary (less than 5 % of the normal length), short length and absent tails. A proximal droplet sur- rounded most rudimentary tails (Fig. 1a-b). The propor- tion of spermatozoa with abnormal heads ranged from 47 to 62 %, which is ten times higher than in normal ejaculates (Table 1). None of the spermatozoa were motile. Histological sections of the testicles revealed a lack of full-length sperm tails in the luminal part of the tubuli seminiferi indicating disturbed spermato- genesis (Fig. 1c-d). Bulls with MMAF have been observed in Holstein- Friesian, Ayrshire and Indobrasil cattle [11–15]. The af- fected bulls were isolated cases within their breeds with- out known relationship among each other indicating a heterogeneous genetic etiology of MMAF across breeds. However, Alanko et al. [16] reported three related bulls from the Ayrshire cattle breed with a sterilizing tail stump sperm defect suggesting that such conditions may be inherited in an autosomal recessive fashion in cattle. Background f l Here we present the phenotypic manifestation and the genetic analysis of a recessively inherited tail stump sperm defect in the Swedish Red cattle breed. The appli- cation of homozygosity mapping facilitated the mapping of the fertility disorder to a short segment on bovine chromosome 13. The analysis of comprehensive whole- genome sequence data revealed a frameshift mutation in ARMC3 that most likely causes the sperm tail disorder in Swedish Red cattle. The analysis of the pedigree records of three affected bulls revealed a common ancestor (born in 1987) in their paternal and maternal path (see Additional file 1). Eighteen male half-sibs of the affected bulls were used for AI. The quality of their ejaculates was normal and their fertility records were within reference ranges indi- cating undisturbed reproductive performance. Based on these findings, an autosomal recessive mode of inherit- ance was assumed for the tail stump sperm defect. 13 Numerous full-length sperm tails are present in the luminal part of the tubuli seminiferi in the control bull, whereas full-length sperm tails are absent in the affected bull Pausch et al. BMC Genetics (2016) 17:49 Page 3 of 9 Page 3 of 9 Table 1 Sperm morphology in fresh ejaculates of three affected AI bulls Phenotype Bull 1 Bull 2 Bull 3 Tail morphology Normal tails 0 % 0 % 0 % Absent tails 2 % 3 % 4 % Rudimentary tails 45 % 63 % 28 % Short straight tails 27 % 15 % 29 % Folded or coiled short tails 26 % 19 % 39 % Head morphology Normal heads 42 % 53 % 38 % Abnormal heads 58 % 47 % 62 % Table 1 Sperm morphology in fresh ejaculates of three affected the identification of the mutation. Deleterious recessive mutations are assumed to have occurred after breed for- mation and are thus likely to be breed-specific. Thus we assumed that the causal mutation should not segregate among the sequenced control animals. Multi-sample variant calling in the 8.42 Mb region of extended homo- zygosity on BTA13 yielded genotypes at 81,925 single nucleotide and short insertion and deletion polymor- phisms (74,385 SNPs, 7540 Indels). In addition, 11,505 structural variants were detected in the genome-wide se- quence data of the affected bull and 226 control animals with genome coverage of at least eight-fold. Seventy-seven variants were compatible with recessive inheritance that is homozygous for the reference allele in 300 control animals and homozygous for the alternate allele in the affected bull. Bioinformatic analysis revealed that 76 variants were located in non-coding regions of the genome and one variant resided in the coding region of the armadillo repeat containing 3-encoding gene (ARMC3, Chr13: 24,301,425 bp, ss1815612719, Fig. 3a, see Additional files 2 and 3). Only two genomic regions were consistently homozy- gous in all affected animals: a 1.13 Mb segment on BTA22 (from 48,349,750 bp to 49,479,051 bp) and an 8.42 Mb segment on BTA13 (from 22,308,682 bp to 30,733,648 bp) (Fig. 2a). The segment on BTA22 was also homozygous in six fertile half-sibs precluding an as- sociation with the tail stump sperm defect. In contrast, the 8.42 Mb segment on BTA13 was never found in the homozygous state in eighteen unaffected half-sibs corre- sponding to an autosomal recessive inheritance (Fig. 2b). 13 To further reduce the number of plausible candidate causal mutations, we exploited whole-genome sequence data of 1147 animals from 29 cattle breeds that had been sequenced for Run4 of the 1000 bull genomes project [17]. Because of the close relationship among animals of three Nordic Red cattle breeds, we excluded 56 sequenced animals from the Ayrshire, Swedish Red and Danish Red cattle breed for variant filtering. Thirty-five out of 77 com- patible variants also segregated among 1009 animals from breeds other than Nordic Red (see Additional file 4). In 13 Three young bulls (11 months) of the Swedish Red cattle breed born in 2008, 2009 and 2012, were reported from an AI center because they produced ejaculates with im- motile spermatozoa during a semen collection period of 5 months. Examination of the bulls’ fresh ejaculates re- vealed a reduced sperm concentration (~140 million spermatozoa per ml) despite normal ejaculate volume To identify the genomic region associated with the tail stump sperm defect, three affected and 18 unaffected male half-sibs were genotyped with the Illumina Bovi- neSNP50 genotyping array. After quality control, geno- types at 46,035 SNPs were screened for the presence of long runs of homozygosity (ROH) in three affected bulls. Fig. 1 Phenotypic manifestation of the tail stump sperm defect. Representative figures of spermatozoa of a control (a) and an affected bull (b). Spermatozoa of affected bulls had multiple aberrations such as short tails (blue star), rudimentary tails with proximal droplet (arrows), rudimentary tails without proximal droplet (yellow triangle) and coiled tails (red star). Histological sections of the testicles of a control (c) and an affected (d) bull. Numerous full-length sperm tails are present in the luminal part of the tubuli seminiferi in the control bull, whereas full-length sperm tails are absent in the affected bull Fig. 1 Phenotypic manifestation of the tail stump sperm defect. Representative figures of spermatozoa of a control (a) and an affected bull (b). Spermatozoa of affected bulls had multiple aberrations such as short tails (blue star), rudimentary tails with proximal droplet (arrows), rudimentary tails without proximal droplet (yellow triangle) and coiled tails (red star). Histological sections of the testicles of a control (c) and an affected (d) bull. Numerous full-length sperm tails are present in the luminal part of the tubuli seminiferi in the control bull, whereas full-length sperm tails are absent in the affected bull Fig. 1 Phenotypic manifestation of the tail stump sperm defect. Representative figures of spermatozoa of a control (a) and an affected bull (b). Spermatozoa of affected bulls had multiple aberrations such as short tails (blue star), rudimentary tails with proximal droplet (arrows), rudimentary tails without proximal droplet (yellow triangle) and coiled tails (red star). Histological sections of the testicles of a control (c) and an affected (d) bull. A 1 bp deletion in ARMC3 is associated with the tail stump sperm defect Blue colour highlights the protein sequence of the tenth ARM repeat, which is absent in the mutated (mt) bovine sequence Fig. 3 A 1 bp deletion in ARMC3 induces premature translation termination. a Snapshot from the Integrated Genomics Viewer (IGV, [51]) showing a homozygous 1 bp deletion on chromosome 13 at 24,301,425 bp in an animal with the tail stump sperm defect. b Genomic structure of bovine ARMC3. Bovine ARMC3 consists of 19 exons (vertical bars) and its translation starts in exon 2. The red vertical bar represents the eleventh exon where the 1 bp deletion is located. The coordinates of ten Armadillo (ARM) repeats were determined using the Simple Modular Architecture Research Tool [50]. Blue arrows represent the position of the start and stop codons. c The bovine ARMC3 protein sequence consists of 876 amino acids and it contains ten ARM repeats (green boxes). The red triangle represents the start of the shift in translation resulting from the 1 bp deletion. d Multi-species alignment of a part of the ARMC3 protein sequence. Blue colour highlights the protein sequence of the tenth ARM repeat, which is absent in the mutated (mt) bovine sequence Fig. 3 A 1 bp deletion in ARMC3 induces premature translation termination. a Snapshot from the Integrated Genomics Viewer (IGV, [51]) showing a homozygous 1 bp deletion on chromosome 13 at 24,301,425 bp in an animal with the tail stump sperm defect. b Genomic structure of bovine ARMC3. Bovine ARMC3 consists of 19 exons (vertical bars) and its translation starts in exon 2. The red vertical bar represents the eleventh exon where the 1 bp deletion is located. The coordinates of ten Armadillo (ARM) repeats were determined using the Simple Modular Architecture Research Tool [50]. Blue arrows represent the position of the start and stop codons. c The bovine ARMC3 protein sequence consists of 876 amino acids and it contains ten ARM repeats (green boxes). The red triangle represents the start of the shift in translation resulting from the 1 bp deletion. d Multi-species alignment of a part of the ARMC3 protein sequence. Blue colour highlights the protein sequence of the tenth ARM repeat, which is absent in the mutated (mt) bovine sequence conclusion, the coding variant in ARMC3 and 41 non- coding variants were considered as candidate causal vari- ants for the tail stump sperm defect. A 1 bp deletion in ARMC3 is associated with the tail stump sperm defect To pinpoint the mutation causing the tail stump sperm defect, the whole genome of an affected bull was se- quenced to an average read depth of 9.29. In addition, we exploited data of 300 previously sequenced animals from eleven cattle breeds other than Swedish Red for Fig. 2 Homozygosity mapping in three animals with a sterilizing tail stump sperm defect. a Shades of blue represent long runs of homozygosity (ROH) in three animals along the 29 autosomes. The red borders highlight two regions on BTA13 and BTA22 with ROH in all affected animals. b Autozygosity mapping on BTA13 in three affected animals. Blue and pale blue represent homozygous genotypes (AA and BB), heterozygous genotypes (AB) are displayed in light grey. White color indicates missing genotypes. The red bar indicates a common 8.42 Mb segment of homozygosity Fig. 2 Homozygosity mapping in three animals with a sterilizing tail stump sperm defect. a Shades of blue represent long runs of homozygosity (ROH) in three animals along the 29 autosomes. The red borders highlight two regions on BTA13 and BTA22 with ROH in all affected animals. b Autozygosity mapping on BTA13 in three affected animals. Blue and pale blue represent homozygous genotypes (AA and BB), heterozygous genotypes (AB) are displayed in light grey. White color indicates missing genotypes. The red bar indicates a common 8.42 Mb segment of homozygosity Pausch et al. BMC Genetics (2016) 17:49 Page 4 of 9 Fig. 3 A 1 bp deletion in ARMC3 induces premature translation termination. a Snapshot from the Integrated Genomics Viewer (IGV, [51]) showing a homozygous 1 bp deletion on chromosome 13 at 24,301,425 bp in an animal with the tail stump sperm defect. b Genomic structure of bovine ARMC3. Bovine ARMC3 consists of 19 exons (vertical bars) and its translation starts in exon 2. The red vertical bar represents the eleventh exon where the 1 bp deletion is located. The coordinates of ten Armadillo (ARM) repeats were determined using the Simple Modular Architecture Research Tool [50]. Blue arrows represent the position of the start and stop codons. c The bovine ARMC3 protein sequence consists of 876 amino acids and it contains ten ARM repeats (green boxes). The red triangle represents the start of the shift in translation resulting from the 1 bp deletion. d Multi-species alignment of a part of the ARMC3 protein sequence. Discussion l h h h Although there is considerable phenotypic variation both in semen quality and insemination success of AI bulls, the genetic determinants underlying male reproductive traits are scarcely understood [18]. Low heritability of fertility traits and small-sized samples complicated the mapping of causal sequence variants in the past. More- over, fertility-associated variants did not reach convin- cing levels of significance in replication studies [19, 20]. Recently, the availability of comprehensive genotype and massive re-sequencing data enabled the identification of a recessively inherited variant of idiopathic male subferti- lity in cattle [21]. However, to our knowledge, our study is the first to reveal a mutation that manifests in morpho- logical aberrations of the spermatozoa in cattle. g p The analysis of pedigree records indicated that the sperm tail disorder is inherited in an autosomal recessive fashion. Sequence variants underlying recessive traits are traditionally identified by comparing allele counts of dense molecular markers in affected and unaffected indi- viduals (e.g., [21]). The likelihood to map a mendelian trait in a genome-wide case/control-association study depends on the number of affected individuals [22]. The tail stump sperm defect is a rare disorder in the Swedish Red cattle breed. Assuming a frequency of the deleteri- ous allele of 12 % in the population, random mating and 100 bulls that are annually purchased by the Swedish AI center, one would expect only one of them to be affected by the tail stump sperm defect. Accordingly, only three affected bulls were recognized in the past 10 years. We genotyped those bulls with a genotyping array and resorted to perform homozygosity mapping, which facili- tates pinpointing genomic regions underlying recessive traits with a small number of affected individuals [23]. Three affected bulls had a common 8.42 Mb segment of extended homozygosity which is a typical length ob- served in studies that are based on few affected animals [23–26]. Compatible with recessive inheritance, none of the fertile half-sibs was homozygous. Next generation se- quencing of an affected bull revealed a frameshift muta- tion in ARMC3 (ss1815612719, c.1350delG, p.A451fs26) that segregated with the tail stump sperm defect. Forty- one variants in non-protein-coding regions were also as- sociated with the disorder. However, we consider the frameshift in ARMC3 as the most likely causal mutation because it is predicted to result in a protein that lacks 401 amino acids. Discussion l h h h The function of the truncated ARMC3 protein may be severely compromised, since it lacks To our knowledge, our study reveals for the first time an association of a mutation in ARMC3 with morpho- logical abnormalities of the sperm flagellum. However, deleterious mutations in other genes encoding armadillo repeat-containing proteins have already been shown to compromise sperm motility [8, 32]. In our study, the spermatozoa of bulls that were homozygous for the frameshift mutation in ARMC3 were immotile because of severe flagellar abnormalities. A previous study dem- onstrated that dysfunction of ARMC4, a paralog to ARMC3, impairs physiological function of the cilia and sperm flagella in humans [33]. Proper function of Gudu, a gene highly homologous to ARMC4, is essential for an undisturbed spermatogenesis in Drosophila melanogaster [34]. Our investigations also evidenced an impaired spermatogenesis in bulls homozygous for the frameshift mutation in ARMC3. Such findings suggest a crucial role of ARMC3 for physiological spermatogenesis. p y g p g The morphological aberrations of the spermatozoa ob- served in our study are similar to those observed in Yorkshire boars with a loss of function mutation in SPEF2 [6]. Both defects manifest in immotile spermato- zoa precluding fertilization in vivo both in natural ser- vice and AI. The phenotypic manifestations of the two defects differ only slightly. Spermatozoa of animals being homozygous for the ARMC3 frameshift mutation mostly lack the midpiece with mitochondria, which is, however, commonly present in spermatozoa of animals homozy- gous for the SPEF2 mutation [6]. A 1 bp deletion in ARMC3 is associated with the tail stump sperm defect from position 451 onwards resulting in a premature translation termination at position 476 (p.A451fs26). The mutated protein should be shortened by 401 amino acids (46 %). Bioinformatic analysis revealed that the protein se- quence of bovine ARMC3 contains ten armadillo/beta-ca- tenin-like (ARM) repeats (Fig. 3c). The deletion variant resides within the highly conserved armadillo repeat con- taining domain. Due to the frameshift with premature translation termination, the mutated protein is expected to lack one ARM repeat (Fig. 3d). The bovine ARMC3 gene consists of 19 exons encod- ing 876 amino acids (Fig. 3b). The variant compatible with recessive inheritance (ss1815612719) is a 1 bp dele- tion in the eleventh exon of ARMC3 affecting the third base of codon 450 (ENSBTAT00000061467:c.1350_1351- delGGinsG). Sanger sequencing confirmed homozygosity for the deletion variant in two bulls with the tail stump sperm defect. The 1 bp deletion is expected to alter the reading frame and to change the amino acid sequence We genotyped 97 AI bulls from the Swedish Red cattle breed with normal fertility at ss1815612719 using Pausch et al. BMC Genetics (2016) 17:49 Page 5 of 9 domains that are likely required for normal protein func- tion [27]. customized genotyping assays. None of the bulls was homozygous for the deletion variant. Seventy-four bulls were homozygous for the reference allele and 23 bulls were heterozygous carriers of the 1 bp deletion yielding a frequency of the deletion of 11.9 %. Absence or impaired function of ARMC3 possibly pre- vents physiological spermatogenesis resulting in mor- phological aberrations of the spermatozoa. The sperm tails of homozygous bulls were severely disorganized and all spermatozoa were immotile precluding successful fertilization in vivo. Apart from immotile spermatozoa, the bulls were healthy. The morphological aberrations of the spermatozoa are similar to those observed in the Ayrshire cattle breed [12–14, 16]. Because Swedish Red cattle are closely related to Ayrshire cattle [28, 29], it is possible that the frameshift mutation in ARMC3 oc- curred in a common ancestor of the two breeds and that it might also be associated with the sperm tail disorder in Ayrshire cattle. However, the genetic underpinnings of apparently similar phenotypes may be completely dif- ferent across breeds (e.g., [24, 30, 31]). In any case, it is recommended to survey sequence variants in ARMC3 in bulls with fertility disorders in cattle breeds other than Swedish Red. Genotyping of affected and unaffected animals Genotyping of affected and unaffected animals Twenty-one bulls (three affected, 18 unaffected) of the Swedish Red cattle breed were genotyped using the Illu- mina BovineSNP50 Bead chip (Illumina, Inc., San Diego, CA, USA). The chromosomal position of the SNPs corresponded to the UMD3.1 assembly of the bovine gen- ome [37]. Mitochondrial, X-chromosomal, Y-chromosomal SNPs and SNPs with unknown chromosomal position were not considered for further analyses. After quality con- trol (per SNP and per individual call-rate higher than 90 %, no deviation from the Hardy-Weinberg equilibrium (P > 0.0001)), 46,035 SNPs were retained for further analyses. Beagle genetic analysis software [38] was used to impute sporadically missing genotypes and to infer haplotypes. Homozygosity mapping Segments of extended homozygosity were identified in three affected bulls using the homozyg-function imple- mented in the whole genome association analysis toolset PLINK [39, 40]. Due to the relatively sparse genome coverage of the genotype data (1 SNP per 56 kb), we re- stricted our analysis to runs of homozygosity (ROH) with a minimum number of 20 contiguous homozygous SNPs and a minimum length of 500 kb. Animal ethics statement All animals were housed at an approved commercial AI center in Örnsro, Sweden. Semen samples were collected by employees of the AI center as part of their regular breeding and reproductive measures in cattle industry. Bulls with the tail stump sperm defect were slaughtered because their semen was not suitable for artificial insem- ination. The decision to slaughter the bulls was made solely by the owner (i.e., AI center) of the bulls. None of the authors of the present study was involved in the de- cision to slaughter the bulls. Testicles of an affected bull were collected after slaughter. Consent from the owner of the bulls was obtained to use the semen and tissue samples for this study. No ethical approval was required for this study. Sperm motility, morphology and testicular histology Sperm motility, morphology and testicular histology We examined ten ejaculates per bull. Aliquots of fresh semen were put into vials to measure sperm concentration using a photometric method and a haemocytometer (Bür- ker chamber). A drop of semen (approximately 7 μl was put on a pre-warmed slide to evaluate sperm morphology. Head and sperm tail morphology of 200 spermatozoa was assessed from slides stained with the Williams stain (bright field microscopy) and from a wet mount formol-saline sample using a phase contrast microscope with 1000× magnification, respectively. Moreover, sperm head morph- ology was assessed in dry smears stained with carbol fuch- sin according to Williams [35] and Lagerlöf [36]. Testicles Conclusions The combination of high-density genotype and whole- genome re-sequencing data revealed a recessively inherited Pausch et al. BMC Genetics (2016) 17:49 Page 6 of 9 frameshift mutation in bovine ARMC3 that most likely causes a sterilizing tail stump sperm defect in Swedish Red cattle. Our findings suggest that impaired function of ARMC3 compromises spermatogenesis and thereby re- sults in severely disorganized sperm tails, which prevents successful fertilization in vivo. Compared to mutations that manifest in idiopathic male sub- or infertility [21], sperm- atozoa of affected animals have striking morphological ab- errations that facilitate to unambiguously identify homozygous bulls at AI centers. However, our findings fa- cilitate to identify affected young bulls before they are pur- chased by AI centers using e.g., genotyping assays on customized genotyping arrays. frameshift mutation in bovine ARMC3 that most likely causes a sterilizing tail stump sperm defect in Swedish Red cattle. Our findings suggest that impaired function of ARMC3 compromises spermatogenesis and thereby re- sults in severely disorganized sperm tails, which prevents successful fertilization in vivo. Compared to mutations that manifest in idiopathic male sub- or infertility [21], sperm- atozoa of affected animals have striking morphological ab- errations that facilitate to unambiguously identify homozygous bulls at AI centers. However, our findings fa- cilitate to identify affected young bulls before they are pur- chased by AI centers using e.g., genotyping assays on customized genotyping arrays. from an affected bull were collected after slaughter. Histo- logical specimens were taken from the testicles, fixed in Bouin’s solution and embedded in paraffin. Sections (5 μm) were cut and stained with haematoxylin and eosin. Animals Genomic DNA of an affected bull was prepared from a semen sample following standard protocols using pro- teinase K digestion and phenol-chloroform extraction. A gDNA sequencing library with 420 bp insert size was prepared using the TruSeq DNA Sample Preparation Kit (Illumina inc., San Diego, CA, USA). The sample was se- quenced on an Illumina HiSeq2500 system using TruSeq SBS v3 chemistry (Illumina inc., San Diego, CA, USA) and the 2x100 bp paired-end read module. The fastq- files were generated with the CASAVA bcl2fastq conver- sion software (version 1.8.3, Illumina inc., San Diego, CA, USA). The alignment of the reads to the University of Maryland reference sequence (UMD3.1, [37]) was per- formed with the Burrows-Wheeler Aligner [41]. The resulting SAM file was converted into a BAM file with SAMtools [42]. Duplicate reads were identified and marked with the MarkDuplicates command of Picard- tools [43]. Three bulls of the Swedish Red cattle breed born between 2008 and 2012 with a sterilizing tail stump sperm defect were included in the study together with 18 unaffected fertile male half-sibs. The bulls were housed in an AI bull center in Örnsro, Sweden. The age of the bulls during semen collection ranged from 11 to 16 months. Em- ployees from the AI center collected semen approximately twice a week as part of their regular practice. Competing interests The authors declare that they have no competing interests Abbreviations f l AI: artificial insemination; ARM: armadillo; MMAF: multiple morphological abnormalities of the flagella; ROH: runs of homozygosity; SNP: single nucleotide polymorphism. Validation of the ss1815612719 polymorphism PCR primers TTCAGTGCCAGGTTCATTGC and TTG GCTGGATGAGGTCAGTT were designed with Primer 3 [48] to scrutinize the ss1815612719 polymorphism by Sanger sequencing in two affected bulls and 97 unaffected artificial insemination bulls of the Swedish Red cattle breed. DNA was extracted from semen samples following standard protocols using proteinase K digestion and phenol-chloroform extraction. Genomic PCR products were sequenced using a 3730x1 DNA Analyzer (Applied Biosystems) and data were analyzed with the Variant Re- porter v1.0 program (Applied Biosystems). Authors’ contributions HP analyzed the SNP and NGS data, participated in the study design and drafted the manuscript. HV participated in molecular genetic analyses and revised the manuscript. KH performed molecular genetic investigations. CW generated NGS data. TIT and AS analyzed SNP data and carefully revised the manuscript. RKV provided SNP data of affected animals. HL contributed to study design and molecular genetic analyses and carefully revised the manuscript. LS identified and examined the three affected bulls and took the photos of the stained spermatozoa and testicles. RF analyzed NGS data. MA conceived the study participated in the study design, sample collection, data analysis and preparation of the manuscript. All authors read and approved the final manuscript. Additional file 2: Sequence variants identified using the SAMtools software package that were compatible with recessive inheritance. Additional file 2: Sequence variants identified using the SAMtools software package that were compatible with recessive inheritance. Grey background indicates 15 sequence variants that were not polymorphic among 1147 animals of the 1000 bull genomes project. Red color indicates a coding variant compatible with recessive inheritance. The functional consequence of the alternative allele was predicted using the Variant Effect Predictor from Ensembl (see Methods). (XLSX 61 kb) Additional file 3: Structural sequence variants that were compatible with recessive inheritance. Grey background indicates an intergenic sequence variant that was not polymorphic among 1147 animals of the 1000 bull genomes project. (XLSX 36 kb) Additional file 4: Genotype distribution of 73 candidate causal mutations for the tail stump sperm defect in 1147 animals from the 1000 bull genomes project. Alternate allele frequency and genotype distribution of 73 variants in 29 breeds (homozygous animals for the reference allele \| heterozygous animals \| homozygous animals for the alternate allele). Grey color indicates variants that were considered as candidate causal mutations. Red color indicates the deletion mutation in the coding sequence of ARMC3. (XLSX 49 kb) Bioinformatic analysis of ARMC3 We thank Auli Himanen, Jonas Krantz, Anders Edman, Hans Stålhammar and Søren Borchersen from Viking Genetics for their invaluable help to obtain biological material for the study. The outstanding laboratory help provided by Mrs. Annika Rikberg and Karin Selin-Wretling at the Sperm Laboratory of the Division of Reproduction at University of Agricultural Sciences (SLU), Uppsala is highly acknowledged. We thank the 1000 Bull Genomes consortium for sharing sequence variants of 1147 animals. This work was supported by the Finnish Veterinary Foundation. y The ARMC3 protein sequence was obtained from ensembl (ENSBTAT00000061467) and the ClustalW2 tool [49] was used for multiple species alignment. The annota- tion of ARMC3 protein domains was carried out using the Simple Modular Architecture Research Tool [50]. Identification of candidate causal variants Single nucleotide and short insertion and deletion poly- morphisms were genotyped in the affected bull together with 300 previously sequenced animals from eleven Pausch et al. BMC Genetics (2016) 17:49 Page 7 of 9 cattle breeds (Gelbvieh (n = 12), Nordic Finncattle (n = 6), Fleckvieh (n = 153), Original Simmental (n = 15), Holstein-Friesian (n = 31), Brown Swiss (n = 50), Murnau- Werdenfelser (n = 2), Ayrshire (n = 2), Red-Holstein (n = 21), Original Braunvieh (n = 8)) other than Swedish Red [44] using the multi-sample approach implemented in the mpileup function of SAMtools [42] and a variant calling pipeline as detailed by Jansen et al. [25]. Larger insertions and deletions and structural rearrangements were identi- fied in the affected animal and 226 sequenced control ani- mals with an average genome coverage above 8-fold using the Pindel software package [45]. To identify mutations compatible with recessive inheritance, all polymorphic sites were filtered for variants that were homozygous for the alternate allele in the affected bull and homozygous for the reference allele in 300 sequenced control animals. Candidate causal variants were annotated using the Vari- ant Effect Predictor tool [46, 47]. Additionally, sequence variants of 1147 animals from 29 breeds that were se- quenced for the 1000 bull genomes project [17] were ana- lyzed to obtain genotypes of compatible variants in a larger cohort. The animals of the 1000 bull genomes pro- ject were mostly influential sires that had been widely used for artificial insemination. cattle breeds (Gelbvieh (n = 12), Nordic Finncattle (n = 6), Fleckvieh (n = 153), Original Simmental (n = 15), Holstein-Friesian (n = 31), Brown Swiss (n = 50), Murnau- Werdenfelser (n = 2), Ayrshire (n = 2), Red-Holstein (n = 21), Original Braunvieh (n = 8)) other than Swedish Red [44] using the multi-sample approach implemented in the mpileup function of SAMtools [42] and a variant calling pipeline as detailed by Jansen et al. [25]. Larger insertions and deletions and structural rearrangements were identi- fied in the affected animal and 226 sequenced control ani- mals with an average genome coverage above 8-fold using the Pindel software package [45]. To identify mutations compatible with recessive inheritance, all polymorphic sites were filtered for variants that were homozygous for the alternate allele in the affected bull and homozygous for the reference allele in 300 sequenced control animals. Identification of candidate causal variants Candidate causal variants were annotated using the Vari- ant Effect Predictor tool [46, 47]. Additionally, sequence variants of 1147 animals from 29 breeds that were se- quenced for the 1000 bull genomes project [17] were ana- lyzed to obtain genotypes of compatible variants in a larger cohort. The animals of the 1000 bull genomes pro- ject were mostly influential sires that had been widely used for artificial insemination. Additional files Additional file 1: Pedigree of three bulls with the tail stump sperm defect. Red and blue color represents three affected bulls and their common ancestor. The drawn pedigree includes only obligate mutation carriers. (PNG 27 kb) Additional file 2: Sequence variants identified using the SAMtools software package that were compatible with recessive inheritance. Grey background indicates 15 sequence variants that were not polymorphic among 1147 animals of the 1000 bull genomes project. Red color indicates a coding variant compatible with recessive inheritance The functional Additional file 2: Sequence variants identified using the SAMtools software package that were compatible with recessive inheritance. Grey background indicates 15 sequence variants that were not polymorphic among 1147 animals of the 1000 bull genomes project. Red color indicates a coding variant compatible with recessive inheritance. 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Availability of supporting data 1Lehrstuhl fuer Tierzucht, Technische Universitaet Muenchen, 85354 Freising, Germany. 2Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, 04920 Saarentaus, Finland. 3Natural Resources Institute Finland (Luke), Green Technology, 31600 Jokioinen, Finland. 4Genoskan A/S, 8830 Tjele, Denmark. 5Department of Veterinary Biosciences and Research Programs Unit, Molecular Neurology, University of Helsinki and Folkhälsan Research Center, 00290 Helsinki, Finland. 6Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden. The data supporting the results of this article are in- cluded within the article and its additional files. Whole- genome sequencing data of a bull with the tail stump sperm defect were deposited in the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under accession number PRJEB12739. Page 8 of 9 Page 8 of 9 Pausch et al. 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Nat Biotechnol. 2011;29:24–6. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2915661233	https://discovery.ucl.ac.uk/id/eprint/10070810/1/2019_JMCA_Enhanced%20performance%20of%20ZnO%20nanoparticle%20decorated%20all-inorganic%20CsPbBr3%20quantum%20dot%20photodetectors.pdf	English	null	Enhanced performance of ZnO nanoparticle decorated all-inorganic CsPbBr<sub>3</sub> quantum dot photodetectors	Journal of materials chemistry. A	2,019	cc-by	7,499	aDepartment of Electronic and Electrical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. E-mail: hao.xu.15@ucl.ac.uk; jiangwu@ uestc.edu.cn bInstitute for Materials Discovery, University College London, Torrington Place, London WC1E 7JE, UK. E-mail: k.choy@ucl.ac.uk cSchool of Energy, Soochow Institute for Energy and Materials Innovations, Key Laboratory of Advanced Carbon Materials and Wearable Energy Technologies of Jiangsu Province, Soochow University, Suzhou 215006, China dDepartment of Chemistry, University College London, 20 Gordon Street, Bloomsbury, London WC1H 0AJ, UK eState Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Information, University of Electronic Science and Technology of China (UESTC), Chengdu, Sichuan, 610054, China fDepartments of Chemistry and Mechanical Engineering, The University of Hong Kong, Hong Kong SAR, China gZhejiang Institute of Research and Innovation, The University of Hong Kong, Qingshan Lake SciTech City, Hangzhou, China hInstitute of Fundamental and Frontier Science, University of Electronic Science and Technology of China, Chengdu 610054, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ta00230h Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence Cite this: J. Mater. Chem. A, 2019, 7, 6134 Kai Shen, a Xiao Li, a Hao Xu, a Mingqing Wang, b Xiao Dai,c Jian Guo, d Ting Zhang,e Shibin Li, e Guifu Zou, c Kwang-Leong Choy,b Ivan P. Parkin, d Zhengxiao Guo, dfg Huiyun Liua and Jiang Wu ah All-inorganic perovskite quantum dots have attracted substantial attention due to their excellent optical properties. However, the surface states of colloidal quantum dots and the insuﬃcient carrier transport in a quantum dot ﬁlm hinder their further development. Here, solution-processed CsPbBr3/ZnO quantum dot/nanoparticle nanocomposites are used to lessen the impact of surface states as well as facilitate charge transport. The blending of ZnO nanoparticles during CsPbBr3 quantum dot synthesis results in improved optical properties as well as ﬁlm formation that enhances charge transport. A photodetector based on the CsPbBr3/ZnO/glassy-graphene heterostructure is fabricated, which exhibits an enhanced photoresponse and distinct self-powered operation with an open-circuit voltage as large as 150 mV. Most importantly, an excellent stability of the hybrid nanoparticle/quantum dot photodetector is reported and consistent high performance with marginal degradation is achieved for more than 7 months. Received 7th January 2019 Accepted 11th February 2019 DOI: 10.1039/c9ta00230h rsc.li/materials-a Received 7th January 2019 Accepted 11th February 2019 using emerging materials as alternatives to conventional ones in the state-of-the-art photodetectors. PAPER Enhanced performance of ZnO nanoparticle decorated all-inorganic CsPbBr3 quantum dot photodetectors† Kai Shen, a Xiao Li, a Hao Xu, a Mingqing Wang, b Xiao Dai,c Jian Guo, d Ting Zhang,e Shibin Li, e Guifu Zou, c Kwang-Leong Choy,b Ivan P. Parkin, d Zhengxiao Guo, dfg Huiyun Liua and Jiang Wu ah aDepartment of Electronic and Electrical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. E-mail: hao.xu.15@ucl.ac.uk; jiangwu@ uestc.edu.cn aDepartment of Electronic and Electrical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. E-mail: hao.xu.15@ucl.ac.uk; jiangwu@ uestc.edu.cn Enhanced performance of ZnO nanoparticle decorated all-inorganic CsPbBr3 quantum dot photodetectors† Cite this: J. Mater. Chem. A, 2019, 7, 6134 Journal of Materials Chemistry A Journal of Materials Chemistry A Cite this: J. Mater. Chem. A, 2019, 6134 Received 7th January 2019 Accepted 11th February 2019 DOI: 10.1039/c9ta00230h rsc.li/materials-a Materials Che PAPER d on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. ensed under a Creative Commons Attribution 3.0 Unported Licence. Introduction Recently, organolead halide perovskites (OHPs), a family of cost-eﬀective and high-throughput materials, have been exten- sively explored for optoelectronic devices, including solar cells, light emitting diodes (LEDs), and photodetectors, owing to their excellent optoelectronic properties, such as tunable direct band gaps, large absorption coeﬃcients, high carrier mobility, and long diﬀusion lengths.8–11 Within a few years, halide perovskite based photovoltaic devices have achieved impressive power conversion eﬃciencies as high as 23.6%.12 However, OHPs are unstable in ambient air and sensitive to moisture and heat, causing signicant degradation and hindering their further development. In comparison, all-inorganic halide perovskite, cesium lead halide (CsPbX3, X ¼ Cl, Br, I), quantum dots (QDs) not only inherit the advantages of OHPs, but also exhibit better thermal stability.9,13 For example, Liu et al. presented photode- tectors made of CsPbCl3 nanocrystals, which exhibited a good photoresponse to UV light.14 Han and co-workers reported CsPbBr3 microcrystal based photodetectors with high on/oﬀ ratios as well as a fast response speed,15 and Fan et al. demon- strated CsPbI3 nanowire congured array-photodetectors, which had a stabilized cubic phase and exhibited high responsivity.16 Apart from that, the excellent emission eﬃciency and tunable emission color of CsPbX3 QDs benet from the quantum size eﬀect and anion exchange, and make them potential candidates in high quantum eﬃciency and low-cost LEDs.13,17,18 Particularly, CsPbBr3 exhibits better environ- mental stability, which does not need to be synthesized in Photodetectors play an important role in a wide range of applications, such as optical communication, environmental monitoring, imaging and biochemical sensing.1–4 Various semiconductors have been employed in photodetectors, such as GaN, Si and InGaAs.5–7 Although the technologies of commer- cial photodetectors are mature and reliable, there has been increasing interest in developing cost-eﬀective photodetectors gZhejiang Institute of Research and Innovation, The University of Hong Kong, Qingshan Lake SciTech City, Hangzhou, China hInstitute of Fundamental and Frontier Science, University of Electronic Science and Technology of China, Chengdu 610054, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ta00230h This journal is © The Royal Society of Chemistry 2019 6134 \| J. Mater. Chem. A, 2019, 7, 6134–6142 Journal of Materials Chemistry A View Article Online View Article Online Paper Journal of Materials Chemistry A Fig. 1d shows the surface morphology evolution of CsPbBr3 QD lms aer introducing ZnO NPs, obtained from scanning electron microscopy (SEM). As shown in Fig. Results and discussion The hybrid of CsPbBr3 QDs/ZnO NPs was synthesized by an optimized one-step injection method at room temperature.26 A Cs+ precursor was prepared by dissolving Cs2CO3 powder in propionic acid (PrAc), and PbBr2 powder was dissolved into a mixture of solvents of butylamine (BuAm), isopropanol (IPrOH) and PrAc to prepare a PbBr2 precursor, as illustrated in the schematic owchart in Fig. 1a. The PbBr2 precursor was then mixed with the Cs+ precursor, hexane (HEX) and PrAc, which immediately turned yellow aer stirring and was centri- fuged and dispersed in toluene 3 times to remove irrelevant solvents. ZnO NPs dispersed in chloroform were injected into CsPbBr3 QD solution. Subsequently, ZnO NP blended CsPbBr3 QD lms were prepared using spin coating followed by annealing. Transmission electron microscopy (TEM) was employed to investigate the crystal quality of the as-prepared perovskite QDs. A representative TEM image of CsPbBr3 QDs is shown in Fig. 1b, in which well-crystallized dots without any lattice distortion or crystal defects were observed. The average size of the as-prepared nanocomposites is measured to be 9 nm (Fig. S1, ESI†). Furthermore, chemical compositions (Cs, Pb, Br, Zn and O) and their distributions were analyzed by energy dispersive X-ray spectroscopy (EDX) in a zoomed-out area as shown in Fig. 1c. Each element, particularly Zn and O, is uniformly and well distributed in the observed area, con- rming that ZnO NPs and CsPbBr3 QDs were uniformly mixed. In order to gain further insight into the optical properties of CsPbBr3 QD lms with ZnO NPs, UV-Vis spectroscopy, photo- luminescence (PL) and time-resolved PL (TRPL) measurements were performed. In Fig. 2b, the steady-state absorption spec- trum of pure CsPbBr3 QDs exhibits a sharp absorption edge at 531 nm, corresponding to a direct bandgap of 2.34 eV, slightly higher than that of their bulk counterparts due to the quantum size eﬀect.9,30 Aer introducing 20 mL to 100 mL of ZnO NPs, the absorption edges gradually shied to shorter wavelengths. This blue shiindicates the formation of CsPbBr3 QDs/ZnO NP nanocomposites and hence stronger quantum connement of charges, i.e., holes, in CsPbBr3 QDs/ZnO NPs. PL spectra excited using a 405 nm laser are illustrated in Fig. 2c, in which an evident peak at 526.9 nm with a narrow FWHM of 29.1 nm was identied for the pure CsPbBr3 QDs. Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In this work, cost-eﬀective and solution-processed CsPbBr3 QDs were synthesized immediately followed by in situ blending of ZnO nanoparticles (NPs) at room temperature without the use of an inert gas. The ZnO NP blended CsPbBr3 QD lm clearly exhibits improvement in crystal quality, in terms of grain boundaries. The blending of ZnO NPs also reduces surface trap states and enables increased control over the balance of charges on the surface of the QDs, which leads to an increase in carrier transport. Photodetectors based on the hybrid CsPbBr3 QDs/ZnO NPs show enhanced performance, including higher photocur- rent, lower dark current, better photoresponsivity and faster response speeds. Most importantly, the photodetectors exhibit excellent stability aer being stored in air for more than 7 months, which is promising for practical perovskite-based devices and further promotes their potential commercialization. The as-prepared materials were studied by powder X-ray diﬀraction (PXRD). In Fig. 2a, CsPbBr3 QD lms exhibit almost unchanged XRD peaks before and aer injecting ZnO NPs. The characteristic peaks at 15.1, 21.45 and 30.7 were assigned to the (110), (112) and (220) planes of the crystalline orthorhombic CsPbBr3 perovskite (ICSD # 97851), respectively, consistent with reported work.26 The dominant peaks with a full-width at half-maximum (FWHM) of 0.5 not only indi- cate the uniformity of QDs but also the high crystallinity of the CsPbBr3 QD lms. Notably, there is an additional peak located at 11.65, which could be attributed to the formation of CsPb2Br5 or excess PbBr2.27,28 By adding ZnO NPs, this peak tends to be reduced due to the capping of CsPbBr3 QDs with ZnO NPs, which protects CsPbBr3 QDs from reacting with excess Pb2+ and Br ions.29 Introduction 1d, the lm surface of pristine QDs without ZnO NPs is comprised of large grains with an average dimension of circa 1 mm, which resulted from clustering or agglomeration of CsPbBr3 QDs. Long grain boundaries and large pinholes were observed as well, indicative of the relatively poor coverage of the lm on the substrate. In contrast, via blending more ZnO NPs by increasing the injection volume from 20 mL to 100 mL but at the same concentration, the perovskite lms possessed smaller grains and fewer pinholes. Additionally, the increase in ZnO NP blending gradually results in a more compact lm and thus better surface coverage, as depicted in Fig. 1d and S2.† This signicant improvement of surface morphology reveals successful binding of CsPbBr3 QDs by ZnO NPs, which is potentially advantageous to reduce grain boundaries and facilitate charge transfer as a result of the high conductivity of ZnO NPs. a glove box with a N2 atmosphere to prevent degradation.18–21 However, CsPbBr3 QDs, the same as general colloidal QDs, are prone to a high density of trap states which are detrimental to device performance.22,23 On the other hand, the insulating barriers, produced by a large number of long ligand chains or dangling bonds in CsPbBr3 QDs, signicantly mitigate eﬃcient carrier transport. Therefore, new ligand or passivation strate- gies are highly desirable and being sought to promote the carrier transport as well as lower surface state densities and associated surface recombination. To date, atomic-ligand and hybrid passivation schemes have been demonstrated by intro- ducing monovalent halide anions and organic crosslinks, but they are time-consuming and the synthesis was at relatively high temperature, not suitable for exible devices.24,25 This journal is © The Royal Society of Chemistry 2019 Results and discussion It was observed that the PL emission peak of the ZnO NP blended CsPbBr3 QD lm shis from 526.9 nm to 514 nm aer increased injection of ZnO NPs, in good agreement with the absorption spectra (Fig. 2b). Moreover, the ZnO NP blended CsPbBr3 QD lms possessed narrower FWHMs of PL spectra, indicating the better unifor- mity of QDs owing to suppressing agglomeration of CsPbBr3 QDs (Fig. S3, ESI†). In order to understand charge carrier dynamics and conrm the benets of adding ZnO NPs, TRPL spectra of CsPbBr3 QDs were obtained (Fig. 2d), which can be This journal is © The Royal Society of Chemistry 2019 J. Mater. Chem. A, 2019, 7, 6134–6142 \| 6135 View Article Online View Article Online Journal of Materials Chemistry A Fig. 1 Synthesis, and structural and ﬁlm properties of the CsPbBr3 QDs blended with ZnO NPs. (a) Schematic representation of the growth process of the CsPbBr3 QDs. (b) High resolution TEM image of the CsPbBr3 QDs. The scale bar is 10 nm. (c) A low magniﬁcation TEM image of the CsPbBr3 QDs and element mapping within this area by EDX. The scale bar in the low magniﬁcation TEM image is 200 nm. (d) SEM images of CsPbBr3 QD ﬁlms with diﬀerent amounts of ZnO NPs: pure CsPbBr3 QDs, and CsPbBr3 QDs with 20 mL ZnO, 40 mL ZnO, 60 mL ZnO, 80 mL ZnO and 100 mL ZnO. The scale bar is 1 mm. Journal of Materials Chemistry A Paper Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Synthesis, and structural and ﬁlm properties of the CsPbBr3 QDs blended with ZnO NPs. (a) Schematic representation of the growth process of the CsPbBr3 QDs. (b) High resolution TEM image of the CsPbBr3 QDs. The scale bar is 10 nm. (c) A low magniﬁcation TEM image of the CsPbBr3 QDs and element mapping within this area by EDX. The scale bar in the low magniﬁcation TEM image is 200 nm. This journal is © The Royal Society of Chemistry 2019 Results and discussion (d) SEM images of CsPbBr3 QD ﬁlms with diﬀerent amounts of ZnO NPs: pure CsPbBr3 QDs, and CsPbBr3 QDs with 20 mL ZnO, 40 mL ZnO, 60 mL ZnO, 80 mL ZnO and 100 mL ZnO. The scale bar is 1 mm. CsPbBr3 QDs. From Fig. 2e and f, it can be seen that the increase of ZnO NPs (>40 mL) clearly leads to reduction of s3 and reduced ratios of surface trap-related decay or increased ratios of radi- ative recombination, suggesting surface passivation of CsPbBr3 QD lms by ZnO NPs. It should be noted that the increase in s2 and relatively low ratio of radiative combination even aer incorporating ZnO NPs is due to the type II CsPbBr3/ZnO band alignment and thus electrons transfer from CsPbBr3 QDs to ZnO NPs. In summary, the ZnO blended CsPbBr3 QDs, exhibit a better surface morphology, higher crystal quality, and lower trap states, and are promising for low-cost and solution- processed optoelectronics. tted with a multi-exponential decay function with three decay components. These three time constants s1, s2 and s3 obtained by the tting are summarized in Table S1.† The average decay lifetimes of CsPbBr3 QDs with an increasing amount of ZnO NPs can be calculated as 13.431, 13.921, 16.530, 11.006, 11.536 and 11.724 ns, respectively. A shorter decay time, beneting from the reduced carrier trapping by surface states, is important for fast photodetectors. To understand the carrier dynamics of CsPbBr3 QDs aer blending ZnO NPs, the decay times and the ratios of each decay component are plotted in Fig. 2e and f. The fastest decay time, s1, and the ratio of this decay component are barely changed. This decay time is most likely associated with non-radiative recombination, as the crystal quality of the CsPbBr3 QDs should not be inuenced by ZnO NPs. The ratio of this decay is the lowest, which again indicates high crystal quality of the CsPbBr3 QDs. The time constants s2 and s3 have a distinct dependence on the amount of ZnO NPs incorporated into the QDs. The former, s2, is about 1–2 ns, which can be assigned to typical radiative recombination.31 The long decay time, s3, of over 10 ns may be caused by the surface trap states of tted with a multi-exponential decay function with three decay components. Results and discussion These three time constants s1, s2 and s3 obtained by the tting are summarized in Table S1.† The average decay lifetimes of CsPbBr3 QDs with an increasing amount of ZnO NPs can be calculated as 13.431, 13.921, 16.530, 11.006, 11.536 and 11.724 ns, respectively. A shorter decay time, beneting from the reduced carrier trapping by surface states, is important for fast photodetectors. To understand the carrier dynamics of CsPbBr3 QDs aer blending ZnO NPs, the decay times and the ratios of each decay component are plotted in Fig. 2e and f. The fastest decay time, s1, and the ratio of this decay component are barely changed. This decay time is most likely associated with non-radiative recombination, as the crystal quality of the CsPbBr3 QDs should not be inuenced by ZnO NPs. The ratio of this decay is the lowest, which again indicates high crystal quality of the CsPbBr3 QDs. The time constants s2 and s3 have a distinct dependence on the amount of ZnO NPs incorporated into the QDs. The former, s2, is about 1–2 ns, which can be assigned to typical radiative recombination.31 The long decay time, s3, of over 10 ns may be caused by the surface trap states of Photodetectors based on the CsPbBr3/ZnO QD/NP nano- composites were fabricated on quartz substrates. The device structure is schematically shown in Fig. 3a. Glassy-graphene thin lms, possessing excellent properties of transparency, conductivity, and chemical inertness, were employed as the electron transport layer and transparent electrode.32,33 Two devices, one based on perovskite QDs with 60 mL ZnO NPs (PGH_1) and the other based on perovskite QDs only (PGH_2), This journal is © The Royal Society of Chemistry 2019 6136 \| J. Mater. Chem. A, 2019, 7, 6134–6142 View Article Online Journal of Materials Chemistry A Paper Paper Journal of Materials Chemistry A Fig. 2 Structural and optical characterization of ZnO NP blended CsPbBr3 QDs. (a) XRD patterns. (b) Absorption spectra. (c) Steady-state PL spectra. (d) Time-resolved PL measurement. Each color represents the diﬀerent quantities of ZnO NPs involved. The excitation laser wavelength is 405 nm. (e) Decay times and (f) decay component ratios extracted from the TRPL spectra in (d) as a function of the amount of ZnO NPs added to the CsPbBr3 QDs. Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. This journal is © The Royal Society of Chemistry 2019 Results and discussion At 405 nm illumination, the photovoltage of PGH_1 can be as high as 150 mV; however, the photovoltaic eﬀect was not observed in the PGH_2 photodetector (Fig. S4, ESI†). This phenomenon can be assigned to the introduction of a built-in electric eld by ZnO nanoparticles. As one of the key gures- of-merit, photoresponsivity, which can be expressed as RA ¼ Iph/P (where Iph denotes the photocurrent and P represents the incident light intensity), was calculated and is plotted in Fig. 3d, as a function of applied voltage.33 The obtained largest photo- responsivity of the PGH_1 device at 1 V bias was 0.4 mA W1 and 0.03 mA W1 when illuminated using 405 nm and 520 nm lasers, respectively. The lower response at 520 nm is due to a lower absorption of photons with energy close to the band edge (Fig. 2b). The RA values of PGH_2 under diﬀerent illumi- nations were both one order smaller than those of PGH_1 measured under the same conditions. The improved photo- current and photoresponse again conrm the advantages of blending ZnO NPs in perovskite QDs. The eﬀects of introducing ZnO NPs on the detector perfor- mance were further investigated by atomic force microscopy (AFM) and conductive atomic force microscope (CAFM). The surface morphologies of the samples prepared with 0–80 mL ZnO NPs are displayed in Fig. 4a, respectively. Similar to the SEM images (Fig. 1d), it was observed that pinholes were gradually lled by ZnO NPs, so the surface of QD lms became much more compact and smoother, consistent with the decreased surface roughness. Surface current maps were ob- tained by CAFM to evaluate ZnO NP treatment induced impacts on the electrical properties of lms, as illustrated in Fig. 4b. Without adding ZnO NPs, higher current is measured for the QD lms. On increasing the amount of ZnO NPs, the current gradually became lower and more uniform across the scanned area. This behavior can be explained by the illustration shown in Fig. 4c. Without ZnO NPs, clustering or agglomeration of CsPbBr3 QDs resulted in rough surfaces along with a high density of pinholes, which induced a high leakage current. These pinholes compromise lateral charge transfer and hence collection of photocurrent. The assumption can be well sup- ported by the I–V characteristics shown in Fig. 3c. Results and discussion Blending ZnO NPs in the CsPbBr3 QDs prevented serious clustering or agglomeration of QDs and hence enabled the formation of compact and smooth lms. Photocarriers generated in CsPbBr3 QDs can be more eﬀectively transferred to the terminals when ZnO NPs bridge the gaps between the QDs. The ZnO NPs also function as a means of passivation. Consequently, the devices were able to have smaller leakage current and lower dark current but deliver higher photocurrent aer ZnO NP treatment. were studied. As shown in the inset of Fig. 3a, a device with glassy graphene electrodes shows good transparency. Fig. 3b shows the current–voltage (I–V) curves of PGH_1 measured in the dark, under white light, and under 405 nm and 520 nm laser illumination at room temperature, respectively. The linear dependence of I–V curves suggests good conductivity and eﬃ- cient charge carrier transport between perovskite QDs and glassy-graphene. This is not the case when no ZnO NP was added, and a higher dark current and lower photocurrent were measured for device PGH_2 as shown in Fig. 3c. Notably, there is a distinct open circuit voltage when illuminated using lasers. At 405 nm illumination, the photovoltage of PGH_1 can be as high as 150 mV; however, the photovoltaic eﬀect was not observed in the PGH_2 photodetector (Fig. S4, ESI†). This phenomenon can be assigned to the introduction of a built-in electric eld by ZnO nanoparticles. As one of the key gures- of-merit, photoresponsivity, which can be expressed as RA ¼ Iph/P (where Iph denotes the photocurrent and P represents the incident light intensity), was calculated and is plotted in Fig. 3d, as a function of applied voltage.33 The obtained largest photo- responsivity of the PGH_1 device at 1 V bias was 0.4 mA W1 and 0.03 mA W1 when illuminated using 405 nm and 520 nm lasers, respectively. The lower response at 520 nm is due to a lower absorption of photons with energy close to the band edge (Fig. 2b). The RA values of PGH_2 under diﬀerent illumi- nations were both one order smaller than those of PGH_1 measured under the same conditions. The improved photo- current and photoresponse again conrm the advantages of blending ZnO NPs in perovskite QDs. Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. Results and discussion 2 Structural and optical characterization of ZnO NP blended CsPbBr3 QDs. (a) XRD patterns. (b) Absorption spectra. (c) Steady-state PL spectra. (d) Time-resolved PL measurement. Each color represents the diﬀerent quantities of ZnO NPs involved. The excitation laser wavelength is 405 nm. (e) Decay times and (f) decay component ratios extracted from the TRPL spectra in (d) as a function of the amount of ZnO NPs added to the CsPbBr3 QDs. Fig. 3 The overview and electrical performance of the CsPbBr3 and glassy-graphene heterostructure device. (a) Scheme of the architecture of the PGH photodetector (quartz/Au/Ni/Glassy-graphene/CsPbBr3/Ni/Au). The inset shows a device placed over the UCL Engineering logo, showing good transparency. (b) I–V characteristic curves of the PGH_1 device in the dark and under white light and diﬀerent illuminations, 405 nm and 520 nm. (c) The photocurrent and dark current of the PGH_1 and PGH_2 photodetectors. (d) The calculated photoresponsivity of the two devices above under 405 nm and 520 nm illumination, respectively. Fig. 3 The overview and electrical performance of the CsPbBr3 and glassy-graphene heterostructure device. (a) Scheme of the architecture of the PGH photodetector (quartz/Au/Ni/Glassy-graphene/CsPbBr3/Ni/Au). The inset shows a device placed over the UCL Engineering logo, showing good transparency. (b) I–V characteristic curves of the PGH_1 device in the dark and under white light and diﬀerent illuminations, 405 nm and 520 nm. (c) The photocurrent and dark current of the PGH_1 and PGH_2 photodetectors. (d) The calculated photoresponsivity of the two devices above under 405 nm and 520 nm illumination, respectively. This journal is © The Royal Society of Chemistry 2019 J. Mater. Chem. A, 2019, 7, 6134–6142 \| 6137 View Article Online Paper ticle Online Paper Journal of Materials Chemistry A Paper were studied. As shown in the inset of Fig. 3a, a device with glassy graphene electrodes shows good transparency. Fig. 3b shows the current–voltage (I–V) curves of PGH_1 measured in the dark, under white light, and under 405 nm and 520 nm laser illumination at room temperature, respectively. The linear dependence of I–V curves suggests good conductivity and eﬃ- cient charge carrier transport between perovskite QDs and glassy-graphene. This is not the case when no ZnO NP was added, and a higher dark current and lower photocurrent were measured for device PGH_2 as shown in Fig. 3c. Notably, there is a distinct open circuit voltage when illuminated using lasers. Results and discussion This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. As displayed in Fig. 5f, the rise time (srise) and fall time (sfall) of the PGH_1 device were determined to be less than 76.5 ms Fig. 5 Electrical properties of the PGH_1 photodetector. (a) I–V curves of the device illuminated using a 405 nm laser with the incident ligh intensity varied from 1.3 mW cm2 to 22.8 mW cm2. (b) The photocurrent measured as a function of incident light intensity and the corre sponding power ﬁtting curve. (c) The transient photocurrent response of the device illuminated using a 405 nm laser with varying power from 1. mW cm2 to 22.8 mW cm2 at 1 V bias. (d) The transient photoresponse curves of the PGH_1 photodetector under 405 nm and 520 nm illu mination with an intensity of 22.8 mW cm2. The applied voltages are 1 V and 1 V, respectively. (e) The transient photoresponse curves of th PGH_1 photodetector under 405 nm illumination at 0 V. (f) The corresponding rise and fall time under 1 V bias voltage. (g) The corresponding ris and fall time at 0 V bias. (h) The long-term stability of the PGH_1 photodetector over 7 months. (i) The transient photocurrent response of th device around the middle and end of the stability test. Fig. 5 Electrical properties of the PGH_1 photodetector. (a) I–V curves of the device illuminated using a 405 nm laser with the incident light intensity varied from 1.3 mW cm2 to 22.8 mW cm2. (b) The photocurrent measured as a function of incident light intensity and the corre- sponding power ﬁtting curve. (c) The transient photocurrent response of the device illuminated using a 405 nm laser with varying power from 1.3 mW cm2 to 22.8 mW cm2 at 1 V bias. (d) The transient photoresponse curves of the PGH_1 photodetector under 405 nm and 520 nm illu- mination with an intensity of 22.8 mW cm2. The applied voltages are 1 V and 1 V, respectively. (e) The transient photoresponse curves of the PGH_1 photodetector under 405 nm illumination at 0 V. (f) The corresponding rise and fall time under 1 V bias voltage. (g) The corresponding rise and fall time at 0 V bias. (h) The long-term stability of the PGH_1 photodetector over 7 months. Results and discussion 4 Surface morphology characterization and passivation process of CsPbBr3. (a) Surface morphology of each sample with an increasing percentage of ZnO from 0 mL to 80 mL by AFM. (b) The corresponding current mapping. (c) Schematic process of applying ZnO NP passivation to the CsPbBr3 QD ﬁlm. Fig. 4 Surface morphology characterization and passivation process of CsPbBr3. (a) Surface morphology of each sample with an increasing percentage of ZnO from 0 mL to 80 mL by AFM. (b) The corresponding current mapping. (c) Schematic process of applying ZnO NP passivation to the CsPbBr3 QD ﬁlm. This journal is © The Royal Society of Chemistry 2019 This journal is © The Royal Society of Chemistry 2019 6138 \| J. Mater. Chem. A, 2019, 7, 6134–6142 View Article Online Paper Journal of Materials Chemistry A Typical I–V curves of PGH_1 photodetector under the 405 nm laser illumination with various power intensities are shown in Fig. 5a, exhibiting the apparent dependence on light intensity. With increasing light intensity, photocurrent was enhanced due to a larger population of photogenerated carriers. Power- dependent photocurrent is plotted in Fig. 5b, tted by the power law (Iph f Pb, where b denotes the Duane plot slope). The best tting gave a non-linear behavior of b ¼ 0.502, which is associated with the increased recombination of photogenerated charge carriers at high light intensity. This indicates presence of some recombination centers within the perovskite.34,35 As shown in Fig. 2f, the non-radiative recombination time constant, s1, barely changed aer adding ZnO NPs, which requires further optimization of QD synthesis. Current–time (I–t) curves of transient photoresponse as a function of incident light intensity were measured and are plotted in Fig. 5c. It was observed that all the dynamic curves exhibited a sensitive and reproducible pho- toresponse, and the photocurrent continuously increased with the irradiation power raised from 1.3 mW cm2 to 22.8 mW cm2. Fig. 5d shows the photoresponsive dynamics of the PGH_1 photodetector at 1 V and 1 V bias illuminated using 405 nm and 520 nm lasers, respectively. A larger photocurrent was obtained at 1 V than 1 V bias, caused by the built-in electric eld. Notably, the PGH_1 photodetector displayed an excellent photoresponse under 0 V bias (Fig. 5e), demonstrating self-powered operating mode enabled by the built-in electric eld. Open Access Article. Published on 25 February 2019. Downloaded on 3/21/2019 6:07:52 PM. This journal is © The Royal Society of Chemistry 2019 Results and discussion (i) The transient photocurrent response of the device around the middle and end of the stability test. This journal is © The Royal Society of Chemistry 2019 J. Mater. Chem. A, 2019, 7, 6134–6142 \| 6139 View Article Online Journal of Materials Chemistry A (99.9%) were purchased from Sigma-Aldrich. Chloroform (99.9%) was purchased from Fisher Chemical. All chemicals were used as received without further purication. and 73.5 ms at 1 V bias, respectively, which reached the steady- state condition faster than the PGH_2 device (Fig. S5 and Table S3, ESI†). This pronounced enhancement was mainly attributed to faster charge separation and more eﬃcient carrier transport at interfaces that resulted from surface passivation by ZnO NPs. As compared with previous reports, the photoresponse time of our ZnO NP blended PGH photodetectors is much shorter by two orders than that of all-inorganic perovskite based and 2-D material heterostructure based photodetectors,36–38 indicating the good light-switching behavior of the as-prepared devices. For self-powered photodetection (Fig. 5g), it was found that the fall time is almost unchanged while the rise time is obviously increased under 0 V bias (tted by a single-exponential decay, Fig. S6 and Table S2, ESI†), which was probably due to the internal electric eld not being strong enough to separate the electron–hole pair eﬃciently. The stability of perovskite-based devices is a critical factor for practical applications. To test the stability of the device reported in this study, the photocur- rent of the PGH_1 photodetector was recorded over a seven- month period, as shown in Fig. 5h. The dark current maintained similar values between 1 nA and 5 nA, and the photocurrent still retained its initial value with negligible degradation aer storing under ambient conditions for more than 7 months. Notably, the PGH_1 photodetector still dis- played good reproducibility, as well as marginal degradation in response speed, aer being stored under ambient conditions for over 7 months, as shown in Fig. 5i, exhibiting outstanding stability. In contrast, PGH_2 photodetectors signicantly suﬀered from degradation, with respect to the response speed (Fig. S7 and Table S3, ESI†). This is mainly ascribed to the passivation of ZnO NPs. By comparing with other reported perovskite-based photodetectors,39–44 the ZnO NP decorated PGH photodetector achieves the best reported stability under ambient conditions. Conclusions In summary, CsPbBr3 QDs decorated by ZnO NPs were successfully synthesized by a simple non-vacuum solution- processed method. By introducing ZnO NPs, a better lm surface morphology with fewer defects at the grain boundaries and fewer pinholes was achieved. Improved optical and elec- trical properties of CsPbBr3 QDs were observed aer adding ZnO NPs. PGH photodetectors based on ZnO/CsPbBr3 hybrids were fabricated. The detectors exhibited excellent responsivity, fast photoresponsive on-oﬀswitching (<76.5 ms), and self- powered operating mode. Moreover, an outstanding stability over 7 months was achieved for our devices reported in this study, which could pave the way for future commercialization of perovskite-based optoelectronic applications. Materials characterization Transmission electron microscopy (TEM) images and energy dispersive X-ray (EDX) mappings were obtained on a JEOL 2100 microscope attached with an X-MaxN detector (Oxford Instru- ments). The surface morphology of the samples was examined using an EVO LS15 scanning electron microscope (ZEISS). X-ray diﬀraction was recorded using a powder X-ray diﬀractometer (PXRD, Bruker D8 Discover) with CuKa1,2 radiation (l ¼ 1.54056 ˚A). Optical absorption spectra were measured using Lambda 750S UV-Vis spectrometers (Perkin Elmer) at room temperature. Steady-state and time-resolved PL spectra were ob- tained on a TCSPC (LifeSpec-ps) from Edinburgh Instruments with an excitation wavelength of 405 nm at 15 C. The current mapping measurements were carried out on a Keysight 5600LS atomic force microscope (AFM) in the current sensing mode in the air. Device fabrication Few-layer glassy-graphene (5 nm) was pre-deposited onto the quartz substrate by a polymer-assisted deposition (PAD) approach.32 The as-synthesized perovskite precursor was then spin-coated on the glassy-graphene coated substrate at 1000 rpm for 60 s. Subsequent deposition cycles were applied to increase the thickness of the absorbing layer. The samples were annealed at 120 C for 1 min aer each spinning, and baked at 120 C for 5 min aer spin coating of the last layer. The total thickness of the active layer was around 150 nm (8 deposition cycles). The heterojunction was formed at the interface where the perovskite and glassy-graphene were overlapped, with the dimension circa 2 mm by 5 mm. The Ni/Au (10/100 nm) elec- trodes were nally deposited through a shadow mask by thermal evaporation. The size of the metal contact pads was circa 1 mm by 4 mm. Material synthesis Procedures involved in the preparation of ZnO NP blended CsPbBr3 QDs are carried out in ambient air at room temperature, as shown in Fig. 1a. A Cs+ precursor was prepared by dissolving Cs2CO3 powder (1.1736 g) in the PrAc (2 mL). Meanwhile, the PbBr2 powder (0.9175 g) was dissolved into a mixture of solvents of BuAm, IPrOH and PrAc (5 mL, 1 : 1 : 1) to form the PbBr2 precursor. Subsequently, the PbBr2 precursor (270 mL) was swily injected into the solution mixture containing the Cs+ precursor (25 mL), IPrOH (5 mL) and HEX (10 mL). This solution turned light yellow within seconds. Subsequently, high-quality CsPbBr3 QDs were obtained by centrifuging and dispersing the as- prepared solution in toluene 3 times, in order to eliminate the trace of other solvents. Diﬀerent centrifugation speeds of 1500 rpm, 2000 rpm and 1500 rpm for 2 min were used each time. Finally, diﬀerent amounts of ZnO NP precursor (0.5 M, dissolved in chloroform) from the top of the clear solution were injected into the as-prepared CsPbBr3 QDs. References 22 M. V. Kovalenko, M. Scheele and D. V. Talapin, Science, 2009, 324, 1417–1420. 1 G. Konstantatos, I. Howard, A. Fischer, S. Hoogland, J. Cliﬀord, E. Klem, L. Levina and E. H. 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https://openalex.org/W2999813277	https://link.springer.com/content/pdf/10.1007/s12520-019-00981-7.pdf	English	null	From petrographic analysis to stereomicroscopic characterisation: a geoarchaeological approach to identify quartzite artefacts in the Cantabrian Region	Archaeological and anthropological sciences	2,020	cc-by	16,009	Introduction artefacts was carried out by the first prehistorians, such as Juan Villanova i Piera or Édouard Lartet (Pelayo López and Gonzalo Gutiérrez 2012). The characterisation of these raw materials has become more detailed and frequent since the 1950s through the use of classic petrological methodologies and because of increasing archaeologist interest in new per- spectives to understand prehistoric societies (Polanyi 1957). Both perspectives converge in the characterisation of obsidian Archaeological and Anthropological Sciences (2020) 12: 32 https://doi.org/10.1007/s12520-019-00981-7 Archaeological and Anthropological Sciences (2020) 12: 32 https://doi.org/10.1007/s12520-019-00981-7 ORIGINAL PAPER 1 Institute of Prehistory and Protohistory, Department of Classical World and Asian Cultures, Friederich-Alexander University of Erlangen-Nuremberg (FAU), Kochstr. 4/18., D-91054 Erlangen, Germany 3 Department of Mineralogy and Petrology, University of the Basque Country (UPV/EHU), Barrio Sarrena s/n, 48940 Leioa, Biscay, Spain Abstract Several isolated studies have tried to understand quartzite from an archaeological perspective by applying two different meth- odological approaches. The first one is based on non-destructive characterisation, aiming to understand human procurement and management of quartzite, without solid geoarchaeological criteria. The second characterised the material from archaeological sites using only petrographic or geochemical perspectives of a limited sample. Currently, both perspectives are unconnected, creating a methodological gap that needs to be solved to study the procurement and management of quartzite in greater depth. The present study, mainly methodological, will explore the gap between petrographic analysis and non-destructive characterisation. Doing so, we could fill this vacuum of information and generate a solid geoarchaeological basis to characterise not only a sample but complete assemblages. To this end, we analyse the lithic assemblages at El Arteu and El Habario, two Middle-Palaeolithic sites in the Cantabrian Region, northern Spain. We summarise the main results derived from petrographic analysis, but especially we will focus on non-destructive criteria to characterise the lithic surfaces of archaeological quartzite using stereoscope micros- copy. This process allows us to understand the complete assemblage but also, through technological characterisation, understand the management of different quartzite petrogenetic types in both sites. Keywords Lithic technology . Management of raw material . Quartzite . Petrology . Stereomicroscope . Keywords Lithic technology . Management of raw material . Quartzite . Petrology . Stereomicroscope . Geoarchaeology * Alejandro Prieto alejandro.de.prieto@fau.de; alejandro.prieto@ehu.eus; alejandropdd@gmail.com From petrographic analysis to stereomicroscopic characterisation: a geoarchaeological approach to identify quartzite artefacts in the Cantabrian Region Alejandro Prieto1,2 & Iñaki Yusta3 & Alvaro Arrizabalaga2 Received: 8 June 2019 /Accepted: 6 November 2019 /Published online: 14 January 2020 # The Author(s) 2020 2 Department of Geography, Prehistory and Archaeology, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 5., 01006 Vitoria-Gasteiz, Spain Quartzite in archaeology The study of raw materials used for lithic implements is a well-known topic of research in prehistoric archaeology. Since the beginning of this scientific discipline, petrological description and classification of rocks transformed into Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12520-019-00981-7) contains supplementary Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12520-019-00981-7) contains supplementary material, which is available to authorized users. material, which is available to authorized users. material, which is available to authorized users. 2 Department of Geography, Prehistory and Archaeology, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 5., 01006 Vitoria-Gasteiz, Spain * Alejandro Prieto alejandro.de.prieto@fau.de; alejandro.prieto@ehu.eus; alejandropdd@gmail.com 3 Department of Mineralogy and Petrology, University of the Basque Country (UPV/EHU), Barrio Sarrena s/n, 48940 Leioa, Biscay, Spain 32 Page 2 of 23 Archaeol Anthropol Sci (2020) 12: 32 as the key raw material to trace incipient commercial routes in the Mediterranean in Recent Prehistory (Binns and McBryde 1969; Dixon et al. 1968). The interesting results, and also the methodologies generated, led to a significant increase in lithic raw material studies for the next two decades, focussed not only in the trade itself, but also in the areas where abiotic resources were extracted by prehistoric societies (Earle and Ericson 1977; Kowalski et al. 1972; Sieveking et al. 1972; Singer and Ericson 1977). In the 1980s and 1990s, raw mate- rial studies increased not only in number, but also in their geographic and chronological frameworks. To do so, different raw materials were analysed and, for Palaeolithic Archaeology in Europe, flint became the best and most widely characterised raw material (e.g. Demars 1980; Floss 1990; Geneste 1985; Luedtke 1979; Morala 1980; Séronie-Vivien and Séronie-Vivien 1987). At the same time, research fo- cussed on economic and social dynamics through the techno-typological characterisation of lithic assemblages has increased our knowledge of the acquisition and management of flint (Andrefsky 1994; Roebroeks 1988; Turq 1996). All these elements create a better understanding on the economy created around raw materials and the complex management and catchment patterns followed by Palaeolithic societies. proposed different procurement and management strategies for quartzite, mainly based on techno-typological analysis, but their conclusions are clearly biased by the lack of accuracy in their descriptions. Currently, both perspectives are uncon- nected, creating a methodological gap that needs to be solved for more in-depth studies of quartzite acquisition and manage- ment strategies, based on a solid geoarchaeological approach. Quartzite in archaeology The goal of this methodological work is to explore the gap between destructive analysis, mainly petrographic study of thin sections, and non-destructive characterisa- tion of quartzite. The information gap might be filled in this way and a solid geoarchaeological basis might be established to characterise not only a sample but complete quartzite assemblages. To this end, we have analysed the lithic collections from El Arteu and El Habario, two Middle-Palaeolithic sites situated in the Central part of the Cantabrian Region, northern Spain. We summarise the main results derived from the petrographic and geo- chemical characterisation of 18 samples recently pub- lished by our research group (Prieto et al. 2019), but es- pecially, we will focus on non-destructive criteria to char- acterise the lithic surfaces of quartzites using stereoscope microscopy and descriptive characterisation. The diage- netic and metamorphic changes which modify the texture and structure of former sedimentary rocks, the grain size and its distribution, and the mineral component itself will be analysed in this research using both approaches. Therefore, the correlation between both techniques (destructive and non-destructive) is the main goal of this research. This process allows us to understand the com- plete assemblage but also, through technological charac- terisation, to understand the management of quartzite in both sites, taking into account production, usage and dis- card of this raw material (Roebroeks 1988; Turq et al. 2013). During the last two decades, several studies have contributed to the knowledge about lithic management strategies developed by Middle-Palaeolithic groups in the Cantabrian Region (e.g. Carrión et al. 2008; Gonzalez-Urquijo et al. 2005; Rios-Garaizar 2012). These researches accomplished not only a description of lithic assemblages using typological characterisation of retouched artefacts, but they also unveiled catchment mechanisms (Carrión and Baena 1999; Castanedo 2001; Manzano et al. 2005), technological procedures (Carrión 2002; Cuartero et al. 2015; Maillo 2007; Santamaría et al. 2010) or use practices (Rios-Garaizar 2010). These stud- ies have opened new perspectives to discover a complex and varied economy from geographic, chronological and human standpoint. The research we present here shows not only the management of quartzite at these two Middle-Palaeolithic sites, but also the new wider perspec- tives this geoarchaeological study can open in archaeolog- ical practice (Dibble et al. 2017; Romagnoli et al. 2018). Quartzite in archaeology Despite being the second most-often-used lithic raw mate- rial in the European Palaeolithic, quartzite has not received the same methodological development from geoarchaeological perspectives as flint or obsidian. This situation creates a lack of information and many biases in understanding the catch- ment and management strategies pursued by prehistoric soci- eties. This is especially important in those areas where flint is not common or in ancient chronologies in areas where non- flint rocks are predominant. However, in recent years, quartz- ite from archaeological deposits has been studied from geoarchaeological perspectives by combining different de- structive methodologies such as petrographic analysis and geochemical procedures (Blomme et al. 2012; Cnudde et al. 2013; Dalpra and Pitblado 2016; Pitblado et al., 2008, 2012; Prieto et al. 2019; Veldeman et al. 2012). These studies reflect the ambiguity of the term quartzite in archaeological literature, where it can refer to different geological origins, from clearly metamorphic to sedimentary. This prompts the need of petrog- raphy to exactly differentiate sedimentary orthoquartzite from metamorphic quartzites. Similar discrepancies between mega- scopic identification and laboratory analysis are also men- tioned by petrologists, especially when quartzite is used as a field name (Howard 2005; Skolnick 1965). Archaeological quartzites have also been studied by other specialists using non-destructive techniques. In the Cantabrian Region, several studies have attempted to study this material by its macroscopic characterisation without examining these rocks from detailed and curated geoarchaeological perspec- tives (e.g. Álvarez-Alonso et al. 2013; Castanedo 2001; Manzano et al. 2005; Sarabia 2000). These studies have Archaeol Anthropol Sci (2020) 12: 32 Page 3 of 23 Page 3 of 23 32 32 Materials Cambrian, Ordovician, Silurian and Devonian strata (Bastida 2004). The second one is the western part of the Basque- Cantabrian Basin, in particular, the Navarro-Cantabrian sul- cus, dominated by sedimentary Mesozoic rocks and small parts of Cenozoic material. Neogene and Quaternary deposits are represented too (Barnolas and Pujalte 2004). In this con- text, quartzite is characterised by multiple and different envi- ronments: as outcrop (quartz arenites), generally related to the older strata; as pebbles and blocks inside carboniferous con- glomerates; and as pebbles in fluvial quaternary deposits. Cambrian, Ordovician, Silurian and Devonian strata (Bastida 2004). The second one is the western part of the Basque- Cantabrian Basin, in particular, the Navarro-Cantabrian sul- cus, dominated by sedimentary Mesozoic rocks and small parts of Cenozoic material. Neogene and Quaternary deposits are represented too (Barnolas and Pujalte 2004). In this con- text, quartzite is characterised by multiple and different envi- ronments: as outcrop (quartz arenites), generally related to the older strata; as pebbles and blocks inside carboniferous con- glomerates; and as pebbles in fluvial quaternary deposits. The sites of El Habario and El Arteu are located in the central part of the Cantabrian Region, in the northwestern part of Spain (Fig. 1). They are in the western part of the Autonomous Community of Cantabria. This area is a moun- tainous region and the sites are located in the basin of the River Deva and its tributaries, in the eastern foothills of the Picos de Europa. This area is characterised by a complex geology determined by the presence of two geological do- mains. The first one is the eastern part of the Cantabrian Zone, mainly composed by Carboniferous materials. Two main provinces can be distinguished, the Pisuerga-Carrión Province, to the South and the Picos de Europa and Ponga Province to the West and North. Both provinces also contain El Habario and El Arteu assemblages have been attributed chrono-culturally to the Mousterian, with discoid reduction methods mostly in quartzite. They appear to form a network of sites together with El Esquilleu rock shelter (Baena et al. 2005, 2012; Carrión 2002; Carrión et al. 2008, 2013; . 1 Location of El Habario, El Arteu and the Deva-Cares Valley. Main chrono-lithological strata are displayed based on a 1:1.000.000 geological m lvaro et al. 1994) Fig. 1 Location of El Habario, El Arteu and the Deva-Cares Valley. Features to characterise, analysis to understand We first studied 18 selected hand samples that were used for thin-section preparation to describe their textural and grain features. Once they were described using a stereomicroscope and the information was related with that obtained by standard petrographic methods on an optical microscope, we described the other 687 quartzite pieces from the complete assemblage. q p p g We also described these pieces according to the basic techno-typological characterisation based on Analytical Typology (Laplace 1972). We used the proposal made by Maite García-Rojas to understand technological products (García-Rojas 2010, 2014). First, we classified lithic imple- ments attending to three categories: (a) cores, characterised by the presence of one or more knapping surfaces and one or more striking platforms; (b) knapping products, characterised by the presence of a ventral and a dorsal surface and the presence of a butt of striking platform; and (c) chunk, material that could not be classified in the two previous categories due to the lack of criteria. Cores are classified as discoid, levallois, irregular or core on flakes attending to their features and mor- phology (Fig. 2). Knapping products are classified as blanks, in case there is only one plane in the dorsal surface (including flakes, blades and bladelets), or core preparation/rejuvenation products, a knapping product with presence of two different planes on its dorsal surface. One of these planes has a percus- sion platform while the other, a dorsal surface. This category includes core tables or core-trimming elements. Finally, blanks are classified into four categories attending to The assemblages studied here are deposited in the Museum of Prehistory and Archaeology of Cantabria (MUPAC). Of the total of artefacts made in quartzite from El Habario (HA) and El Arteu (ATS), we sampled a set of representative pieces for thin section analysis (n = 17) and X-ray fluorescence analysis, as described in Prieto et al. (2019). We also select another sample from a Deva river beach. Petrographic analysis in- cludes packing, texture and quartz grain feature description, also grain size characterisation based on image processing and, finally, mineral characterisation. The results point to a high variability of archaeological quartzite according to the genesis of the material. The 18 quartzites were classified into seven main types according to their petrogenesis as described by thin section, especially through the analysis of textures, packing and quartz grain features, and secondly by quartz grain size and morphology. Materials The interpretation of El Arteu for hunting activities inside a complex residential model in the area was suggested by Baena et al. (2005). Radiolarite (n = 11), limestone (3), lutite (1) and hydrothermal quartz (1) are represented in addi- tion to archaeological quartzite (n = 237). The assemblages studied here are deposited in the Museum of Prehistory and Archaeology of Cantabria (MUPAC). Of the l f f d i i f l b i ( A) d understanding them as cumulative features to the main types defined. The petrogenetic types used here are the following (Table 1): for sedimentary quartzites, clastic fabric with matrix or non-quartz cement quartz arenite (MA), clastic quartz arenite (CA), syntaxially overgrown orthoquartzite (OO) and sutured grain orthoquartzite (SO); and for truly metamorphic quartzites another three types depending on the textural changes on quartz grains: bulging recrystallised quartzite (BQ), subgrain rotation recrystallised quartzite (RQ) and grain boundary migration recrystallised quartzite (MQ). This classi- fication is based on the geological work of several researchers (Bastida 1982; Folk 1974; Howard 2005; Wilson 1973). In order to readily identify and manage in large databases, a two-letter code is used for every petrogenetic type. When possible, we try to relate the first letter with a relevant microtextural feature of the rock, e.g. presence of matrix (M) or syntaxially quartz overgrowths (O); whereas the second capital letter applies to the specific rock type, precisely sand- stones are labelled as arenites (A) or orthoquartzite (O), and metamorphic quartzites with the letter (Q). Manzano et al. 2005).The site of El Habario is an open-air site in a mountainous area, close to Carboniferous conglomerate bedrock units (X = 368.973 Y = 4.784.861, 30T ETRS-1989). Two quite distinct levels were differentiated during excava- tion. The superficial level (El Habario A) is a sedimentary packet or level altered by bioturbation. The underlying, El Habario B, is a fertile level apparently in a primary position. The lithic assemblage was attributed to a single phase within the Mousterian and show techno-typological coherence. Despite there are no numerical dates, El Habario B was assim- ilated to the Middle Palaeolithic, mainly based on typological and technological studies (Carrión and Baena 1999, 2005). The latest researches concluded that the dominant lithic reduc- tion model was based on hierarchical centripetal reduction processes aimed at flake production. Materials All these reasons, as well as the proximity to the Remoña Conglomerates, identify this site as a workshop of quartzite, probably related with the cen- tral layers of the sequence of El Esquilleu and El Arteu. The collection analysed here comes from El Habario B and the number of pieces is 467. All except three lithics are quartzites. p p q The archaeological site of El Arteu is situated in a small rock shelter in a rugged high-mountain area near the Deva and its confluence with the River Cares (X = 368.247 Y = 4.793.505, 30T ETRS1989). The artefacts were collected after they had fallen from a section, but they display industrial coherence. The lithic assemblage (n = 255) was attributed to a single phase within the Mousterian (Carrión 2002; Carrión et al. 2008). Technological characterisation of this assemblage showed that lithic reduction was done under Levallois or dis- coid lithic method and it was aimed at obtaining pointed flakes. The interpretation of El Arteu for hunting activities inside a complex residential model in the area was suggested by Baena et al. (2005). Radiolarite (n = 11), limestone (3), lutite (1) and hydrothermal quartz (1) are represented in addi- tion to archaeological quartzite (n = 237). Materials Main chrono-lithological strata are displayed based on a 1:1.000.000 geologica (Álvaro et al. 1994) Fig. 1 Location of El Habario, El Arteu and the Deva-Cares Valley. Main chrono-lithological strata are displayed based on a 1:1.000.000 geological map (Álvaro et al. 1994) o, El Arteu and the Deva-Cares Valley. Main chrono-lithological strata are displayed based on a 1:1.000.000 geological map Fig. 1 Location of El Habario, El Arteu and the Deva-Cares Valley. Main chrono-lithological strata are displayed based on a 1:1.000.000 geological map (Álvaro et al. 1994) 32 Page 4 of 23 Archaeol Anthropol Sci (2020) 12: 32 32 Page 4 of 23 Manzano et al. 2005).The site of El Habario is an open-air site in a mountainous area, close to Carboniferous conglomerate bedrock units (X = 368.973 Y = 4.784.861, 30T ETRS-1989). Two quite distinct levels were differentiated during excava- tion. The superficial level (El Habario A) is a sedimentary packet or level altered by bioturbation. The underlying, El Habario B, is a fertile level apparently in a primary position. The lithic assemblage was attributed to a single phase within the Mousterian and show techno-typological coherence. Despite there are no numerical dates, El Habario B was assim- ilated to the Middle Palaeolithic, mainly based on typological and technological studies (Carrión and Baena 1999, 2005). The latest researches concluded that the dominant lithic reduc- tion model was based on hierarchical centripetal reduction processes aimed at flake production. All these reasons, as well as the proximity to the Remoña Conglomerates, identify this site as a workshop of quartzite, probably related with the cen- tral layers of the sequence of El Esquilleu and El Arteu. The collection analysed here comes from El Habario B and the number of pieces is 467. All except three lithics are quartzites. The archaeological site of El Arteu is situated in a small rock shelter in a rugged high-mountain area near the Deva and its confluence with the River Cares (X = 368.247 Y = 4.793.505, 30T ETRS1989). The artefacts were collected after they had fallen from a section, but they display industrial coherence. The lithic assemblage (n = 255) was attributed to a single phase within the Mousterian (Carrión 2002; Carrión et al. 2008). Technological characterisation of this assemblage showed that lithic reduction was done under Levallois or dis- coid lithic method and it was aimed at obtaining pointed flakes. Fig. 2 Technological products identified at both sites: a levallois core (El Habario), b irregular core (El Habario), c discoidal core (El Habario) and d core on flake core (El Arteu) Features to characterise, analysis to understand Finally, the metrical characterisation of the pieces was done using their weight. and orientation and (c) characterisation of non-quartz ele- ments (Fig. 4). Features to characterise, analysis to understand The variability in mineral charac- terisation and geochemical composition was only used to un- derstand similarities between quartzites at both sites, Page 5 of 23 3 Archaeol Anthropol Sci (2020) 12: 32 32 Table 1 Summarised characterisation of thin section from the samples of El Arteu and El Habario, derived from Prieto et al. 2019. ++ is for higher presence Petrogenetic type Texture Packing Quartz grain features Grain size, morphology and orientation Samples Matrix or cemented Quartz arenite MA Clastic Floating Punctual Detrital quartz grains, abundant matrix and/non-quartz cement Variable quartz grain size and morphology, preferential orientation? Geological origin Clastic rained quartz arenite CA Clastic Tangential Complete Detrital grains, concave-convex boundaries Variable quartz grain size and morphology, preferential orientation? ATS-002 ATS-151 Syntaxially overgrown Ortho-quartzite OO Clastic Tangent-Complete Syntaxially overgrowth, concave-convex boundaries, un- dulatory extinction Bigger quartz grains Homogeneous distribution Well-rounded grains ATS-001 ATS-007 ATS-023 ATS-308 HA-5855 Sutured grain Ortho-quartzite SO Clastic Complete Suturated Suturated quartz grains Undulatory extinction Deformation lamellae Smaller quartz grains Preferential orientation Irregular quartz grains ATS-190 ATS-302 ATS-310 HA-5827 Bulging recrystallised Quartzite BQ Mortar Suturated Recrystallised quartz grains Suturated quartz grains Deformation lamellae Two mode size and morphological distribution: Small & rounded + bigger and irregular grains Preferential orientation ATS-016 ATS-072 HA-5847 HA-5848 Subgrain rotation recrystallised Quartzite RQ Mortar Suturated Recrystallised quartz grains ++ Suturated quartz grains Deformation lamellae Two modes on grain morphology One mode and negative kurtosis size distribution Preferential orientation ATS-195 Grain boundary migration recrystallised Quartzite MQ Clastic/ foam Suturated Suturated gran limits Deformation lamellae ++ One mode distribution with negative kurtosis for size and morphology HA-5842 Fig. 2 Technological products identified at both sites: a levallois core (El Habario), b irregular core (El Habario), c discoidal core (El Habario) and d core on flake core (El Arteu) Fig. 2 Technological products identified at both sites: a levallois core (El Habario), b irregular core (El Habario), c discoidal core (El Habario) and d core on flake core (El Arteu) 32 Page 6 of 23 Archaeol Anthropol Sci (2020) 12: 32 the quantity of scars on the dorsal surface. In addition, they are classified into another four categories attending to the quantity of cortex on dorsal surfaces (Fig. 3). Retouch is analysed as an accumulative feature on technological products, and we have only counted the number of primary types on each piece (Fernández-Eraso and García-Rojas 2013; Laplace 1987). Fig. 3 Technological products identified at both sites: a blank without scars and more than 66% of cortex on it (El Arteu), b blank with one negative scar and more than 66% of cortex on it (El Habario), c blank with two negative scars with less than 33% of cortex on it (El Arteu), d blank with three or more negative scars and cortex between 33 and 66% of dorsal surface (El Arteu), e core preparation/rejuvenation product (El Arteu) and f chunk (El Arteu) Qualitative characterisation of texture, packing and quartz grain features We qualitatively systematised lustre to the naked eye on the surface of quartzites in four categories: (a) no lustre, (b) low lustre, (c) medium lustre and (d) high lustre. The non-destructive petrological characterisation of quartz- ites in both assemblages was based on four different scales of observation. The first one is based on naked-eye description. The second one, uses two × 10 and × 20 hand magnifiers, a stereomicroscope and the Dino-Lite digital microscope to the same magnifications. The third scale employs × 50 magnifi- cation provided by the Dino-Lite digital microscope and by stereomicroscopes. The fourth applies × 250 magnification with the first instrument. Most of the pieces were photographed in a flat position to × 50 and × 250 magnifica- tion to create a library of reference pictures. We preferentially used Dino-Lite model AD7013MZT with polarized light to eliminate most of the surface lustre. The microscope was han- dled with the MS35B vertical stand. We used the software Dino-Capture 2.0. The stereomicroscope used was the Nikon SMZ800, with up to × 120 magnification. Non- destructive rock characterization is based on (a) qualitative characterisation of texture and quartz grain features, (b) quantitative-qualitative characterisation of quartz grain size Density of surface micro-cracks/chips in the surface of quartzite to naked eye. These areas are characterised as lighter and sparkling elements with scale morphology that appears on the surface of quartzites (Fig. 5). We systematised their pres- ence using the following criteria: (a) absence, (b) small, (c) medium or (d) high presence of micro-cracks. Quartz grain boundaries definition/recognisability was defined using a surface section of approximately 1.6 × 1.3 mm at × 250 magnification as (a) very easy, when at least 25 quartz grains were recognisable; (b) easy, when between 15 and 25 quartz grains were observable; (c) difficult, when between 5 and 15 quartz grains could be recognised; and (d) impossible, when fewer than 5 quartz grains were recognisable. We distinguished five different types of packing in hand specimens according to the categories used for thin section and the following criteria (Fig. 6): Fig. Archaeol Anthropol Sci (2020) 12: 32 Archaeol Anthropol Sci (2020) 12: 32 1. Floating, if the quartz grains are separated one from each other by matrix or cement. 2. Punctual or isolated, when the quartz grains are one near another and the contacts between them are restricted to single points. At ranges between × 50 and × 250, the grains are closer to each other, but contacts are very small or absent. 3. Tangential, in case the grains are joined together, but ce- ment is still present. At × 50–× 250 magnification, the cement is restricted to a thin layer between the limits of the quartz grains. 4. Complete, where there is a very small content of cement or matrix and the grains create an almost complete texture. Matrix or cement is almost absent, limited to few accu- mulations in small confined areas between grains. They are delimited by very weak, fine and straight contours. 5. Suturated, when the limits between grains generate a com- plete and deformed texture. Matrix or cement is restricted Fig. 4 Non-destructive characterisation scheme, showing the features observed in different scales of observation Fig. 4 Non-destructive characterisation scheme, showing the features observed in different scales of observation cement is restricted to a thin layer between the limits of the quartz grains. 1. Floating, if the quartz grains are separated one from each other by matrix or cement. 1. Floating, if the quartz grains are separated one from each other by matrix or cement. cement is restricted to a thin layer between the limits of the quartz grains. 2. Punctual or isolated, when the quartz grains are one near another and the contacts between them are restricted to single points. At ranges between × 50 and × 250, the grains are closer to each other, but contacts are very small or absent. 4. Complete, where there is a very small content of cement or matrix and the grains create an almost complete texture. Matrix or cement is almost absent, limited to few accu- mulations in small confined areas between grains. They are delimited by very weak, fine and straight contours. 3. Tangential, in case the grains are joined together, but ce- ment is still present. At × 50–× 250 magnification, the 3. Tangential, in case the grains are joined together, but ce- ment is still present. At × 50–× 250 magnification, the 5. Fig. 5 Surface micro-cracks on the lithic artefact ATS-190. To the naked eye, white, irregular and lighter superficial areas are identified. At × 20, × 50 and × 250 magnification surface micro- cracks are also present and recognised by similar features Qualitative characterisation of texture, packing and quartz grain features 3 Technological products identified at both sites: a blank without scars and more than 66% of cortex on it (El Arteu), b blank with one negative scar and more than 66% of cortex on it (El Habario), c blank with two negative scars with less than 33% of cortex on it (El Arteu), d blank with three or more negative scars and cortex between 33 and 66% of dorsal surface (El Arteu), e core preparation/rejuvenation product (El Arteu) and f chunk (El Arteu) Archaeol Anthropol Sci (2020) 12: 32 Page 7 of 23 32 Archaeol Anthropol Sci (2020) 12: 32 Suturated, when the limits between grains generate a com- plete and deformed texture. Matrix or cement is restricted Fig. 5 Surface micro-cracks on the lithic artefact ATS-190. To the naked eye, white, irregular and lighter superficial areas are identified. At × 20, × 50 and × 250 magnification surface micro- cracks are also present and recognised by similar features 32 Page 8 of 23 Archaeol Anthropol Sci (2020) 12: 32 Fig. 6 Microphotographs of the surface of five different archaeological quartzites at 250x magnification attending to the packing criteria. From left to right Rb_01 (River beach) (floating), ATS-002 (punctual), ATS-151 (tangential), ATS007 (complete) and ATS-072 (suturated) Fig. 6 Microphotographs of the surface of five different archaeological quartzites at 250x magnification attending to the packing criteria. From left to right Rb_01 (River beach) (floating), ATS-002 (punctual), ATS-151 (tangential), ATS007 (complete) and ATS-072 (suturated) to small points on quartz limits, as in the previous packing type. c. Compact and grainy texture is defined by the presence of a very soft granulated touch on the surface of the sample. There is no presence of matrix or cement. To the naked eye, the colour is more homogeneous and micro-cracks are recognisable. At low magnification, the relief is gen- tler than that in the previous texture and some of the sur- faces show successive planes of squamous surfaces. At middle magnification, most of the grains are still recognisable, especially the lighter grains which show clear and curved outlines, although some parts of them cannot be appreciated. In a same way, at high magnifica- tion, grains are recognisable, especially those with thicker, brighter and curved outlines. Other grains or parts of the outlines are vague or diffused. The previously commented lighter grains are visible (Fig. 7). We distinguished six different types of texture, not only attending to the perception of the surface relief or the feel (touch) of the quartzite surface, but also using criteria observ- able by stereomicroscope. Texture categories are the following: a. Saccharoid texture is defined by the generalised presence of matrix or a carbonate cement on the surface of the samples. The touch is granular and sandy and it is usually heterogeneously coloured to the naked eye. At low mag- nification, slopes and rough relief are appreciated. Archaeol Anthropol Sci (2020) 12: 32 At mid- dle magnification, it is possible to observe the presence of isolated to tangential quartz grains surrounded by large amounts of matrix and secondary diagenetic cement, cre- ating a rough relief. Some quartz grains can sparkle, but the lustre is not homogeneously distributed. At high mag- nification, quartz grains tend to be isolated and, again surrounded by matrix, generally as the sum of small specks that cover the surface of quartz and other mineral grains (Fig. 7). d. Fine and grainy texture is defined by a smooth touch and a moderate lustre. In general, grains are difficult to observe completely. Although the touch is fine, small rough areas could be observed, mainly generated by the presence of secondary ferruginous, siliceous or car- bonate precipitates or by the presence of joints. To the naked eye, colour is relatively homogeneous, except in micro-cracks, which are generally brighter. At low magnification, the relief is soft without roughness, al- though some surfaces of the quartzite show squamous surfaces. At middle magnifications, grains are hardly recognisable and there is a thin and bright lustre cov- ering the surface of the rock. At high magnification, grains are almost unrecognisable and only some out- lines can be appreciated. Small specks are visible on the outlines of the quartz grains and they seem to be on the same level as the rest of the surface, covered by the previously mentioned thin and bright lustre (Fig. 8). b. Granular texture is defined by a clear granular touch on the surface of the sample. The quantity of matrix or ce- ment is reduced and its presence is restricted to small areas or the surroundings of the edges of the grains. To the naked eye, it generally shows a heterogeneous colour dis- tribution. At low magnification, softly rough to flat relief is appreciated. At middle magnification, it is possible to recognise quartz grains and some of them can sparkle, but lustre is not homogenous. At high magnification, grains of these rocks are also recognisable and small quantities of cement or matrix (mainly formed by the sum of small specks) fill the small empty areas between quartz grains (Fig. 7). b. Granular texture is defined by a clear granular touch on the surface of the sample. Fig. 7 Microphotograph of the surface of three quartzites. On the left, × 50 magnification picture and, on the right, × 250 magnification one. From top to bottom, ATS-164 possesses a saccharoid texture; ATS-080 exhibits granular texture; and, finally, ATS-304 represents compact and grainy texture Fig. 8 Microphotograph of the surface of three quartzite pieces taken at × 50 magnification (left) and at × 250 magnification (right). From top to bottom, ATS- 079 shows fine and grainy texture; ATS-072 exhibits fine texture; and, finally, HA-5842 represents soapy texture Archaeol Anthropol Sci (2020) 12: 32 The quantity of matrix or ce- ment is reduced and its presence is restricted to small areas or the surroundings of the edges of the grains. To the naked eye, it generally shows a heterogeneous colour dis- tribution. At low magnification, softly rough to flat relief is appreciated. At middle magnification, it is possible to recognise quartz grains and some of them can sparkle, but lustre is not homogenous. At high magnification, grains of these rocks are also recognisable and small quantities of cement or matrix (mainly formed by the sum of small specks) fill the small empty areas between quartz grains (Fig. 7). b. e. Fine texture is defined by a really smooth touch and a shiny/brilliant lustre. In general, grains cannot be ob- served and only some small relicts from their outlines are seen. The touch is very fine and, as in the previous Page 9 of 23 32 Archaeol Anthropol Sci (2020) 12: 32 Fig. 7 Microphotograph of the surface of three quartzites. On the left, × 50 magnification picture and, on the right, × 250 magnification one. From top to bottom, ATS-164 possesses a saccharoid texture; ATS-080 exhibits granular texture; and, finally, ATS-304 represents compact and grainy texture Fig. 8 Microphotograph of the surface of three quartzite pieces taken at × 50 magnification (left) and at × 250 magnification (right). From top to bottom, ATS- 079 shows fine and grainy texture; ATS-072 exhibits fine texture; and, finally, HA-5842 represents soapy texture 32 Page 10 of 23 Archaeol Anthropol Sci (2020) 12: 32 recognisable, not by the outlines but by a small, thin and almost unrecognisable bulging that creates a wavy micro-relief. Hardly any small specks are appreciated (Fig. 8). texture, some areas can be rougher due to cement pre- cipitations. To the naked eye, colour is homogenous and shows a high lustre. Micro-cracks are much more limited than those in the previous texture, although they are present. At low magnification, the relief is very soft and no rugosity is observed; neither is there a large quantity of squamous surfaces, which are re- duced to very small areas. At middle magnification, no grains are recognisable and only small specks are visible. Thin and bright lustre is observed and it clearly covers the entire surface of the quartzite. Fig. 9 Microphotograph of the surface of some quartzite pieces at × 250 magnification. The main quartz grain features are a quartz grains with plain and angular limits (arrows on clear angular outlines of the quartz grains), from the sample ATS-089. b Quartz grains with plain and rounded limits, from sample HA- 5632. c Quartz grains with ruffled and irregular limits generated by the presence of matrix or cement, from sample ATS-339. d Appearance of regrowth of syntaxial quartz cement (arrow on syntaxial quartz cement in the direction of its growth), from sample ATS-308. e Quartz grains with ruffled irregular and thin limits on flat surface, from sample HA-5500. f Quartz grains with no apparent boundaries detected, reduced to small alignments of specks, from sample HA-5519 Archaeol Anthropol Sci (2020) 12: 32 At high mag- nification, no grain is observed and only small specks or associations of them are recognised on the surface, reduced to small areas (Fig. 8). texture, some areas can be rougher due to cement pre- cipitations. To the naked eye, colour is homogenous and shows a high lustre. Micro-cracks are much more limited than those in the previous texture, although they are present. At low magnification, the relief is very soft and no rugosity is observed; neither is there a large quantity of squamous surfaces, which are re- duced to very small areas. At middle magnification, no grains are recognisable and only small specks are visible. Thin and bright lustre is observed and it clearly covers the entire surface of the quartzite. At high mag- nification, no grain is observed and only small specks or associations of them are recognised on the surface, reduced to small areas (Fig. 8). Quartz grain features were determined by applying mor- phological features of the border of the quartz grains. We reduced the variability of each quartzite to the two prevailing categories. These were: 1. Quartz grains with plain and angular limits (Fig. 9a). 2. Quartz grains with plain and rounded limits (Fig. 9b). 3. Quartz grains with ruffled and irregular limits generated by the effect of matrix or cement (Fig. 9c). f. . Soapy texture is quite similar to the preceding one. The touch is soapy, even smoother than that in the previous texture, and no grains are observed. To the naked eye, colour is really homogeneous, very bright, and micro- cracks are limited. At low magnification, the relief is plain and soft and there is almost no plain squamous surface. At middle magnifications, no grains are recognisable and neither are the small specks appreci- ated in the preceding texture. Previous thin and bright lustre is extended onto the entire surface of the quartz- ite. At high magnifications, only a few grains are 4. Quartz grains with appearance of regrowth of quartz syntaxial cement, recognisable by the partial or complete dual grain outline that creates a lighter, glossy and curved space between both lines, generally in concavo-convex lines between grains (Fig. 9d). 5. Quartz grains with ruffled, irregular and thin limits and surfaces with flat relief (Fig. 9e). 6. Quartz grains with no boundaries detected, where the limits are reduced to small alignments of specks or small saturated lines (Fig. 9f). Archaeol Anthropol Sci (2020) 12: 32 Fig. 9 Microphotograph of the surface of some quartzite pieces at × 250 magnification. The main quartz grain features are a quartz grains with plain and angular limits (arrows on clear angular outlines of the quartz grains), from the sample ATS-089. b Quartz grains with plain and rounded limits, from sample HA- 5632. c Quartz grains with ruffled and irregular limits generated by the presence of matrix or cement, from sample ATS-339. d Appearance of regrowth of syntaxial quartz cement (arrow on syntaxial quartz cement in the direction of its growth), from sample ATS-308. e Quartz grains with ruffled irregular and thin limits on flat surface, from sample HA-5500. f Quartz grains with no apparent boundaries detected, reduced to small alignments of specks, from sample HA-5519 Archaeol Anthropol Sci (2020) 12: 32 Page 11 of 23 32 32 Qualitative characterisation of quartz grain size and orientation On the other hand, we completed the qualitative analysis of quartz grains by describing their sorting degree using the fol- lowing categories: homogeneous, bimodal or heterogeneous quartz grain according to the size distribution. As commented before, we tried to recognize in hand spec- imens the mineral and textural features observed in thin sections, as a way to link the information from destructive and non-destructive methodologies. Nevertheless, due to the lower resolution of stereomicroscopes and the restric- tions imposed by the lack of polarizing light to properly recognize grain features, the amount of information gener- ated, and especially the possibility of applying different techniques, here we measure quartz grain size using a qual- itative scale. This allows us to analyse a large number of rock implements using only two criteria that systematise the grain size of quartzites. We also noted the presence of bedding planes on the sur- face of quartzites, both to the naked eye and using low mag- nification, according to the following criteria: (a) absence of bedding, (b) non-clear bedding and (c) clear bedding. Finally, we tried to recognize foliation features on at least one surface of the sample, although this is not an easy task in hand specimens of highly compact quartzites (Fig. 10). Qualitative characterisation of non-quartz mineral In addition to quartz, archaeological quartzites contain other minerals in very small proportions. Up to three of the most representative minor minerals were identified in every sample. The observed minerals are the following: as primary phases (a) white mica, (b) feldspars, (c) pyrite and (d) undetermined black minerals, and as alteration phases (e) Fe and/or Mn oxides (Fig. 11). On one hand, we determined mean grain size using × 50 and × 250 magnification, obtaining the approximate measure- ment of the secondary axis of the particles. The measurement was performed with the measuring mode provided by the software Dino-Capture 2.0. In general, the number of mea- sured grains per sample is higher than 20. So we used three categories, based on the Udden-Wentworth scale (Wentworth 1922) and modified for their application to quartz grains ac- cording to the conclusion reached by Prieto et al. 2019: (a) Coarse quartz grain size, used for quartzites containing quartz grains bigger than fine sand (> 0.25 mm); (b) medium quartz grain size, used for quartz grains between coarse silt and fine sand (0.031 to 0.25 mm); and (c) fine quartz grain size, used for quartzites with grains smaller than coarse silt (< 0.062). For heterogeneous samples, the biggest grain determined was assigned as the size of the quartzite. From petrographic analysis to binocular characterisation The groups and types established through petrographic anal- ysis of thin sections were used for non-destructive character- isation of hand specimens by using similar qualitative criteria Fig. 10 Microphotograph of the surface of two quartzite pieces at × 50 and × 250 magnification. On the left side, ATS-205 exhibits clear bedding of the surface with grains preferentially oriented and silica matrix filling the empty space between them. No grain is deformed. On the right side, sample HA-5847 shows clear fo- liation. It is easy to distinguish schistosity from bedding owing to the modification of quartz grains in the former, while in the latter no modification of quartz grains is observable (both observations at × 250 magnification) Fig. 10 Microphotograph of the surface of two quartzite pieces at × 50 and × 250 magnification. On the left side, ATS-205 exhibits clear bedding of the surface with grains preferentially oriented and silica matrix filling the empty space between them. No grain is deformed. On the right side, sample HA-5847 shows clear fo- liation. It is easy to distinguish schistosity from bedding owing to the modification of quartz grains in the former, while in the latter no modification of quartz grains is observable (both observations at × 250 magnification) 32 Page 12 of 23 Archaeol Anthropol Sci (2020) 12: 32 Fig. 11 Microphotograph of the surface of six quartzite pieces. a ATS-001 to × 250 magnification shows orange/red irregular iron oxides. b HA-5814 at × 50 magnification shows black and irregular manganese oxides. c HA-5654 at × 250 magnification shows two black, plain and subrounded undetermined heavy minerals. d HA-5715 at × 250 magnification shows very small, black, plain and angular pyrites. There are also iron oxides near the pyrites. e ATS-002 at × 50 shows some small, white and bright exfoliating feldspars. f ATS-308 at × 250 magnification shows some small, white and bright mica with changeable structures CA type (clastic quartz arenite) is characterised through thin section by the association of clastic grained texture, tan- gent or tangent-to-complete packing, and the presence of clas- tic grains delimited by concave-convex quartz limits. This is reflected through non-destructive techniques in the associa- tion of granular texture, tangent or sometimes complete pack- ing, and flat and rounded or angular quartz grain limits. Lustre is present, but it is of low intensity. There is a small increase in the presence of micro-cracks. From petrographic analysis to binocular characterisation In order to avoid duplicated information, features of quartz grains present in previous phases do not appear in the following types. P: packing; T: texture; F: quartz grain feature; M: morphology of quartz grains. In lustre and micro-cracks (M. crack), 0 means absence, 1 low, 2 medium and 3 high lustre intensity or density of micro- cracks/scale variable. The presence of micro-cracks is also variable. It is easy to recognise grains, although not as easy as in the previ- ous sedimentary quartz arenites (Fig. 14). characteristics on the quartzite surfaces, such as fine texture, suturated packing, flat and ruffled limits of quartz grains and the non-detection of quartz grains. The quantity of recognisable grains is smaller than five and the presence of surface micro-cracks is again variable. The intensity of lustre is high. Finally, and as in the previous type, foliation is obvi- ous in most of the samples (Fig. 15). SO type (sutured grain orthoquartzite) is characterised through thin section by the association of clastic grained tex- ture, suturated grained packing, stylolitic boundaries, high presence of undulatory extinction and occasional presence of recrystallised grains or deformation lamellae. In hand speci- mens, there is no boundary detection, but somehow it is relat- ed to fine and grainy texture, suturated packing and flat and ruffled grain boundaries. As in the previous type, a variable lustre is common, as well as the presence of surface micro- cracks. Grains are less easily recognisable than those in the previous type. Foliation structures are easy to recognise on some of the samples (Fig. 14). RQ type (subgrain rotation recrystallised quartzite) is characterised through thin section by the high presence of recrystallised quartz grains together with mortar texture, suturated packing and presence of stylolitic quartz grains. On the quartzite surface, this type is associated with a soapy texture and absence of evident quartz grain boundaries and few suturated borders. The intensity of lustre is high. As in previous types, foliation is obvious on some samples. There are almost no micro-cracks on the surface (Fig. 15). BQ type (bulging recrystallised quartzite) is characterised through thin section by the association of mortar texture, suturated packing and presence of stylolitic quartz grains, de- formation lamellae, clear undulatory extinction and significant presence of recrystallised grains. From petrographic analysis to binocular characterisation Bedding appears in some of the studied samples and it is generally easy to recognise > 25 quartz grains in a 1.6 × 1.3-mm surface sector (Fig. 13). for the assessment of the implement surface. The description of all these features during the data acquisition process, the comparison with the original rough samples subjected to thin section analysis and the comparison with the reference collec- tion of microphotographs helped us to assign petrogenetic groups and types to every artefact. Figure 12 schematically relates both approaches. MA type (clastic fabric with matrix or non-quartz cement quartz arenite) is characterised through thin section by the association of clastic and cemented texture and floating or punctual packing, as well as by the presence of clastic quartz grains. This is reflected in hand specimen (non-destructive) characterisation through the association between saccharoidal texture and floating or punctual packing. Moreover, by the presence of either flat or irregular outlines on angular or rounded quartz grains because of matrix or cement. There is no presence of lustre or of micro-cracks. Some quartzites show bedding planes on their surfaces. In general, in this type, it is easy to recognise at least 25 different grains, although sometimes this task is complicated by the high presence of matrix or non-quartz cement (Fig. 13). OO type (syntaxially overgrown orthoquartzite) is characterised through thin section by the association of clastic grained texture, complete packing, the presence of evenly dis- tributed quartz with undulatory extinction, concavo-convex quartz limits and chiefly by syntaxial quartz overgrowths over the former quartz grains. This is reflected on the quartzite surface by the association between compact and grainy tex- ture, complete packing and flat and rounded quartz grain limits, surrounded by the halo of quartz overgrowth. Most of the samples have medium lustre, while others are more Page 13 of 23 32 Archaeol Anthropol Sci (2020) 12: 32 Fig. 12 Schematic representation of the features that define the groups and types of quartzites, showing the relationship between the petrographic and non- destructive petrological characterisation. In red, textural and packing and characterisation. In blue, the features of quartz grain and morphology. In black, other diagnostic features. The solid lines that link different columns represent the association of similar characteristics. The dashed lines represent unclear or infrequent relationships. Blue triangles represent relationships where the presence of a quartz grain feature conditions texture or packing. From petrographic analysis to binocular characterisation It is associated with MQ type (grain boundary migration recrystallised quartz- ite) is characterised through thin section by the association of clastic/foam texture, suturated packing, concavo-convex quartz grain limits, presence of deformation lamellae and 32 Page 14 of 23 Archaeol Anthropol Sci (2020) 12: 32 Fig. 13 Pictures at different magnifications of quartz arenites. From top to bottom, samples BR_01 (Beach river 01), an example of an MA type and ATS-002, a CA type. From left to right, the first two pictures are stereomicroscopic images at × 50/× 20 and × 250. The images on the right are thin sections under crossed polarisers Fig. 13 Pictures at different magnifications of quartz arenites. From top to bottom, samples BR_01 (Beach river 01), an example of an MA type and ATS-002, a CA type. From left to right, the first two pictures are stereomicroscopic images at × 50/× 20 and × 250. The images on the right are thin sections under crossed polarisers Fig. 14 Pictures at different magnifications of orthoquartzites. From top to bottom, samples ATS-023, an example of an OO type and ATS-302, a SO type. From left to right, the first two pictures are stereomicroscopic images at × 20 and × 250. The images on the right are thin sections under crossed polarised light Fig. 14 Pictures at different magnifications of orthoquartzites. From top to bottom, samples ATS-023, an example of an OO type and ATS-302, a SO type. From left to right, the first two pictures are stereomicroscopic images at × 20 and × 250. The images on the right are thin sections under crossed polarised light Page 15 of 23 32 Archaeol Anthropol Sci (2020) 12: 32 Fig. 15 Pictures at different magnifications of pure quartzites. From top to bottom, samples HA-5847, an example of an BQ type; ATS-195, an example of RQ sample; and HA-5842, an example of MQ sample. From left to right, the first two pictures are stereomicroscopic images at × 50/ 10/× 50 and × 250. The images on the right are thin sections und crossed polarised light Archaeol Anthropol Sci (2020) 12: 32 Page 15 of 23 3 Fig. 15 Pictures at different magnifications of pure quartzites. From top to bottom, samples HA-5847, an example of an BQ type; ATS-195, an example of RQ sample; and HA-5842, an example of MQ sample. From petrographic analysis to binocular characterisation From left to right, the first two pictures are stereomicroscopic images at × 50/× 10/× 50 and × 250. The images on the right are thin sections under crossed polarised light geochemical XRF data show any clear relationship with pet- rogenetic types. The influence of the mineralogy of the former sediment and post-depositional weathering could explain the variability of non-quartz minerals. It is important to highlight the limited accuracy of mineral detection just by observing the quartzite surface and also the higher influence of post- depositional weathering on the surface than in inner parts. stylolitic quartz grain limits. On the quartzite surface, it is related with soapy texture and the absence of quartz grain boundaries. Intensity of lustre is high, and there is an almost complete absence of micro-cracks on the surface. Foliation is not present (Fig. 15). Due to methodological limitations, grain size as observed with stereomicroscopy does not show any correlation with sizes measured in thin sections so we could only approximate a general increase in size for the type OO. Therefore, grain size determination is only used as a cumulative feature to describe variability within the types, as a possible conse- quence of the internal characteristics of the former sediment from which the quartzite was created. Management of quartzite in El Habario In addition, there is a moderate increase in the frequency of retouched artefacts with the increase in deformation/ metamorphic processes throughout the OO, SO and BQ types. We identified 71 cores. The most frequent is the irregular one, followed by discoid, core on flake, levallois and prismatic-shaped core (Online Resource 1). There is no clear correlation between type of core and petrogenetic quartzite type, even though most levallois cores are made on SO type. In addition, the representation of core on flake is different between petrogenetic types, especially comparing the orthoquartzite and quartzite groups. p p g Q yp There are clear differences in weight based on the tech- nological categories and petrogenetic types (Fig. 16). They are clear in core category because of the absence of OO and SO lighter than 40 g. This is especially significant when these cores are compared with those in RQ quartzite, all lighter than that weight. In addition, cores heavier than 600 g are limited to those made on OO and BQ petrogenetic types. Moreover, the mean weight indicates that heavier cores are made on OO and SO types, followed by BQ. Finally, RQ cores are the lightest. There are also differences in weight of knapping product. In general, there is a clear decrease in weight from CA quartz arenite to RQ quartzite. We identified 355 knapping products. The most frequent type is blank. Core preparation/rejuvenations products are scarce, forming less than 2% of the assemblage and they are only made on SO, BQ and MQ petrogenetic types. Regarding blanks, there are some differences between petrogenetic types and the quantity of negative scars on them, especially in the Table 2 Frequency table of petrological features identified in El Habario based on binocular characterisation. Management of quartzite in El Habario The seven proposed petrogenetic types could be identified by stereomicroscopy in the quarzitic implements (n = 467) from El Habario. Metamorphic quartzite is the best represented group owing to the high quantity of the BQ petrogenetic type, with > 50% of the assemblage. Sedimentary orthoquartzite is As discussed by Prieto et al. (2019) for the samples studied in thin sections, neither non-quartz mineralogy nor 32 Page 16 of 23 Archaeol Anthropol Sci (2020) 12: 32 comparison between CA and MQ types with all other types. In the first two types, all blanks possess at least two negative scars, while on the other types, each category is represented. There are more blanks with high quantity of negative scars on SO and RQ types than on BQ and OO types. The latter type is the quartzite with greatest frequency of blanks without nega- tive scars (Online Resource 2). The extension of cortex on dorsal surfaces is different on each petrogenetic type, especial- ly because of the absence of cortical surfaces on MQ blanks and the larger extension of them on CA blanks. In addition, blanks with broad cortex of more than 66% are more frequent in the OO type than in SO, BQ and RQ (Online Resource 3). the second-best represented group, and both petrogenetic types (OO and SO types) are similarly represented. Finally, the group of quartz arenite is underrepresented (Table 2). The most frequent grain size category is heterogeneous distribu- tion, in 47% of the pieces, even though homogeneous distri- bution is also well represented, in 37% of them. Regarding quartzite types and size varieties, nine preferential varieties are associated with OO, SO and BQ types and fine and medium grain sizes. The most frequent technological category is knapping product, followed by core, and chunk. Cores are restricted to orthoquartzite and quartzite groups (Table 3). Cores made on orthoquartzite and BQ type appear in similar percentages. There is a higher representation of knapping products in the OO, SO and BQ types than in RQ and MQ quartzites. Finally, chunks are only represented in MA, OO, SO and BQ types. According to the presence of retouch on different petroge- netic types, the CA and RQ types are, on proportion, more retouched than OO, SO, BQ and MQ types (Online Resource 4). Columns are petrogenetic types and rows contain the characteristics of grains according to size, classified first by distribution and second by size itself. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Management of quartzite in El Habario Petrogenetic type MA CA OO SO BQ RQ MQ Unknown Total % % % % % % % % % Grain size characterization Homogeneous and one mode distribution Fine grain 12 11 19 24 67 28 4 21 1 25 103 22 Medium grain 19 17 3 4 43 18 4 21 69 15 Coarse grain Heterogeneous and two modes distribution Fine grain 11 10 7 9 12 5 30 6 Medium grain 1 25 11 10 4 5 13 5 2 11 31 7 Coarse grain 1 1 1 2 0 Heterogeneous distribution Fine grain 25 23 30 38 41 17 5 26 3 75 3 30 107 23 Medium grain 1 100 2 50 30 27 15 19 60 25 4 21 112 24 Coarse grain 1 25 2 2 2 3 1 0 6 1 Unknown 7 70 7 1 Total 1 0 4 1 111 24 80 17 238 51 19 4 4 1 10 2 467 100 Columns are petrogenetic types and rows contain the characteristics of grains according to size, classified first by distribution and second by size itself. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Table 2 Frequency table of petrological features identified in El Habario based on binocular characterisation. Columns are petrogenetic types and rows contain the characteristics of grains according to size, classified first by distribution and second by size itself. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Columns are petrogenetic types and rows contain the characteristics of grains according to size, classified first by distribution and second by size itself. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Management of quartzite in El Habario The columns % are the percentage of each petrogenetic type in relation to each category of knapping product, while the columns rel.% are the percentage of each category of knapping product in relation to each petrogenetic type of quartzite. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases In addition, the weight of orthoquartzites and BQ type is more variable than RQ and MQ types. Finally, chunk weight also differs between the OO, SO and BQ petrogenetic types due to the decrease in mean weight and variability as defor- mation increases. the second most important one. Finally, the group of metamor- phic quartzite is the least frequent (19%) (Table 4). The most frequent grain size distribution category is homogeneous distri- bution around one mode in 46% of cases, even though general heterogeneous distribution is also well represented. Regarding grain size, the most frequent categories are fine and medium grain sizes. We identified six preferential varieties: three belong to OO type, one with fine size and homogeneous distribution and another two medium-sized varieties with homogeneous and heterogeneous distribution. Another two varieties are BQ and SO types with fine grain size and homogeneous distribution. The last one is the CA type with heterogeneous distribution and medium quartz grain sizes. Management of quartzite in El Habario Finally, values in bold italics are the categories representing between 1 and 5% of cases Page 17 of 23 32 Archaeol Anthropol Sci (2020) 12: 32 Archaeol Anthropol Sci (2020) 12: 32 Table 3 Frequency table of main technological categories identified in El Arteu grouped by petrogenetic types of quartzite Technological order Cores Knapping prdct. Chunk Total ∑ % rel.% ∑ % rel.% ∑ % rel.% ∑ % MA 1 2 100 1 0 CA 4 1 100 4 1 OO 14 20 13 90 25 81 7 17 6 111 24 SO 9 13 11 63 18 79 8 20 10 80 17 BQ 40 56 17 180 51 76 18 44 8 238 51 RQ 6 8 32 13 4 68 19 4 MQ 1 1 25 3 1 75 4 1 Undetermined 1 1 10 2 1 20 7 17 70 10 2 Total 71 15 355 76 41 9 467 100 Columns are the categories of knapping products and the total of items belonging to each petrogenetic type. The columns % are the percentage of each petrogenetic type in relation to each category of knapping product, while the columns rel.% are the percentage of each category of knapping product in relation to each petrogenetic type of quartzite. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Archaeol Anthropol Sci (2020) 12: 32 Page 17 of 23 32 Columns are the categories of knapping products and the total of items belonging to each petrogenetic type. The columns % are the percentage of each petrogenetic type in relation to each category of knapping product, while the columns rel.% are the percentage of each category of knapping product in relation to each petrogenetic type of quartzite. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Columns are the categories of knapping products and the total of items belonging to each petrogenetic type. Management of quartzite in El Arteu Six petrogenetic types have been identified in the quarzitic im- plements (n = 237) from El Arteu. Sedimentary orthoquartzite (47%) is the best represented group and quartz arenite (21%) is Fig. 16 Boxplot showing the distribution of weight (grams) of all lithic remains grouped first by technological order and second by petrogenetic type. There are another five quartzites heavier than 650 g not shown in the chart: one core and one knapping product on OO orthoquartzite weighing 1124.7 and 952.8 g and another three BQ quartzites cores with weights of 804.45, 722.62 and 689.34 g Themostfrequenttechnologicalcategoryisknappingproduct, followedbychunkandcore.ExceptfortheRQquartzite,coresare represented in every petrogenetic type (Table 5). Finally, chunks are well representedinthe MA, RQ and OO types, while they are underrepresented in the SO and CA petrogenetic types. We identified 17 cores in the whole collection. The most frequent type is core on flake, with eight, followed by discoid and irregular ones, represented by five and four items, respec- tively. There is only one prismatic-shaped core. There is no clear correlation between type of core and petrogenetic types of quartzite (Online Resource 5). One hundred seventy-four knapping products were identi- fied in the lithic assemblage from El Arteu. The most frequent type is blank, amounting to over 97% of the items. Core preparation/rejuvenation products are scarce and they are only represented on CA, OO and SO types. There is a great vari- ability in the number of negative scars on blanks depending on the quartzite types because of the smaller presence of negative Fig. 16 Boxplot showing the distribution of weight (grams) of all lithic remains grouped first by technological order and second by petrogenetic type. There are another five quartzites heavier than 650 g not shown in the chart: one core and one knapping product on OO orthoquartzite weighing 1124.7 and 952.8 g and another three BQ quartzites cores with weights of 804.45, 722.62 and 689.34 g Archaeol Anthropol Sci (2020) 12: 32 32 Page 18 of 23 Table 4 Frequency table of petrological features identified in El Arteu based on binocular characterisation. From a sample to the whole: losing precision to gain information Textural and quartz grain features through microscopic non- destructive techniques show an analogous association of The relationship between petrogenetic types and technology through weight demonstrates clear differences (Fig. 17). Cores Table 5 Frequency table of the categories of knapping products identified in El Habario grouped by the petrogenetic types of quartzite Technological order Cores Knapping product Chunk Total ∑ % % rel ∑ % % rel ∑ % % rel ∑ % MA 4 24 18 11 6 50 7 15 32 22 9 CA 1 6 3 25 14 86 3 7 10 29 12 OO 6 35 5 80 46 71 26 57 23 112 47 SO 2 12 9 19 11 86 1 2 5 22 9 BQ 2 12 5 30 17 77 7 15 18 39 16 RQ 5 3 71 2 4 29 7 3 Undetermined 2 12 33 4 2 67 6 3 Total 17 7 174 73 46 19 237 100 Columns are the main technological categories and the total of items of each raw material. The columns % are the percentage of each raw material in relation to each technological category, while the columns % rel. are the percentage of each technological category in relation to each type. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Technological order Cores Knapping product Chunk Total ∑ % % rel ∑ % % rel ∑ % % rel ∑ % MA 4 24 18 11 6 50 7 15 32 22 9 CA 1 6 3 25 14 86 3 7 10 29 12 OO 6 35 5 80 46 71 26 57 23 112 47 SO 2 12 9 19 11 86 1 2 5 22 9 BQ 2 12 5 30 17 77 7 15 18 39 16 RQ 5 3 71 2 4 29 7 3 Undetermined 2 12 33 4 2 67 6 3 Total 17 7 174 73 46 19 237 100 Columns are the main technological categories and the total of items of each raw material. The columns % are the percentage of each raw material in relation to each technological category, while the columns % rel. Management of quartzite in El Arteu Petrogenetic type MA CA OO SO BQ RQ MQ Unknow Total % % % % % % % % % Grain size characterisation Homogeneous and one mode Fine grain 3 10 41 37 13 59 20 51 2 29 79 33 Medium grain 1 3 25 22 1 5 3 8 1 17 31 13 Coarse grain Heterogeneous and two modes Fine grain 1 5 1 3 6 5 1 3 1 14 10 4 Medium grain 5 23 2 7 3 3 1 5 1 3 12 5 Coarse grain 8 36 2 7 1 14 1 17 12 5 Heterogeneous distribution Fine grain 1 5 2 7 10 9 2 9 7 18 2 29 24 10 Medium grain 5 23 16 55 22 20 5 23 6 15 1 17 55 23 Coarse grain 2 9 2 7 5 4 1 14 10 4 Unknown 1 3 3 50 4 2 Total 22 9 29 12 112 47 22 9 39 16 7 3 6 3 237 100 Columns are petrogenetic types and rows contain the characteristics of grains according to size, classified first by distribution and second by size itself. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Table 4 Frequency table of petrological features identified in El Arteu based on binocular characterisation. Columns are petrogenetic types and rows contain the characteristics of grains according to size, classified first by distribution and second by size itself. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases made on MA type are heavier than cores made on other petro- genetic types. Knapping products and chunks follow similar patterns, and there are none lighter than 5 g in the MA type. scars on the quartz arenite group than on orthoquartzites (Online Resource 6). Cortical areas on blanks are more fre- quent in the CA type, while they are uncommon in the others, especially in SO type (Online Resource 7). Management of quartzite in El Arteu SO orthoquartzites and the group of quartzite are, in general, more retouched than other types of quartzites. In addition, OO orthoquartzite is more retouched than the quartz arenite group (Online Resource 8). The OO type, followed by the SO and the BQ ones, are the petrogenetic types with the highest represen- tation of blanks with multiple primary types. From geoarchaeological characterisation to human knowledge features and forces to those appreciated through petrographic characterisation. Nevertheless, the diagenetic or metamorphic changes (the forces) that can be more accurately determined by petrographic analysis are not easily appreciated on the present surface of the rock as they are in thin section. However, the correlation between destructive petrography and stereomicroscope characterisation allows us to recognise some geologic processes in the rocks and, therefore, the pet- rogenetic types. In this way, it lets us extrapolate the informa- tion from the petrographic characterisation of a representative sample to the complete collection that we study by non- destructive methods. In El Habario site, the best represented quartzite type is BQ, followed by OO and SO types. Except for the OO type, with- out core preparation/rejuvenation products, all technological products are represented, indicating complete chaînes opératoires in all three types. The degree of exploitation of SO type and the quartzite group seems to be greater than of OO type (Fig. 18). The representation of quartz arenites is residual, showing only partial processes. MQ and RQ types are also slightly represented with a partial process of lithic reduction. Nevertheless, these chaînes opératoires are more complex, and they are more related with the last stages of the shaping processes. In quartz arenites, grains are easy to recognise and the borders from the former clastic sediment are well defined. They are only modified by the presence of matrix or cement. In this group, compaction and cementation are the diagenetic processes that consolidated former sediments into sedimenta- ry rocks. In the group of orthoquartzites, grain borders are not so easy to observe in hand specimens, since they are modified by silica overgrowth (more evident in the OO type) and the deformation of quartz grains. For these reasons, and also be- cause of the increase in undulatory extinction on quartz grains, lustre is more intense. The presence of micro-cracks is more frequent, due to the increase of compactness. In SO type, foliation is also a consequence of the deformation. Overgrowth and deformation are the main forces that affect and create these features on rock surfaces. In the group of metamorphic quartzites, individual grains could not be recognised due to high deformation, but also because of the presence of new small recrystallised grains that create a bright and crystalline surface. From a sample to the whole: losing precision to gain information are the percentage of each technological category in relation to each type. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Columns are the main technological categories and the total of items of each raw material. The columns % are the percentage of each raw material in relation to each technological category, while the columns % rel. are the percentage of each technological category in relation to each type. Values in italics are the categories representing more than 10% of the total cases. Values in bold are the categories representing between 5 and 10% of cases. Finally, values in bold italics are the categories representing between 1 and 5% of cases Archaeol Anthropol Sci (2020) 12: 32 Page 19 of 23 32 Fig. 17 Boxplot showing the distribution of weight in grams of all lithic remains grouped first by technological order and second by raw material in the chart on the left, and by petrogenetic type in the chart on the right. There are another four MA petrogenetic quartzite type heavier than 60 g, not shown in the chart (a 722-g chunk, a 296-g blank and two cores 192 and 263 g in weight) recognise the original quartz grains in hand specimens and sometimes also in thin sections. Despite the limitation imposed by grain size observation on quartzite surfaces, we could accomplish grain size measure- ment of each lithic piece using qualitative categories based on descriptive criteria for medium size and quartz grain distribu- tion. These criteria are based on petrographic characterisation of samples using a large quantity of high-resolution measure- ments (Prieto et al. 2019). These categories allow us to under- stand preferential varieties selected by prehistoric societies. Finally, mineral characterisation was not correlated with the information provided by other criteria. As discussed above, the influence of the mineralogy of the former sediment and especially post-depositional weathering could explain the variability of non-quartz minerals. Fig. 17 Boxplot showing the distribution of weight in grams of all lithic remains grouped first by technological order and second by raw material in the chart on the left, and by petrogenetic type in the chart on the right. From a sample to the whole: losing precision to gain information There are another four MA petrogenetic quartzite type heavier than 60 g, not shown in the chart (a 722-g chunk, a 296-g blank and two cores 192 and 263 g in weight) The proposed methodology helped us to understand not only a few samples of the quartzites, but the complete assemblage of these two Middle-Palaeolithic sites, and therefore, we were able to propose human activities carried out in both places. From geoarchaeological characterisation to human knowledge The progression of the degree of meta- morphism generates more crystalline structures, resulting in very smooth and flat surfaces that make it impossible to This information points to different management strategies depending on each quartzite type and related with the proper- ties of each one. We observe fragmented chaînes opératoires reflecting the creation of storage and toolkits on determined quartzite types, associated with the conglomerate outcrops near El Habario site, which contain rock pebbles of the petro- genetic types MA, OO, SO and BQ. BQ and SO type could be the main product of exploitation, while OO type could be a secondary product. We also observed the discarding of previ- ously stocked raw material and partial reparation and partial discard of toolkits made on materials unrepresented in the adjacent quartzite conglomerate (RQ and MQ types). In El Arteu, there is a higher presence of quartz arenites and OO type and a decrease in BQ and SO types. Complete chaînes opératoires on quartz arenites are clear although they are weakly exploited. The high representation of OO type and the presence of all technological products show that this quartzite was knapped in the site as the main raw material. 32 Page 20 of 23 32 Page 20 of 23 Archaeol Anthropol Sci (2020) 12: 32 Fig. 18 Systematic management strategies followed by human populations who inhabited El Habario and El Arteu, grouped by raw material and petrogenetic types. For abbreviations: MA for clastic fabric with matrix or non-quartz cement quartz arenite, CA for clastic quartz- arenite, OO for syntaxially overgrown orthoquartzite, SO for sutu grain orthoquartzite, BQ for bulging recrystallised quartzite, RQ subgrain rotation recrystallised quartzite and MQ for grain boundary gration recrystallised quartzite 32 Page 20 of 23 Archaeol Anthropol Sci (2020) 12 arenite, OO for syntaxially overgrown orthoquartzite, SO for sutured grain orthoquartzite, BQ for bulging recrystallised quartzite, RQ for subgrain rotation recrystallised quartzite and MQ for grain boundary mi- gration recrystallised quartzite Fig. 18 Systematic management strategies followed by human populations who inhabited El Habario and El Arteu, grouped by raw material and petrogenetic types. For abbreviations: MA for clastic fabric with matrix or non-quartz cement quartz arenite, CA for clastic quartz- et al. 2009; Turq et al. 2013, 2017). We also observed the use of CA quartz arenites and the OO type, probably obtained in the fluvial deposits near El Arteu. From geoarchaeological characterisation to human knowledge Their use is probably related to a versatility phenomenon rather than an expedient one (Vaquero and Romagnoli 2017). Its degree of exploitation shows that it was used intensively, similar to SO and BQ types, although they are less represent- ed. RQ type seems to be also intensively exploited, although not every technological product is represented (Fig. 18). This information points to different management strategies than at the previous site, probably due to the availability of different quartzite types. We observe fragmented chaînes opératoires which reflects the use of storage and toolkits, like those created in El Habario, certifying the fragmented charac- ter of Middle Palaeolithic stone tool technology (Meignen These data underline the complex management of raw ma- terial made by Middle-Palaeolithic societies to exploit their habitat in the Cantabrian Region, as it was also suggested by other studies (Carrión et al., 2008, 2013; Gonzalez-Urquijo et al. 2005). Once we could individualise each type of Archaeol Anthropol Sci (2020) 12: 32 Page 21 of 23 32 32 quartzite, we observed different managements guided by their features for knapping, their availability and probably by hu- man decisions on changeable circumstances. The coexistence of multiple methods of lithic reduction in the same layer could be the consequence of these issues, as it was also suggested by other researches along the Cantabrian Region (Carrión and Baena 1999; Baena et al. 2012; Maillo 2007; Rios-Garaizar 2010). The use of different environments where different types of quartzite (or other raw materials) was caught/ selected also contributes to this variability, as it was suggested by other authors (Castanedo 2001; Manzano et al. 2005; Rios- Garaizar 2012; Roy et al. 2017). Finally, lithic assemblages also show a complex management as a consequence of the different human decisions for taking advantage in the change- able environment where societies inhabited (Carrión et al. 2013; Cuartero et al. 2015; Santamaría et al. 2010). We would like to underline that these human and socio-economic stand- points based on complex management of lithic resources in Middle-Palaeolithic contexts are being proposed not only in the Cantabrian Region, but also in other parts of Europe (Richter 2016; Turq et al. 2013, 2017). All these data are transforming our chrono-cultural perspectives of lithic vari- ability into a more complex human, social and economic perspectives. we have filled the gap between petrographic and non- destructive characterisation using a solid geoarchaeological procedure. Conclusions Acknowledgements The authors thank Professor Javier Baena and MUPAC (Archaeological and Prehistorical Museum of Cantabria) for permission to study and sample the lithic archaeological material. The Department of Mineralogy and Petrology of UPV/EHU for access to the laboratories, thin-section preparation and X-ray fluoresce. The authors would like to thanks the two anonymous reviwers for their constructive comments which greatly improve this manuscript. A. Prieto is a collabo- rator in the project HAR2016-76760-C3-2-P funded by the Spanish Ministry of Science and FEDER funds. Our main goal in this paper was to explore and fill the gap between petrographic and non-destructive characterisation of quartzite. The starting point of this research is the characteri- sation of 18 samples through thin-section analysis (Prieto et al. 2019). This characterisation was based on qualitative (also quantitative) description of texture, packing and quartz grain features; the quantitative characterisation of size, morphology and orientation of quartz grains; and the determination of non- quartz minerals. This information helped us to understand the variability of the term quartzite but also to adopt seven petro- genetic types that cover the wide spectrum of rocks used under this term, from the pure sedimentary arenites, the more de- formed quartz arenite and the truly metamorphic quartzite types, all of them exploited as raw materials in Prehistory. This information is used to understand different features on the quartzite surface, such as texture, packing, quartz grain morphologies, lustre, presence of micro-cracks, bedding and foliation, and also, the grain size and the non-quartz minerals on the rock surfaces. All these characteristics have been pre- cisely described and they have been associated with the infor- mation derived by petrographic analysis. This procedure helps us to classify the different types of quartzite, not only for a sample of the assemblage but for the whole collection. In addition, we also establish different grain size varieties. Despite the lower resolution of the non-destructive approach, Funding information Open Access funding provided by Projekt DEAL. This research was partially supported by the project HAR2017-82483- C3-1-P financed by the Spanish Ministry of Science and the Consolidated Research Group in Prehistory of the Basque Country University (IT- 1223-19). A. Prieto is funded by the Education Department of the Government of the Basque Country through a postdoctoral fellowship (POS_2018_1_0021). From geoarchaeological characterisation to human knowledge An increase in the number of quartzites analysed, the application of data mining and machine learning software on micro-pictures or even better recognition of weathering effects could improve the presented methodology (Bustos- Pérez et al. 2019). This study also contributes to the knowledge of raw mate- rial management strategies during the Middle Palaeolithic in the Cantabrian Region. Management strategies are related with the creation of stores of blanks or cores of a determined type, their discard in a situation of abundance of raw material, their exploitation depending on the quartzite qualities or toolkit production and maintenance of determined types. All these behaviours are inserted in a fragmented management strategy taking into account the geographic and temporal var- iables. These data point at the variability and especially the versatility of management of quartzites by Middle Palaeolithic people in a complex economy and society (Carrión et al. 2013; Turq et al. 2013). The data suggest hidden strategies that could only be analysed by the geoarchaeological characterisation of quartzites. The application of the criteria to characterize the materials in potential source areas would enable the determi- nation of the mechanisms used for the selection of raw mate- rials and/or the short- and long-distance mobility circuits and territories of the societies that inhabited this area. References In: Vera JA (ed) Geología de España. SGE-IGME, Madrid, pp 25–49 García-Rojas M (2014) Dinámicas de talla y gestión de las materias primas silíceas a finales del Pleistoceno en el País Vasco. 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https://openalex.org/W2110354204	https://europepmc.org/articles/pmc4379615?pdf=render	English	null	In vitro antibacterial activities of compounds isolated from roots of Caylusea abyssinica	Annals of clinical microbiology and antimicrobials	2,015	cc-by	6,407	© 2015 Edilu et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Caylusea absyssinica, a plant used as vegetable and for medicinal purposes was selected for in vitro antibacterial evaluation in this study. The main aim of this study was to isolate compounds from the plant roots and evaluate their antibacterial activities on clinical bacterial test strains. Methods: Compounds from roots of Caylusea absyssinica (fresen) were identified based on observed spectral (1H-NMR, 13C-NMR and IR) data and physical properties (melting point) as well as reported literature. Disk diffusion method was employed to evaluate the antibacterial activities of the isolated compounds on four test bacterial strains namely, Staphylococcus aureus (ATCC25903), Escherichia coli (ATCC25722), Pseudomonas aeruginosa (DSMZ1117) and Salmonella thyphimurium (ATCC13311). Results: Two compounds, CA1 and CA2 were isolated from the methanol crude extract of the roots of Caylusea absyssinica (fresen). The compounds were identified as β-sitosterol and stigmasterol, respectively. Evaluation of antibacterial activities revealed that the compounds are active against all the bacterial strains in the experiment, showing inhibition zones ranging from 12 mm-15 mm by CA1 and 11 mm-18 mm by CA2 against the different test strains. However, the compounds were less active than the reference drug (Gentamycine), which showed minimum inhibition zone of 21 mm (Pseudomonas aeruginosa) and maximum of 28 mm (Escherichia coli) inhibition zone. Discussion and conclusion: The isolation of the compounds is the first report from roots of Caylusea abyssinica and could be potential candidates for future antibacterial drug development programs. Keywords: Disk diffusion method, β-sitosterol, Secondary metabolite, Stigmasterol Keywords: Disk diffusion method, β-sitosterol, Secondary metabolite, Stigmasterol Eastern African countries where the plant is found in abundance [2,3]. Abdissa Edilu1, Legesse Adane1 and Delelegn Woyessa2* Abdissa Edilu1, Legesse Adane1 and Delelegn Woyessa2* Abdissa Edilu1, Legesse Adane1 and Delelegn Woyessa2* * Correspondence: woyessa@yahoo.com 2Department of Biology, College of Natural Sciences, Jimma University, P.O. Box 5140 Jimma, Ethiopia Full list of author information is available at the end of the article Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 DOI 10.1186/s12941-015-0072-6 Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 DOI 10.1186/s12941-015-0072-6 Open Access Introduction Medicinal plants have been used as important drug sources and also to treat various microbial infections [1]. The search for alternative drug sources is currently re- ceiving due attention to tackle the problem of increasing multiple drug resistant microorganisms. Caylusea Absys- sinica (fresen) is a plant that belongs to the family of Resedaceae. It is an erect herb with height up to 1.5 m tall, and with slightly woody taproot and glabrous stem. This plant mostly grows in open grass land, fields, road sides and rocky areas at 1500–2750 m above sea level. It is distributed in Mediterranean region, and Northern and Eastern Africa. Sudan, Ethiopia, Kenya, Uganda, Rwanda, Burundi, Tanzania and Malawi are some of the Previous report indicates the use of Caylusea absyssi- nica as vegetable and medicinal plant [4]. For instance, in Tanzania and Ethiopia, its leaves and stems are eaten alone or as vegetables [2,5]. The plant is also known for its medicinal use by people living in areas where it is growing. Its leaves are used to treat stomachache, skin diseases diabetes mellitus and amoebiasis [6-8]. Simi- larly, its roots are used to treat abdominal pain impo- tency and Scabies diarrhea and expel intestinal parasites in humans [9-11]. In Ethiopia, the plant is traditionally used to treat internal diseases, fever, shivering and skin diseases of domestic animals [6,12]. There are some attempts to explore the potentials of crude extracts of Caylusea abyssinica against different human diseases [13]. In the report, 80% methanolic ex- tract of leaves of the plant showed antidiabetic and oral * Correspondence: woyessa@yahoo.com 2Department of Biology, College of Natural Sciences, Jimma University, P.O. Box 5140 Jimma, Ethiopia Full list of author information is available at the end of the article Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 Page 2 of 8 glucose tolerance improving actions, particularly at the dose of 200 mg/kg in experimental animals. The report also supported the prevailing traditional claims of the leaves of Caylusea abyssinica for management of dia- betes mellitus [7]. voucher number AE 001 was deposited at the Herbarium of the Department of Biology, Jimma University. The col- lected plant material was chopped into small pieces and air-dried under shade without exposing it to direct sun- light and the dried plant material was grounded to 0.5 μm sizes. Introduction Isolation of some compounds from different morpho- logical parts of the plant has been reported. For instance, isolation of 3-(3-carboxyphenyl) alanine, (3-carboxyphe- nyl) glycine,3-(3-carboxy-4-hydroxyphenyl) alanine, and (3-carboxy-4-hydroxyphenyl)-glycine, in low concen- tration 2-aminoadipic acid, saccharopine [(2S, 2′S)-N6- (2-glutaryl)lysine] and some γ-glutamyl peptides have been reported from the leaves of Caylusea abyssinica [14]. Phytochemical screening of leaves of 80% metha- nolic crude extract of Caylusea abyssinica revealed the presence of various secondary metabolites such as al- kaloids, cardiac glycosides, reducing sugars, steroidal compounds and phenolic compounds, tannins, sapo- nins and flavonoids [13]. However, there is a dearth of study regarding evaluation of compounds from Caylu- sea abyssinica on bacterial pathogens. To the best of our knowledge, there is no report so far on isolation of compounds from the roots of this plant for antibacter- ial activity tests. This work is, therefore, initiated to isolate compounds from roots of Caylusea abyssinica and test their antibacterial activity on four test bacter- ial strains. Chemicals and apparatus General laboratory grade solvents (methanol, acetone, chloroform, petroleum ether and ethyl acetate) were used for gradient extraction and column elution. The materials used for chromatographic analyses were silica gel (60–120 mesh size) and pre-coated TLC (silica gel, UV254). A standard antibiotic disc (Gentamycin 10 μg) was used as a reference drug, and Mueller Hinton agar and Nutrient broth were used for preparation of culture media for the antibacterial activity test (experiment). 1H- NMR, 13C-NMR and DEPT-135 were recorded using Bruker Advance 400 MHz spectrometer. CDCl3 was used as a solvent in all NMR spectroscopic analyses. The Infrared (IR) spectra (KBr) data were obtained from Perkin-Elmer BX infrared spectrometer (400–4000 cm−1). Melting point apparatus (Griffin) was used for melting point determination. Extraction For preliminary antibacterial activity test, 100 g of the powdered plant material was sequentially extracted with petroleum ether (least polar), chloroform, acetone and methanol (most polar) using maceration technique with continuous shaking (at 25°C for 72 hrs) using a shaker (GSL 400). The extracted matter from each solvent was filtered first using a cotton plug followed by Whatman No 1 filter paper. The filtrates were concentrated using rotary evaporator (Laborota 4000) under reduced pres- sure. The resulting crude extracts were weighed and stored in refrigerator at 4°C. After comparing the anti- bacterial activities of the crude extracts of the solvent systems [15], the methanol extract was chosen for fur- ther study based on its better inhibitory effect against test strains for chromatographic isolation of its constitu- ents. Then, a bulk of the powdered material (1000 g) was subjected to extraction employing the same proced- ure (gradient extraction) to afford 28.2 g crude methanol extract. Isolation and characterization of compounds ff l f l h Isolation and characterization of compounds Effective solvent system for column chromatography was selected for elution after carrying out the TLC of the methanol extract in variable combinations of sol- vents like petroleum ether, ethyl acetate, chloroform and methanol alternatively. Among all combination of sol- vents, petroleum ether:ethyl acetate combination showed superior resolution of the components of the extract on TLC plate. Therefore, these combinations of solvents of varying polarity were used for elution of column chro- matography. About 12 g of methanol crude extract of the roots Caylusea abyssinica was subjected to column chromatography (CC) that was packed with silica gel to isolate compounds. A glass column was packed with 120 g silica gel slurry dissolved in petroleum ether. The crude material was adsorbed onto 12 g of dry silica gel. Then the solvent was allowed to evaporate, and the dry sample adsorbed to the silica gel was applied into the column that was already packed with silica gel. The col- umn was then eluted with a mixture of petreoleum ether:ethyl acetate gradually increasing the polarity (i.e., 100:0%, 98:2%, 96:4%, up to 80:20%). A total of 293 frac- tions each with 20 ml were collected and solvent was re- moved under reduced pressure using rota vapor (under reduced pressure). The developed spots on TLC plates were visualized under UV light at 254 and 365 nm and Evaluation of antibacterial activity Staphylococcus aureus (ATCC25903), Escherichia coli (ATCC25722), Pseudomonas aeruginosa (DSMZ1117) and Salmonella thyphimurium (ATCC13311) were clin- ical test isolates used for antibacterial activity tests. All the test strains were from the Post graduate and Re- search Laboratory of Biology Department, Jimma Uni- versity. The antibacterial activity tests were carried out using a standard procedure [16]. All bacterial cultures were first grown on 5% sheep red blood agar Petri plates at 37°C for 24 hrs prior to inoculation onto the nutrient agar. Few colonies (4 to 5) of similar morph- ology of the respective bacteria were transferred with a sterile inoculating loop to a nutrient broth liquid medium and this liquid culture was incubated until ad- equate growth of turbidity equivalent to McFarland 0.5 turbidity standard was obtained. The turbidity of the actively growing broth culture was adjusted with sterile saline solution to obtain turbidity optically comparable to that of the 0.5 McFarland standard that was resulted in a suspension containing approximately 1–2 × 108 CFU/ ml for the test strains. The respective bacterial culture was streaked onto the Muller-Hinton agar Petri plates using a sterile swab to ensure thorough coverage of the plates and a uniform thick lawn of growth following incubation. Then 6 mm diameter sterile discs (Whatmann No.3 paper) were placed on the surface of the inoculated agar approximately at equal distance of corners in Petri plates in triplicates and 50 mg/ml concentration of the test solutions that were prepared by dissolving 500 mg of isolated compounds in 10 ml of DMSO were also ap- plied onto the discs using micropipette. After addition of test solutions on the discs, they were allowed to dif- fuse for 5 minutes and the Petri plates were then kept in an incubator at 37°C for 24 hrs. The antibacterial ac- tivity was evaluated after 24 hrs by measuring the diameter of zone of growth inhibitions surrounding the discs (in mm) using transparent ruler. In this ex- periment, Gentamycin (10 μg) and dimethyl sulfoxide (DMSO) were also used as positive and negative con- trols respectively Two compounds (compound CA1 and CA2) were iso- lated from 12 g of methanol crude extract of the roots of Caylusea abyssinica using column chromatographic separation. Compound CA1 and compound CA2 were obtained by combining fractions 43–56 and 61–73, re- spectively, of the column chromatography (Figure 1). Evaluation of antibacterial activity Further characterization of the compounds revealed the compounds to be β-sitosterol [12,13] and stigmasterol [17,25,26], respectively. Collection and preparation of plant material The root of Caylusea absyssinica was collected in November 2012 from the area surrounding of King Abba Jifar Palace, near Jimma town, Ethiopia. The town’s geographical coordinates are approximately 7°41′N lati- tude and 36°50′E longitude. Botanical identification of the plant was made by a Botanist, and a specimen with Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 Page 3 of 8 Page 3 of 8 bacterial activity test of compounds CA1 and CA2 Antibacterial activity test of compounds CA1 and CA2 Subjecting the compounds to antibacterial activity tests indicated that the antibacterial activities of the isolated compounds were lower than that of the reference drug (Gentamycin 10 μg) against all the bacterial strains used in the experiment and their growth inhibition values were also comparable to each other (Table 1). p As revealed from the current study, the growth inhibi- tory activities of compound CA1 are almost the same against the four bacterial strains used in the experiment. i.e., 15 mm, 13 mm, 13 mm and 12 mm for S. aureus, E. coli, P. aeruginosa and S. thyphimurium, respectively (Table 1). The corresponding activity of Gentamycin against the test strains was found to be 20 mm, 28 mm, 21 mm and 23 mm, respectively. The observed antibac- terial activity data of the compound CA1 are in good agreement with corresponding reported antibacterial ac- tivity of β-sitosterol against the same bacterial strains. The reported growth inhibitions were 11 mm, 13.5 mm, 8.5 mm and 12 mm against S. aureus, E. coli, P. aerugi- nosa and S. thyphimurium, respectively [17]. There are also reports that discuss low/moderate antibacterial ac- tivity of β-sitosterol against several bacterial species such as S. aureus, E. coli, and P. aeruginosa [18-22]. Similarly, the observed antibacterial activities of compound CA2 against the three test bacterial strains (S. aureus, P. aeru- ginosa and S. thyphimurium) were found to be compar- able to each other. The corresponding growth inhibitory activities (in mm) were 12, 11 and 13 against S. aureus, P. aeruginosa and S. thyphimurium, respectively. How- ever, it was found to show relatively superior antibacter- ial activity (i.e., 18 mm) against E. coli. (Table 1). The results of the present study are consistent with previous reports showing low to moderate antibacterial activity of stigmasterol against S. aureus, E. coli, P. aeruginosa and S. thyphimurium [18,23,24]. For instance, it has been re- ported that growth inhibition values (in mm) of stigmas- terol were 13.5, 14, 9.5 and 13 against S. aureus, P. aeruginosa and S. thyphimurium, respectively [25]. Results and discussion then by exposure to iodine chamber. The fractions that showed the same TLC development profiles (color and Rf) were combined and concentrated to dryness under reduced pressure using rotary evapor- ator. The structures of the compounds were elucidated based on combined spectral data which include infra red, nuclear magnetic resonance (1H-NMR, 13C-NMR and DEPT-135) spectra data and melting point values as well as comparison of these data with reported data in literature. All the spectroscopic analyses were car- ried out at the Department of Chemistry, Addis Ababa University. Structural elucidation of the isolated compounds Structural elucidation of compound CA1 thyphimuriu 1 Compound CA1 15 13 13 12 2 Compound CA2 12 18 11 13 3 DMSO - - - - 4 Gentamycin 20 28 21 23 Table 1 Inhibition zone (in mm) of the test compounds (compound CA1 and compound CA2) at 5 reference compound (Gentamycin 10 μl) on zone (in mm) of the test compounds (compound CA1 and compound CA2) at 50 mg/L and the ound (Gentamycin 10 μl) e (in mm) of the test compounds (compound CA1 and compound CA2) at 50 mg/L and the could indicate alcoholic C-O stretching. The observed IR spectrum of compound CA1 agrees with the re- ported IR spectra of β-sitosterol [12,13]. This claim is further confirmed by NMR spectroscopic and physical property data. The 1H-NMR spectrum of compound CA1 (Additional file 2), showed the presence of six me- thyl groups corresponding to peaks at δ0.6 (3H, CH3-18), 0.82 (3H, CH3-29), 0.84 (3H, CH3-26), 0.86 (3H, CH3-27), 0.88 (3H, CH3-21) and 1.02 (3H, CH3-19). The peak at δ2.25 (2H, CH2-4) was attributed to CH2 hydrogen atoms on the fourth carbon. The peak at δ3.53 shows the presence of a proton attached to hydroxyl group bearing carbon (i.e., C-3). The peak at δ5.37 indicates presence of a proton attached to olefinic C-C bond (i.e., C-6) (Figure 2) as reported in [26]. chromatographic elution by petroleum ether: ethyl acet- ate (98:2%). Its Rf value was determined to be 0.49 in petroleum ether:ethyl acetate (90:10%) as a solvent system. The IR spectrum of compound CA1 (Additional file 1) showed a broad band at 3438 cm−1 that indicated the presence of -OH group. There is no broad band in the range of 3400–2400 cm−1 to be associated with -OH group of carboxylic acids. Moreover, absence of a band at about 1700 cm−1 also confirmed that the compound is not a carbonyl compound (or carboxylic acids). There- fore, the band at 2996 cm−1 could indicate C-H stretching of alkenes whereas bands at 2853 cm−1 and 2938 cm−1 could be attributed to C-H stretching of CH3 and CH2 groups, respectively. The presence of a band at 1174 cm−1 Caylusea absyssinica (1000 g) soacked with 5 L pet. ether for 72 hrs over shaker bath at 25oC. Structural elucidation of the isolated compounds Structural elucidation of compound CA1 Compound CA1 was obtained as white crystal that was isolated from the combined fractions 43–56 of column Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 Page 4 of 8 Page 4 of 8 chromatographic elution by petroleum ether: ethyl acet- ate (98:2%). Its Rf value was determined to be 0.49 in petroleum ether:ethyl acetate (90:10%) as a solvent system. The IR spectrum of compound CA1 (Additional file 1) showed a broad band at 3438 cm−1 that indicated the presence of -OH group. There is no broad band in the range of 3400–2400 cm−1 to be associated with -OH group of carboxylic acids. Moreover, absence of a band at about 1700 cm−1 also confirmed that the compound is not a carbonyl compound (or carboxylic acids). There- fore, the band at 2996 cm−1 could indicate C-H stretching of alkenes whereas bands at 2853 cm−1 and 2938 cm−1 could be attributed to C-H stretching of CH3 and CH2 groups, respectively. The presence of a band at 1174 cm−1 Table 1 Inhibition zone (in mm) of the test compounds (comp reference compound (Gentamycin 10 μl) S. no. Compounds S. aureus 1 Compound CA1 15 2 Compound CA2 12 3 DMSO - 4 Gentamycin 20 Caylusea absyssinica (1000 g) soacked with 5 L pet. e over shaker bath at 25o Marc 1 Soaked w for 72 hrs Extract A 9.2 g Extract B 15.3 g Mar Extract C 19.4 g Extract D 28.2 g 100% 98:2 96:6 94:4 92: 8 Fr 43 56 Fr 61 73 Table 1 Inhibition zone (in mm) of the test compounds (compound CA1 and compound CA2) at 50 mg/L and the reference compound (Gentamycin 10 μl) S. no. Compounds S. aureus E. coli P. aeruginosa S. thyphimurium 1 Compound CA1 15 13 13 12 2 Compound CA2 12 18 11 13 3 DMSO - - - - 4 Gentamycin 20 28 21 23 Table 1 Inhibition zone (in mm) of the test compounds (compound CA1 and compound CA2) at 50 mg/L and the reference compound (Gentamycin 10 μl) S. no. Compounds S. aureus E. coli P. aeruginosa S. Structural elucidation of the isolated compounds Structural elucidation of compound CA1 Marc 1 Soaked with 5 L of Chloroform for 72 hrs over shaker bath at 25oC Extract A 9.2 g Extract B 15.3 g Marc 2 Extract C 19.4 g Soaked with 5 L of acetone for 72 hrs over shaker bath at 25oC Soaked with 5 L of methanol for 72 hrs over shaker bath at 25oC Extract D 28.2 g Discarded Mark 100% 98:2 96:6 94:4 92: 8 90:10 88:2 86:4 84:6 Fr 43-56 Compound CA 1 Fr 61-73 compound CA-2 Mark Figure 1 General procedures followed in the extraction and isolation of compounds from the roots of Caylusea abyssinica. Caylusea absyssinica (1000 g) soacked with 5 L pet. ether for 72 hrs over shaker bath at 25oC. rc 1 Soaked with 5 L of Chloroform for 72 hrs over shaker bath at 25oC Marc 1 Marc 2 Soaked with 5 L of acetone for 72 hrs over shaker bath at 25oC Mark Soaked with 5 L of methanol for 72 hrs over shaker bath at 25oC Fr 61 73 compound CA-2 General procedures followed in the extraction and isolation of compounds from the roots of Caylusea abyssinica. Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 Page 5 of 8 HO 1 2 3 4 5 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 6 8 Figure 2 The proposed chemical structure compound CA1 (i.e., β-sitosterol). The observed NMR data of compound CA1 and that of reported data of β-sitosterol are given in Table 2. The 13C-NMR and DEPT-135 spectra of compound CA1 (Additional files 3 and 4) revealed that the compound CA1 possesses a total of 29 carbons atoms. Among these carbons, eleven carbons were found to be CH2 carbons whereas nine CH and six CH3 carbons. The peaks at δ140.8 and 121.7 can be assigned to highly deshielded carbon atoms of C = C double bond. Thus, peaks values at δ140.8 and 121.7 can be assigned to C-5 and C-6, re- spectively [17-19]. The peak at δ71.8 can be assigned to carbon atom bearing hydroxyl group (i.e., C-3) [19]. Moreover, the DEPT-135 spectrum confirmed that the peaks at δ140.7, δ36.5 and δ42.3 indicate quaternary carbon atoms. The observed 13C-NMR and DEPT-135 Figure 2 The proposed chemical structure compound CA1 (i.e., β-sitosterol). Structural elucidation of the isolated compounds Structural elucidation of compound CA1 Table 2 13C-NMR, 1H-NMR and DEPT-135 data of CA1 in comparison with reported data of β-sitosterol C. no. 13C-NMR data of compound CA1 Reported 13C-NMR data of β-sitosterol [12-14,17] 1H-NMR data of compound CA1 Reported 1H-NMR data of β-sitosterol [12-14,17] DEPT-135 of compound CA1 Nature of C atoms based on DEPT-135 1 37.25 37.3 37.25 CH2 2 31.64 31.6 31.64 CH2 3 71.82 71.8 3.54 3.53 71.83 CH 4 42.28 42.2 42.28 CH2 5 140.74 140.8 - C 6 121.74 121.7 5.37 5.37 121.74 CH 7 31.93 31.9 31.93 CH2 8 31.93 31.9 31.93 CH 9 50.12 51.2 50.12 CH 10 36.51 36.5 - C 11 21.08 21.1 21.08 CH2 12 39.76 39.8 39.77 CH2 13 42.28 42.3 - C 14 56.76 56.8 56.76 CH 15 24.31 24.3 24.31 CH2 16 28.26 28.3 28.26 CH2 17 56.04 56.0 56.05 CH 18 11.98 11.9 0.69 0.69 11.99 CH3 19 19.41 19.4 1.02 1.02 19.41 CH3 20 36.51 36.2 36.56 CH 21 18.78 18.8 0.88 0.88 18.79 CH3 22 33.93 33.9 33.94 CH2 23 26.04 26.1 26.04 CH2 24 45.82 45.9 45.82 CH 25 29.13 29.2 29.13 CH 26 19.83 19.8 0.84 0.84 19.83 CH3 27 19.41 19.3 0.86 0.86 19.41 CH3 28 23.06 23.1 23.06 CH2 29 12.27 12.2 0.82 0.81 12.20 CH3 d DEPT-135 data of CA1 in comparison with reported data of β-sitosterol Table 2 13C-NMR, 1H-NMR and DEPT-135 data of CA1 in comparison with reported data of β-sitost ble 2 13C-NMR, 1H-NMR and DEPT-135 data of CA1 in comparison with reported data of β-sitostero Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 Page 6 of 8 HO 1 2 3 4 5 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 6 8 Figure 3 The chemical structure of compound CA2 (i.e., Stigmasterol). spectral data of compound CA1 (Table 2) are also con- sistent with the literature reported data β-sitosterol [17,25,26]. The observed melting point value (133–135°C) was also comparable with the reported melting point value of β-sitosterol (i.e. 135–136°C) [27]. Therefore, based on the observed spectral and melting point data, compound CA1 is most likely β-sitosterol. Structural elucidation of CA2 Compound CA2 was obtained as a white crystalline solid by combining fractions 61–73 of column chromato- graphic separation that was eluted by a solvent system of petroleum ether:ethyl acetate (96:4%). Its Rf value was determined to be 0.60 in petroleum ether:ethyl acetate (80:20%) solvent system (Table 2). Figure 3 The chemical structure of compound CA2 (i.e., Stigmasterol). Table 3 The observed 13C-NMR, DEPT-135 and 1H-NMR data of compound CA2, and the reported 13C-NMR and 1H-NMR data of stigmasterol 13C-NMR, DEPT-135 and 1H-NMR data of compound CA2, and the reported 13C-NMR and 1H-NMR ble 3 The observed 13C-NMR, DEPT-135 and 1H-NMR data of compound CA2, and the reported 13C- ta of stigmasterol Table 3 The observed 13C-NMR, DEPT-135 and 1H-NMR data of compound CA2, and the reported 13C-NMR and 1H-NMR data of stigmasterol C. no. 13C-NMR data of compound CA2 The reported 13C-NMR data of stigmasterol [12-14,17] DEPT-135 data of compound CA2 1H-NMR data of compound CA2 The reported 1H-NMR data of stigmasterol [12-14,17] Dept-135 based nature of the carbon 1 37.2 37.3 37.2 CH2 2 31.6 31.6 31.6 CH2 3 71.8 71.8 71.8 3.44 3.45 CH 4 42.2 42.3 42.3 CH2 5 140.7 140.8 - C 6 121.7 121.7 121.7 5.26 5.33 CH 7 31.9 31.9 31.9 CH2 8 31.9 31.9 31.9 CH 9 50.1 51.2 50.1 CH 10 36.5 36.5 - C 11 21.0 21.1 21.0 CH2 12 39.7 39.7 39.7 CH2 13 42.3 42.3 - C 14 56.8 57.9 56.7 CH 15 24.3 24.4 24.4 CH2 16 28.9 28.4 28.2 CH2 17 55.9 56.1 55.9 CH 18 12.2 11 12.2 0.61 0.68 CH3 19 19.4 21.2 19.4 0.93 1.02 CH3 20 40.5 40.5 40.5 CH 21 21.1 21.2 21.1 0.76 1.01 CH3 22 138.3 138.3 138.3 5.06 5.12 CH 23 129.2 129.3 129.2 4.96 4.98 CH 24 51.2 51.2 51.2 CH 25 33.9 31.9 33.7 CH 26 19.0 19.0 19.0 0.72 0.86 CH3 27 21.2 21.2 21.2 0.74 0.71 CH3 28 25.4 25.4 25.4 CH2 29 12.0 12.1 11.8 0.60 0.78 CH3 Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 Page 7 of 8 Page 7 of 8 Page 7 of 8 Edilu et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:15 The IR (KBr) spectra of compound CA2 (Additional file 5) showed a broad band at 3424 cm−1 which indi- cates the presence of -OH group. Structural elucidation of CA2 Absence of a broad band in between 3400-2400 cm−1 and a strong band at around 1700 cm−1 indicated that the compound is not a carboxylic acid (carbonyl compound). Thus, compound CA2 is most likely an alcohol. The band at 2996 cm−1 could indicate C-H stretching of alkenes whereas bands at 2860 cm−1 and 2924 cm−1 could be attributed to C-H stretching of CH3 and CH2 groups, respectively. The ab- sorption band at 1657 cm−1 is assigned C-C stretching band of C = C double bond. The band at 1465 cm−1 could be due to the C-H bending of CH2, and the band at 1375 cm−1 represents C-H bending of CH3. The ab- sorption band at 1055 cm−1 corresponds to C-C stretch- ing. The observed IR, 1H-NMR and 13C-NMR spectral data (Figure 3) and literature reported data suggested that compound CA2 is most likely stigmasterol [17,25,26]. Conclusions In vitro antibacterial activity test results against the four bacterial strains (S. aureus, E. coli, P. aeruginosa and S. thyphimurium) used in the experiment, both compounds (CA1 and CA2) showed lower (but moderate) antibac- terial activities than the reference drug (Gentamycin). Though lower than that of the reference drug, the ob- served antibacterial activities of the isolated compounds could give insight about the potentials the compounds as lead compound in development of antibacterial drugs. However, further tests are recommended on large num- ber of bacterial strains to decide their potential as candi- dates in development of antibacterial drugs. Authors’ contributions AE participated on designing the study, carried out the research, collected and analyzed data and wrote the manuscript. LA and DW participated in planning and designing of the study, supervised field and laboratory data collection and analysis, wrote up of the manuscript and prepared for publication. All authors have read and approved the final manuscript. Competing interests h h d l h p g The authors declare that they have no competing interests. The observed 13C-NMR (and DEPT-135) data were also found to be consistent with that of stigmasterol (Table 3). 13C-NMR and Dept-135 spectra of compound CA2 (Additional files 7 and 8) showed presence of a total of 29 carbons atoms in the structure. The peaks were related to six methyl, nine methylene, eleven me- thane and three quaternary carbon atoms. The peaks at δ140.7, 121.7, 138.3 and 129.2 in the 13C-NMR spectrum are assigned to C-5, C-6, C-22 and C-23 of C = C double bonds, respectively. The peaks at δ140.7 and 121.7 are assigned to C = C double bond carbons (C-5 and C-6, respectively) in the cyclic structure of the compound. Reports showed that the existence of unsaturation be- tween C-5 and C-6 introduces easily recognizable signals at δ141.2 ± 0.8 and 121 ± 0.4 [14]. The peaks at δ138.3 and 129.2 assignable to the external C = C double bond carbon atoms, and the peak at δ71.8 is associated to the β hydroxyl carbon of C-3 [13,14]. The DEPT-135 spectrum of compound CA2 also confirmed that the peaks at δ140.7, δ36.5 and δ42.3 (Table 2) indicate quaternary carbon atoms. Thus, based on the above spectral data, and also comparing with literature reports, compound CA2 is found to be identical with stigmasterol (Figure 3). The observed mp value (i.e., 173–176°C) was also found to be comparable with the reported mp value (i.e., 174–176°C) [28]. This is the first report on isolation of stigmasterol from Caylusea abyssinica as well as its family. Additional files Additional file 1: IR spectrum of compound CA1. Additional file 2: The 1H-NMR spectrum of compound CA1. Additional file 3: The 13C-NMR Spectra of compound CA 1. Additional file 4: The DEPT-135 spectrum of compound CA 1. Additional file 5: The IR spectrum of compound CA2. Additional file 6: The 1H-NMR spectrum of compound CA2. Additional file 7: The 13C-NMR spectrum of compound CA2. Additional file 8: The DEPT-135 spectrum of compound CA2. The 1H-NMR spectrum of compound CA2 (Additional file 6) showed methyl proton peaks at δ0.60 (3H, CH3-29), 0.61 (3H, CH3-18), 0.72(3H, CH3-26), 0.74 (3H, CH3-27), 0.76 (3H, CH3-21) and 0.93 (3H, CH3-19). The peak at δ3.53 indicates proton of hydroxyl group attached to C-3 (i.e., 1H, −OH on C-3). 1H-NMR spectrum also showed peaks at δ4.98, 5.06, and 5.26 indicating presence of three protons corresponding to that of a trisubstituted and a di- substituted C = C bonds (Table 3). Acknowledgment The authors are thankful to the Department of Chemistry (Addis Ababa University) for providing spectral analyses service, and the Department of Biology (Jimma University) for providing test strains and lab facilities to conduct antibacterial activity tests. Jimma University deserves acknowledgement for financial support of the research work. Author details 1 1Department of Chemistry, College of Natural Sciences, Jimma University, Jimma, Ethiopia. 2Department of Biology, College of Natural Sciences, Jimma University, P.O. Box 5140 Jimma, Ethiopia. Received: 3 December 2014 Accepted: 27 February 2015 Received: 3 December 2014 Accepted: 27 February 2015 References Mesfin F, Demissew S, Teklehaymanot T. An ethnobotanical study of medicinal plants in Wonago Woreda, SNNPR, Ethiopia. J Ethnobiol Ethnomed. 2009;5:5–28. 12. Tesfaye A, Demissew S. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 24. 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https://openalex.org/W3159717549	https://www.researchsquare.com/article/rs-429333/latest.pdf	English	null	Long-term cognitive deficits after traumatic brain injury associated with microglia activation	Clinical immunology	2,021	cc-by	10,612	Long-Term Cognitive Deficits After Traumatic Brain Injury Associated With Microglia Activation Esber Saba American University of Beirut Mona Karout American University of Beirut Leila Nasralla American University of Beirut Firas Kobeissy American University of Beirut Hala Darwish American University of Beirut Samia Khoury (  sk88@aub.edu.lb ) American University of Beirut Research Article Posted Date: June 30th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-429333/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Clinical Immunology on September 1st, 2021. See the published version at https://doi.org/10.1016/j.clim.2021.108815. Page 1/27 Abstract Traumatic Brain Injury (TBI) is the most prevalent of all head injuries, and based on the severity of the injury, it may result in chronic neurologic and cognitive deficits. Microglia play an essential role in homeostasis and diseases of the central nervous system. We hypothesize that microglia may play a beneficial or detrimental role in TBI depending on their state of activation and duration. In the present study, we evaluated whether TBI results in a spatiotemporal change in microglia phenotype and whether it affects sensory-motor or learning and memory functions in male C57BL/6 mice. We used a panel of neurological and behavioral tests and a multi-color flow cytometry-based data analysis followed by unsupervised clustering to evaluate isolated microglia from injured brain tissue. We characterized several microglial phenotypes and their association with cognitive deficits. TBI results in a spatiotemporal increase in highly activated microglia that correlated negatively with spatial learning and memory at 35 days post-injury. These observations could define therapeutic windows and accelerate translational research to improve patient outcomes. Introduction Microglia are the central nervous system (CNS) resident innate immune cells that play critical physiological roles in the healthy and injured brain. They detect and rapidly respond to any disruption in the status quo of the CNS, including infections or tissue injury, and often act to remove cellular debris 1. The activation of microglia from their resting surveillance state occurs within minutes of the injury and is critical for recovery. However, prolonged activation may be detrimental and contribute to secondary damage. When the microglia are activated in response to any disruption, this activation alters their gene expression and morphology 2. Although they have distinctive lineage, microglia resemble blood-derived macrophages that infiltrate the CNS from the periphery in response to tissue damage 3. Microglia are usually classified into the classical M1 phenotype that is considered proinflammatory, and the M2 anti- inflammatory is thought to be involved in neural repair 4. However, more recent data suggest that microglia's activation status is on a continuum between anti- and pro-inflammation rather than a rigid dichotomous phenotype 5. Chronic neuroinflammatory response to an acquired brain insult such as a TBI contributes to the injury and lengthens or halts recovery 6. In chronic neuroinflammation, microglia remain activated for an extended period during which the production of repair mediators is sustained longer than usual 7. Of note, chronic microglial activation has also been linked to most, if not all, neurodegenerative diseases like Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease 8–10. In humans with traumatic brain injury (TBI), microglial activation has been reported as early as 72 hours after injury 11, and it can persist for months after injury 12. TBI is the most prevalent of all CNS injuries and results in chronic neurologic and cognitive deficits 13. TBI causes cell death and neurologic dysfunction through secondary injury mechanisms characterized by edema, neuronal cell death, glial activation, and infiltration of peripheral immune cells 14. Extensive research has been conducted investigating the role of microglia after head injury and its interaction with the neural microenvironment suggesting a role for microglia in the injury process as well as in neurotransmission and maintenance of synaptic integrity 15. TBI initiates a neuroinflammatory cascade that persists over time and may lead to prolonged cognitive deficits. The regional distribution of microglial activation is yet to be determined. Introduction Some studies found chronic activation in the thalamus, others in the hippocampus in addition to the injury site 16. Despite evidence showing a significant role for microglia in the pathogenesis of TBI, no studies to date have examined the spatiotemporal microglial activation response after injury and explored its potential correlation with cognitive deficits 17–19. Here we hypothesized that TBI would result in phenotypic changes in the microglial cell population that persists for a long time after the injury. We also hypothesized that chronic microglial activation is associated with cognitive deficits. We analyzed the spatiotemporal course of microglia changes isolated from the injured brain up to 35 days after controlled cortical impact (CCI) in mice. We used conventional flow cytometry techniques followed by bioinformatics-based multi-parametric methods that are not constrained by assumptions or bias. We observed heterogeneity in microglia phenotypes and temporal changes in microglia subpopulations following TBI. A particular subpopulation that we called hyperactivated microglia was correlated with chronic deficits in learning and spatial memory after injury. Neurological tests A total of 30 mice were used, randomized into 3 groups: sham, mild TBI (mTBI), and severe TBI (sTBI), of 10 mice each. Mice (~ 20 g, 6 weeks old) were allowed two weeks of habituation after arrival and handled for 7 consecutive days before surgery. After the injury (D0), the neurological and cognitive tests were performed, using: Pole climbing, adhesive removal test, spontaneous object and location recognition memory tests, and the Morris water maze, at three different time-points: acute 48 hours, subacute 7 days, and chronic 35 days. Animals were sacrificed and used for microglia isolation. Animals CCI in the somatosensory area was produced with an imp mm depth into the cortical tissue. The animals were rapid and kept in a holding cage on a heating pad until recovery mice, anesthesia was given, and a craniotomy was perfor lesion volume, a series of the five brain sections of each a (H & E staining) and analyzed using a 1.6 × objective and Each hemisphere's volume was calculated by measuring area from each section, and multiplying by the section th Paxinos and Watson atlas of the mouse brain (Fig. 9). Pole climbing test This test assesses motor performance and coordination in mice 66. Each mouse was placed head up on the top of a vertical rod (height = 60 cm, diameter = 1 cm). The mouse is supposed to turn and descend by gripping without slipping. The time needed for the mouse to reach the bottom (t-total) was recorded; also, the t-turn (time at which the mouse turned head down on the rod), the t-half (time at which the mouse reached half of the rod), and the t-stop (Amount of time the mouse stopped on the rod), were recorded. Animals The Institutional Animal Care and Utilization Committee (IACUC) of the American University of Beirut (AUB) approved this study. The study is reported in accordance with ARRIVE guidelines64. C57BL/6 mice were obtained from the Animal Care Facility of the American University of Beirut and housed in a controlled environment (12 h reverse light/dark cycles, 22 ± 2°C). All efforts were made to reduce the number of animals used and their suffering. All animals were handled and fed regular chow and water ad libitum. Controlled cortical impact injury (CCI) model. Controlled cortical impact injury (CCI) model. Open head injury was performed to induce TBI in mice, using the electromagnetic controlled cortical impact device (Leica Impact One Angle with Leica Angle Two™ Stereotaxic Instrument, Biosystems, Buffalo Grove, IL, USA), as described previously 65. Briefly, adult (8 weeks old) C57BL/6 mice (~ 22–25 g, n = 30) were anesthetized with a mixture of Ketamine (50 mg/kg, Panpharma) and Xylazine (15 mg/kg, Interchemie), injected intra-peritoneally. Animals were placed in a stereotactic frame in a prone position and secured by ear and incisor bars. The target site was set parasagittal between Bregma and Lambda (somatosensory area of the parietal cortex) with standard coordinates (+ 1.0 mm AP, + 1.5 mm ML, and − 2 mm DV), where craniotomy was made. Bone was removed using a drill, and the injury was induced by impacting the left cortex with a pneumatic piston containing a 1 mm diameter tip at a rate of 4 m/s. Mild Page 3/27 Page 3/27 CCI in the somatosensory area was produced with an impact of 0.5 mm depth, whereas severe CCI with 2 mm depth into the cortical tissue. The animals were rapidly removed from the stereotaxic frame, sutured, and kept in a holding cage on a heating pad until recovery from anesthesia. In sham-operated control mice, anesthesia was given, and a craniotomy was performed, but no injury was induced. To estimate the lesion volume, a series of the five brain sections of each animal were stained with hematoxylin and eosin (H & E staining) and analyzed using a 1.6 × objective and a computer image analysis system ImageJ. Each hemisphere's volume was calculated by measuring the hemisphere's area, subtracting the lesion area from each section, and multiplying by the section thickness and the sampling interval based on Paxinos and Watson atlas of the mouse brain (Fig. 9). Adhesive removal test The adhesive removal test, a sensory measure, was performed 48h, 7 days, and 35 days post-TBI. Three consecutive experimental trials were conducted for each mouse. Briefly, a strip of tape (0.5 cm x 0.5cm dimensions) was placed on the mouse snout, and the time needed for each mouse to sense (Time-to- contact) and remove (Time-to-remove) the adhesive tape was recorded. Morris Water Maze (MWM) To test spatial learning and memory, the Morris Water Maze was used. The animals learn the location of a submerged platform using constellations of external cues. The MWM was conducted as previously described 68. A fixed submerged platform was placed within the pool for the animals to use as an escape On Day 31 post-trauma, all the animals were placed in a circular dark-colored water tank, 0.55-meter depth and 1-meter diameter, filled with room temperature water (25–26°C) made opaque by adding Tempera® washable, non-toxic white paint. An invisible transparent escape platform of 30 cm height and 10 cm diameter was placed in the mid-NE quadrant, 1.5 cm below the water's surface. The pool was in a large testing room, where there were at least 3 external and visible cues such as pictures. The cues and the experimenter's location (North-East quadrant) were kept constant throughout the experiment. Each mouse was tested for 4 consecutive days, 3 trials per day, and on day 35, only 1 probe trial was performed; the platform is removed from the pool, and the time the animals spend searching where the platform was located is measured. Typically, the normal animal will spend more time searching in the quadrant where the platform was located, indicating spatial memory. The pool was divided into four equal quadrants designated as North East, South East, North West, and South West. At the start of each trial, each mouse was placed in one of the four quadrants. The starting point was fixed in the South quadrant, followed by North, then East, and West. Only on the first testing day, a one-minute trial 0 was performed before the learning trials. Trial 0 was conducted with a visible flag attached to the visible platform to guide the animal to the platform and ensure the animals do not have any visual deficits. Following trial 0, the platform was submerged, and the animals were expected to locate the platform based on their spatial learning and memory. The mice were then placed in each quadrant facing the tank's wall and allowed to swim until they found the platform or for a maximum of 60 seconds. Once the animals reached the platform, they were left on it for 30 seconds before being returned to their cage for an inter-trial interval of 60 seconds. If the animal was unable to find the platform within 60 seconds, the trial was terminated. Spontaneous object and location recognition memory (OR) Spontaneous object and location recognition memory (OR) Spontaneous object recognition depends on the innate capacity of rodents to discriminate a novel from a familiar object (previously encountered). The test was performed as described previously 67. Briefly, mice were placed in the center of an open field at the beginning of each trial and freely explored the open field and objects. At the end of each trial (5min), mice were removed from the open fields and placed in their home cages next to each testing area for the inter-trial interval (ITI-5 min). The open fields and objects were cleaned with 70% ethanol during the ITI. During Trial 1, the animal explored an empty open field. During trials 2 to 4, three identical objects were placed near the corners of each open field. The objects' Page 4/27 Page 4/27 configuration remained unchanged during trials 2 to 4 to allow the mice to encode the objects' shape and location. Object recognition was examined in Trial 5 and 6 after replacing one familiar object with a novel object. Object recollection or response to spatial novelty was examined in Trial 7–8 after moving the novel object to a new location in the open field. All data were recorded by a video camera suspended above the 4 open fields and connected to image analyzer software (Any Maze-IL-USA®). The zones were located 5 cm around each object on the software, and the software recorded the time the animal spent in each zone with their head directed toward the object. configuration remained unchanged during trials 2 to 4 to allow the mice to location. Object recognition was examined in Trial 5 and 6 after replacing object. Object recollection or response to spatial novelty was examined in novel object to a new location in the open field. All data were recorded by above the 4 open fields and connected to image analyzer software (Any M located 5 cm around each object on the software, and the software record each zone with their head directed toward the object. Morris Water Maze (MWM) In this case, the experimenter guided the animal to the platform and allowed it 30 seconds of exploration before returning to its' cage. Time spent to find the platform (latency to escape- NE) during the testing days was used to evaluate spatial learning. Time to reach the platform or escape latency is commonly used to assess spatial learning; here, we slightly changed the presentation of the results by calculating cumulative (total of 3 trials/day) latency to the platform that accounts for acquisition and incremental spatial learning of all trials. On day 35 (probe trial), the percentage time traveled in the target zone was used as an index of spatial memory. A video camera recorded the Page 5/27 Page 5/27 performance, and the time spent in each quadrant was acquired and analyzed using an automated tracking system (Any Maze-IL-USA®). Microglia isolation and Flow Cytometry After completing the neurological and learning and memory tests, the mice were sacrificed at acute (48hrs), sub-acute (7-days), and chronic (35-days) time points. Brains were collected and dissected to isolate the different regions of interest: the hippocampus, the thalamus, and the cortex from the contralateral and the ipsilateral hemispheres 69. Briefly, after brain dissociation, immune cells were enriched over a percoll gradient to remove myelin and other debris, protocol adapted from 70. The cells were then labeled with antibodies against CD11b, CD45, F4/80, CX3CR1, CD68, CD206, IA/AE, and TNF-α, then measured by fluorescence-activated cell sorting (FACS) (Table 1). Table 1 Fluorochrome-conjugated antibodies clones. Fluorochrome-conjugated antibody Clone PerCP-Cy5.5 anti- mouse CD45 30-F11 BV421/50 anti- mouse CD11b M1/70 APC anti-mouse F4/80 BM8 PE-Cy7 anti-mouse CD206 C068C2 PE anti- mouse CD68 FA-11 FITC anti-mouse CX3CR1 SA011F11 BV610/20 anti-mouse TNFa MP6-XT22 BV710/50 anti-mouse IA/IE M5/114.15.2 Table 1 Fluorochrome-conjugated antibodies clones. Fluorochrome-conjugated antibody Clone PerCP-Cy5.5 anti- mouse CD45 30-F11 BV421/50 anti- mouse CD11b M1/70 APC anti-mouse F4/80 BM8 PE-Cy7 anti-mouse CD206 C068C2 PE anti- mouse CD68 FA-11 FITC anti-mouse CX3CR1 SA011F11 BV610/20 anti-mouse TNFa MP6-XT22 BV710/50 anti-mouse IA/IE M5/114.15.2 Flow Cytometry Standard file was combined into a single flow cytometry standard file to define spatially distinct populations. Mean fluorescence intensity (MFI) is presented on a “logical” scale, as previously described 71, and viable cells were selected from all events. We next applied t-distributed stochastic neighbor embedding (TSNE), a nonlinear dimension reduction method that projects data into a lower- dimensional space. The algorithm represents the distance between any two points by the probability of these two points being neighbors 72. We used the R package R-tsne, default parameters for TSNE implementation (iterations = 1000, perplexity = 30, θ = 0.5). Because we are interested in changes in cell populations' density across different conditions, the TSNE was followed by the unsupervised K-means clustering to cluster cells based on different markers' expression. K-means is a commonly used clustering algorithm for single-cell analysis after dimensionality reduction 73. Traumatic brain injury affects motor and sensory performance. We evaluated the performance of mice on the pole climbing test. At 48h, mice with mild and severe TBI performed significantly worse than sham-injured mice as indicated by the total time taken by the animals to descend the pole (mild:11.7 ± 0.6 and severe: 21.4 ± 3.6 vs. 6.4 ± 0.8 seconds, P < 0.001 and P < 0.0001 respectively). Less time is indicative of better motor coordination and balance. Both TBI groups showed recovered motor performance at 7- and 35-days post-injury (dpi) in comparison to their performance at 48h after injury (7d: 7.1 ± 0.4 and 8.7 ± 1, P < 0.05 and P < 0.01 respectively; 35d: 8.9 ± 2 and 8.1 ± 1, P < 0.01) (Figure 1, a-d). Furthermore, we found group differences among the sham, mTBI, and sTBI groups in time to sense and remove the stimuli on the adhesive removal test (Figure 1, e-f). At 48h post-TBI, the mean time to sense and remove the tape was significantly higher in mild and severe-TBI mice than the sham group (mild:21.4 ± 3 and severe: 25.9 ± 4 vs. sham: 8.4 ± 1.5 seconds, P < 0.05 and P < 0.01 respectively). The sensory deficit persisted on day 7 in both injury groups (mild:17.7 ± 5.8, and severe: 20.9 ± 3.5 vs. sham: 8.2 ± 3.7 seconds, P < 0.01). However, 35 days post-TBI, only the group that received severe injury showed persistent sensory deficits compared to sham (15.4 ± 2.5 vs. 7.9 ± 3.5 seconds, P < 0.05). Neurological and cognitive assessment Neurological and cognitive assessment Statistical analysis Statistical analysis Page 6/27 Unless otherwise indicated, all values presented are mean ± SEM. Fisher exact test was used to calculate the significance of categorical variables. A paired wise χ2 test with Bonferroni correction is used to compare the different clusters of cells per group and conditions after K-means clustering. We have used the Kruskal-Wallis test followed by Dunnett's post-test or ANOVA followed with Bonferroni corrections for multiple comparisons. We have used linear regression to measure the association between microglia frequencies and latency to the platform on the MWM. The two-tailed p-value was considered significant when < 0.05. The bioinformatics analysis was done using the SPSS ver.19 and Graph-pad prism 5. Unless otherwise indicated, all values presented are mean ± SEM. Fisher exact test was used to calculate the significance of categorical variables. A paired wise χ2 test with Bonferroni correction is used to compare the different clusters of cells per group and conditions after K-means clustering. We have used the Kruskal-Wallis test followed by Dunnett's post-test or ANOVA followed with Bonferroni corrections for multiple comparisons. We have used linear regression to measure the association between microglia frequencies and latency to the platform on the MWM. The two-tailed p-value was considered significant when < 0.05. The bioinformatics analysis was done using the SPSS ver.19 and Graph-pad prism 5. The Morris water maze test Both the sham and mTBI mice showed similar learning curves 31-35 days post-injury. However, as expected, a longer latency to the platform was found after a severe TBI compared to the Sham and mTBI on all days. The group with severe TBI showed little learning during the four trial days, indicating impaired spatial learning that persisted up to 35 days (Figure 3, a). When examining the percentage time spent in the target quadrant and the latency to correct quadrant (data not shown), the animals with mild injury were similar to the sham group, unlike the animals post severe TBI who remained significantly impaired. The same was found when we examined the percentage of time spent swimming in the correct quadrant on the probe trial (Figure 3, b) (day 5). The animals with mTBI performed like the sham and remembered where the platform was located, whereas the animals with a severe injury did not, indicating impaired spatial memory (Figure 3, c). Spontaneous object recognition test During the sample phase (learning trial), the time spent acquiring information about the objects, such as size and shape, was measured as exploration time. To exclude position preference, the mean time spent exploring each object in the 3 corners of the box within each group was compared (phase 1), and no significant differences were found (data not shown). When comparing the time spent exploring the different objects during this phase, the sham group spent more time exploring all the objects at 48 h compared to animals with mild and severe TBI: (sham total exploratory time 24.9 ± 3.8 vs. mild 7.4 ± 2.7 and severe 12.9 ± 2.5 seconds, P < 0.0001 and P < 0.01 respectively). Page 7/27 On the Novelty test (phase 2), one familiar object was replaced with a novel one. The exploration time of the remaining two familiar objects and the novel one was compared. At all time-points, the sham animals spent more time exploring the novel object than animals with mild and severe TBI (Figure 2, a, c and e). The same novel object was then moved to a novel position on the novel location test (phase 3). At all time-points, the sham animals spent more time exploring the novel object/location than the animals with mild and severe TBI (Figure 2, b, d, and f). Animals with mild and severe TBI, at 7- and 35-days post- trauma, showed persistent impaired spontaneous object recognition compared to the sham animals. Conventional Flow cytometry analysis identified microglia and infiltrating macrophages. To investigate the dynamics of microglial activation following TBI, we isolated microglia from different brain regions: hippocampus, thalamus, and cortex ipsilateral and contralateral to the injury site. Live cells were stained with anti-CD11b, CD45 and CX3CR1. Figure 4 a-b shows representative flow cytometry scatter plots that were used to measure the frequency of microglia in the different parts of the brain. Interestingly, starting at 48 hours post-TBI the frequency of isolated microglia (CX3CR1hi) increased in all regions studied in both mild and severe TBI groups. In the mild TBI group the increase in microglia was significant in the ipsilateral cortex at 48 hours, and in both ipsi- and contralateral cortices at 7 days. While in the severe TBI group, a significant increase was noted in all investigated regions at 48 hours. This increase in frequency remained significant in the cortical areas and hippocampi on days 7 and 35 (except for the ipsilateral cortex on day 35). The thalamus showed significantly increased microglia frequency at 48 hours bilaterally, but only the increase in the ipsilateral thalamus remained significant at day 7 and the contralateral thalamus on day 35. Furthermore, myeloid cells (CD45hi CX3CR1lo) likely representing infiltrating macrophages were detected at 48h in the ipsilateral side in the three investigated brain regions of the severe TBI group only. These cells were not detected at subacute and chronic time points. The increase of microglia numbers in both hemisphere of injured mice reflects a diffuse injury that extends beyond the injury site. To investigate the dynamics of microglial activation following TBI, we isolated microglia from different brain regions: hippocampus, thalamus, and cortex ipsilateral and contralateral to the injury site. Live cells were stained with anti-CD11b, CD45 and CX3CR1. Figure 4 a-b shows representative flow cytometry scatter plots that were used to measure the frequency of microglia in the different parts of the brain. Page 8/27 Microglia activation markers, mainly CD11b, CD206, TNFa and AIEI, were found upregulated at 48h post- injury. Even though the markers were increased in both hemispheres, the highest MFIs were noted on the ipsilateral side. As one would expect, microglia markers followed the same dynamics as observed for the cell frequencies in figure 4 c. almost all activation markers that were upregulated at the early time point return to baseline on day 7. Conventional Flow cytometry analysis identified microglia and infiltrating macrophages. However, on day 35 we observed a re-emergence of these markers in the brains of the severe TBI group. The increase of IAIE, which is considered a marker of activation in microglia 20, at this later time point suggests that microglia are chronically activated. The activation was noticeable at the level of the ipsilateral Hippocampus and the contralateral thalamus and cortex (figure 5). TNFa, the inflammatory cytokine, was found to follow the same pattern as Class II, suggesting that the chronically activated microglia are likely proinflammatory (Data not shown). Non-linear Dimensionality Reduction Reveals Multiple Distinct Microglia Cell States To investigate the phenotypic heterogeneity and dynamics of cells after traumatic brain injury, we used the combination of 8 markers projected into the two-dimensional space followed by cluster analysis. This method allowed the characterization of several subpopulations of cells. The markers CD11b and TNFa contributed the most to the model as was shown by the sum of square error (Figure 6, a). We identified a population of infiltrating macrophages (CD45hi, CD11bhi, CX3CR1lo, CD68-, F4/80-, CD206hi, TNFa-, IA/IEhi) and three populations of microglia that were characterized by a relatively high expression of CX3CR1 and CD68. These cells could be divided into three clusters: microglia cluster 1 (CD45+, CD11bhi, CX3CR1++, CD68++, F4/80lo, CD206+, TNFa-, IA/IE+) microglia cluster 2 (CD45+, CD11blo, CX3CR1++, CD68++, F4/80lo, CD206+, TNFa+, IA/IE+), and highly activated microglia (CD45++, CD11bhi, CX3CR1hi, CD68hi, F4/80+, CD206 hi, TNFa++, IA/IE ++) (Figure 6, b). Clusters 1 and 2 are similar for most markers, except for significantly higher CD11b and lower TNFa expression in cluster 1 compared to cluster 2. The third cluster, representing highly activated microglia, is characterized by increased expression of almost all the markers, especially TNFa and IAIE (Figure 6, c). In the non-injured brains, the relative proportions of identified clusters were different between brain regions. The predominant population is from cluster 1, with a smaller percentage of cluster 2 cells. Interestingly, highly activated microglia are also found at a very low frequency throughout the different parts of the control brains (Figure 7). At 48 hours after TBI, we observed an increase in highly activated microglia (cluster 3) in the cortex of mild TBI animals and all brain regions of severe TBI animals. These cells resolved by day 7 in both TBI groups. Interestingly, cluster 3 cells reappeared on day 35 in the cortex and subcortical area of severe TBI mice. Furthermore, we have documented a switch in abundance between cluster 1 and cluster 2 at the injury site 48 hours dpi (Figure 7). Although cluster 2 decreases at the later time points in animals with severe TBI, these cells persist with a frequency significantly higher than in the sham group. Also, we noted an equivalent increase of cluster 2 in both the mild TBI and severe TBI 7 days post injury. Non-linear Dimensionality Reduction Reveals Multiple Distinct Microglia Cell States However, the level of cluster 2 goes down in the mTBI group at 35 days but remains significantly higher in the severe TBI group compared to the rest of the groups. The increase in In the non-injured brains, the relative proportions of identified clusters were different between brain regions. The predominant population is from cluster 1, with a smaller percentage of cluster 2 cells. In the non-injured brains, the relative proportions of identified clusters were different between brain regions. The predominant population is from cluster 1, with a smaller percentage of cluster 2 cells. Interestingly, highly activated microglia are also found at a very low frequency throughout the different parts of the control brains (Figure 7). At 48 hours after TBI, we observed an increase in highly activated microglia (cluster 3) in the cortex of mild TBI animals and all brain regions of severe TBI animals. These cells resolved by day 7 in both TBI groups. Interestingly, cluster 3 cells reappeared on day 35 in the cortex and subcortical area of severe TBI mice. Furthermore, we have documented a switch in abundance between cluster 1 and cluster 2 at the injury site 48 hours dpi (Figure 7). Although cluster 2 decreases at the later time points in animals with severe TBI, these cells persist with a frequency significantly higher than in the sham group. Also, we noted an equivalent increase of cluster 2 in both the mild TBI and severe TBI 7 days post injury. However, the level of cluster 2 goes down in the mTBI group at 35 days but remains significantly higher in the severe TBI group compared to the rest of the groups. The increase in Page 9/27 Page 9/27 cluster 2 types of microglia and highly activated microglia at the acute time point suggests a proinflammatory environment early after the injury in both mild and severe TBI. The highly activated microglia correlate with learning deficit. To explore the association between the changes in microglial phenotype with spatial learning and memory, we performed the Morris water maze on day 31 to 34 followed by microglia isolation in 24 mice included in the four experimental groups, normal, sham, mild TBI and severe TBI. The aim here was to investigate microglial phenotype within the hippocampus and thalamus as they are critical in the formation and processing of spatial learning and memory 21. As expected, mice with severe TBI showed significant spatial learning and memory deficits (Figure 8, a). Furthermore, these mice showed increased highly activated microglia at 35 days post-injury (Figure 8, b). Using general linear regression, we found that the frequency of the highly activated microglia explained up to 47% of the variability observed in the cumulative latency to the platform, indicating a possible relationship between the existence of activated microglia and spatial learning/memory deficit chronically after the injury (Figure 8, c & d). Microglia clusters 1 and 2 did not contribute much to the model and showed no correlation with cumulative latency to platform. Discussion In the present study, we investigated whether CCI traumatic brain injury results in a spatiotemporal phenotypic change in microglia and if these changes are linked to a neurologic and cognitive deficit in a mouse model. Microglia are the brain immune cells, and they are implicated in almost all physiological processes in the CNS and play an important role in several inflammatory and neurodegenerative diseases. Chronic changes in microglia phenotype and function, support the notion that chronic microglial dysfunction may contribute to the chronic progressive neurodegeneration that are observed years after head injury 19. However, the precise role of microglia in neuroinflammation following TBI and leading to neurological, sensory-motor and cognitive, deficits at acute, subacute, and chronic time points after TBI remain to be defined. Here, we provided insights into the dynamics of several microglial marker expression after injury using multi-color flow cytometry coupled with an unbiased bioinformatics approach to characterize microglia in the different brain compartment after injury. We used CX3CR1 and the classical CD11b and CD45 22 markers to identify microglia although we have also verified the cell lineage with microglia-specific marker TMEM119, and macrophage-specific marker CCR2 23 (data not shown). At 48 hrs after CCI, both the mild and severe TBI groups showed statistically significant sensory-motor and cognitive deficits compared to the sham animals 24. At this acute time point, the number of microglia increased in the ipsilateral cortex in the mild injury group, while the number doubled in the severe TBI group in both hemispheres suggesting diffuse activation. Similar studies done in rodents described an increase from 2 to 20-fold of the number of cells in the injured brain 25,26. An increase in the numbers of Page 10/27 Page 10/27 Page 10/27 isolated cells is attributed to resident microglia proliferation and expansion and peripheral cell infiltration 25,27. It is reported that infiltrating monocytes and neutrophils respond quickly by crossing the blood-brain barrier and penetrating the injured tissue 27,28. We found that infiltrating macrophages were only observed in the ipsilateral hemisphere of severe TBI (Figure 4) 29. Surprisingly, infiltrating macrophages were not proinflammatory as they do not express TNFa and have a very high expression of CD206 the mannose receptor that is usually expressed on M2 macrophages, an observation in line with a previous report showing that macrophages in TBI have a mixed M1/M2 profile30. Discussion Interestingly, these cells were not found when the injury was mild, suggesting a possible threshold for the impact to solicit peripheral cells infiltration. Probably the mild injury produced here didn’t result in the disruption of the blood-brain barrier which leads to limited recruitment of circulating myeloid cells to the injured site 31. Although neutrophils have an important role post-injury as they are the first responders, we elected to focus on microglia as the cells most likely responsible for the long-term damage 32. When we looked at microglia markers at 48 hours post-injury, we found that almost all markers were upregulated mainly on the ipsilateral side suggesting activation of these cells. Microglia activation corresponds to new biological functions 25. These changes are readily visualized in immunohistochemistry imaging, previously reported in rodents 33–36. Early activation of microglia after TBI has been observed in animal models 37–39 and in humans 40. Persistent inflammation is also known to occur for up to one year after injury in rodents 41 and for several years in humans 42. While there is agreement about the persistent activation of microglia after TBI, the phenotype and functional aspects of these microglia remain less clear. Earlier studies have focused on the increased number of microglia at different time point after injury 43 and morphological evidence of activation 44. Using the M1/M2 phenotype scheme adapted from peripheral macrophages, researchers reported dysregulation of the anti-inflammatory M2 phenotype in association with chronic microglial activation 41,45,46. However, mass cytometry and single-cell transcriptomic analysis reveal that microglia often present mixed phenotypes 47,48 and that the M1/M2 scheme may be an oversimplification49. An upregulation of Class II MHC and CD11b in chronically activated microglia 50 is consistent with our observations. High levels of CD11c and CD14 expression have been described in geriatric microglia, and chronic neuroinflammation may predispose to other neurologic disorders such as Alzheimer’s disease 51. In normal brains, our results showed that microglia are predominantly of cluster 1 with a ratio close to 2 to 1 in comparison to cluster 2, this was seen in all investigated regions. At 48 hours after injury, all the identified microglial populations increased in the injured brains as an indication of active proliferation. We noted at this time point a shift in the predominance between Cluster 1 and 2, suggesting that the injury microenvironment at 48 hours is pro-inflammatory as cluster 2 express significantly higher TNFa. Discussion As for cluster 3, these cells were found highly upregulated only in injured brain, even in mice that underwent the sham procedure. Page 11/27 At seven days post injury, the animals with mTBI showed complete recovery of motor deficits but not the sensory functions. Both sensory-motor functions remained impaired after the severe TBI. Both animal groups showed persistent spontaneous object recognition deficits post injury52,53. The highly activated microglia were nearly undetected at this time point with the persistence of the inflammatory cluster 2 in the cortex but not in the subcortical areas. Interestingly, at the chronic time point, sensory-motor and cognitive deficits were sustained post severe TBI and highly activated cells reappeared in injured brains along with persistence of cluster 2 cells suggesting an increase in the inflammatory background. An interesting observation in this study is the resolution of the highly activated microglia population at day 7 and its reappearance at the chronic time points. Previous studies have shown a bimodal change in microglia morphology 54 in the mouse model. This observation raises the possibility that after resolution of the acute injury, the second wave of activation may be triggered by late neuronal or axonal injury. This is in line with the delayed appearance of neurofibrillary tangles observed in humans after a single TBI 40. In this study, we did not observe any patterns in the spatial distribution of the microglia populations in the severe TBI group suggesting that activation of microglia is already diffuse and includes ipsilateral, contralateral as well subcortical regions at 48 hours. In the mild TBI group, the proliferation of microglia was mostly observed on the ipsilateral cortex at 48hrs and ipsilateral cortex and hippocampus at 7 days. In this study, we did not observe any patterns in the spatial distribution of the microglia populations in the severe TBI group suggesting that activation of microglia is already diffuse and includes ipsilateral, Experimental studies in models of traumatic brain injury have shown that the majority of microglia and recruited macrophages at the site of injury have mixed pro and anti-inflammatory-like activation profiles, but that the anti-inflammatory-like response is short-lived and there is a phenotypic shift towards a proinflammatory-like dominant response within one week of injury 54. Others have demonstrated prolonged activation of microglia in the subcortical areas at 28 days after injury 43,55. Here, the identified macrophages profile fits an anti-inflammatory response at 48h. Discussion Whether macrophages and their cross- talk with microglia have a role in downregulating inflammation at 7 days is difficult to judge in our experimental setting 56. Since both pro-and anti-inflammatory paths associated with overexpression of the same markers, it may be necessary to add additional parameters, such as cytokine expression 57. In- depth characterization of the phenotype is also important for therapy after TBI as the timing of attenuating therapy delivery is critical for success 58. Sensory-motor and cognitive deficits are prevalent post traumatic brain injury of all intensities and based on severity may persist for years after the injury52,59 . Spontaneous object recognition and spatial learning and memory deficits found in this research are in line with our previous and other research studies60,61. Yet, to our knowledge few research characterized the cognitive deficits systematically over time, at acute, sub-acute and at chronic time points. The different tests used in this research study allowed for the early detection of the sensory motor and cognitive deficits and monitoring its persistence up to 35 days post injury while examining the course of microglial activation. The deficits seen after a TBI of mild intensity are often subtle and harder to detect in small animals. We have uncovered the sensory- motor and cognitive deficits at the acute and sub-acute time points in the animals with mild TBI. We have also found activation of the pro-inflammatory microglia at these same time points which warrant further Page 12/27 Page 12/27 investigation and analysis of these early intervals, as it may shed light into a possible therapeutic window for TBI management of any intensity. Finally, to link microglial phenotypes to cognitive deficit we have investigated the microglia response within the subcortical areas, as they are critical in the formation and processing of spatial learning, memory and relaying sensory and motor signals. The deficits seen on the MWM were increasing in relation to the severity of the injury as previously reported 62. We found that each 1 % increase in the frequency of highly activated microglia at day 35 corresponds to an approximately 6-second increase in the cumulative latency to platform on the MWM indicating a decrease in the learning curve. Whether the presence of highly activated microglia is the cause of the spatial learning deficit cannot be concluded from our studies. The microglia may become activated by injured neurons or axons and requires further investigation. Discussion We also need to explore the same microglial activation pattern while using both cognitive tests, the spontaneous object recognition and the MWM at the acute phases in both mild and severe TBI. Our study has some limitations; first, the panel of antibodies and the numbers of markers included are limited by the capacity of the conventional flow cytometer. Future studies using high throughput mass cytometry will be of interest. Second, sex differences are also reported comparing activation of microglia following TBI in adult female and male mice 63; in our study, we used only male mice. In conclusion, using a nonbiased high-dimensional immune profiling of microglia in TBI, we revealed the immune landscape of sub-acute and chronic microglial activation. These results significantly extend our knowledge of how TBI affect long term deficits and will serve ultimately to define regions of interest and treatment windows to evaluate the efficacy of therapy. Page 13/27 Abbreviations CCI: Controlled Cortical Impact CNS: Central Nervous System DPI: Days Post Injury MFI : Mean Fluorescence Intensity MWM: Maurice Water Maze TBI: Traumatic Brain Injury TSNE: t-distributed stochastic neighbor embedding Declarations Author contribution CCI: Controlled Cortical Impact Author contribution Page 13/27 Page 13/27 Concept and design: SJK, HD, FK Acquisition, analysis, and interpretation of data: ESS, MK, LN, HD, SJK Drafting of the manuscript: ESS, HD, SJK Statistical analysis: ESS, MK Additional Information This study was funded by the Office of Naval Research (ONR), ONRG - NICOP - N62909-17-1 2059 AWARD. The funders had no role in study design, data collection, analysis, decision to publish, or manuscript preparation. All animals were treated in accordance with the International Guidelines for animal research. All procedures involving animals were approved by the Institutional Committee for the Care and Use of Laboratory Animals at the American University of Beirut The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials All data generated or analyzed during this study are included in this published article. References 1. Wolf, S. A., Boddeke, H. W. G. M. & Kettenmann, H. Microglia in Physiology and Disease. Annu. Rev. Physiol. 79, 619–643 (2017). 2. Yeh, H. & Ikezu, T. Transcriptional and Epigenetic Regulation of Microglia in Health and Disease. Trends Mol. Med. 25, 96–111 (2019). 2. Yeh, H. & Ikezu, T. 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Res. 9, 2579–2605 (2008). Figures Page 19/27 Figure 1 Figure 1 Neurological tests: (a-d) Pole climbing test. (a) t-total: Time needed to reach the bottom of the vertical rough-surfaced pole. (b) t-half: time needed to reach half of the pole. (c) t-turn: time at which the mous turned head down on the pole. (d) t-stop: Amount of time mouse stopped on the pole. (e-f) Adhesive removal test. (e) The time needed to sense (time-to-contact) and (f) to remove (time-to-remove) the tap Figure 1 Neurological tests: (a-d) Pole climbing test. (a) t-total: Time needed to reach the bottom of the vertical rough-surfaced pole. (b) t-half: time needed to reach half of the pole. (c) t-turn: time at which the mouse turned head down on the pole. (d) t-stop: Amount of time mouse stopped on the pole. (e-f) Adhesive removal test. (e) The time needed to sense (time-to-contact) and (f) to remove (time-to-remove) the tape. Page 20/27 Kruskal-Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < 0.01, p 0.001, *p < 0.0001 Figure 2 Time spent exploring the novel versus familiar objects during phase 2 (novel object) and phase 3 ( location) of spontaneous object and location recognition memory test at 48h (A-B), 7 days (C-D) an days (S-F). Kruskal-Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < p < 0.001, **p < 0.0001. Kruskal-Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < 0.01, p < 0.001, p < 0.0001 0.001, p < 0.0001 Figure 2 Figure 2 Time spent exploring the novel versus familiar objects during phase 2 (novel object) and phase 3 (novel location) of spontaneous object and location recognition memory test at 48h (A-B), 7 days (C-D) and 35 days (S-F). Kruskal-Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Figure 2 Figure 2 Figure 2 Time spent exploring the novel versus familiar objects during phase 2 (novel object) and phase 3 (novel location) of spontaneous object and location recognition memory test at 48h (A-B), 7 days (C-D) and 35 days (S-F). Kruskal-Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Page 21/27 Figure 3 Morris water maze at 35 days (a) Escape latency to reach the platform between different groups presented as a cumulative time to the platform. Figures (b) Representative Morris water maze performance in different groups, n = 10/group. (c) In the MWM probe trial, time spent in the target quadrant. Kruskal- Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Figure 3 Morris water maze at 35 days (a) Escape latency to reach the platform between different groups presented as a cumulative time to the platform. (b) Representative Morris water maze performance in different groups, n = 10/group. (c) In the MWM probe trial, time spent in the target quadrant. Kruskal- Wallis test followed by Dunnett's multiple comparison post‐test. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Page 22/27 Figure 4 Figure 4 Flow cytometry staining of microglia and other myeloid cells. (a) Representative scatter plot using microglial marker CD45 and CX3CR1. (b) Representative scatter plot using microglial marker CD45 and CD11b. (c) Percentage of cells for each of the experimental group at the various time points in the 3 brain regions. Bold numbers in red are considered significant (p < 0.05) by fisher exact test in comparison to the sham group of the same time point. Page 23/27 Figure 5 Microglial IAIE mean fluorescence intensity detected by FACS presented per group, time points, regions, and sides. Significance is calculated in reference to the sham of the same condition using ANOVA followed by Bonferroni correction for multiple comparisons p < 0.05, *p < 0.01. Figure 5 Microglial IAIE mean fluorescence intensity detected by FACS presented per group, time points, regions, and sides. Significance is calculated in reference to the sham of the same condition using ANOVA followed by Bonferroni correction for multiple comparisons p < 0.05, *p < 0.01. Microglial IAIE mean fluorescence intensity detected by FACS presented per group, time points, regions, and sides. Significance is calculated in reference to the sham of the same condition using ANOVA followed by Bonferroni correction for multiple comparisons p < 0.05, *p < 0.01. Page 24/27 Page 24/27 Page 24/27 Figure 6 Non-linear dimensionality reduction using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis. (a) Sum of the squared error to identify the markers with the highest variability among clusters. (b) TSNE plot showing the four different clusters. (c) The distributions of the different markers in relation to the four identified clusters. Figure 6 Non-linear dimensionality reduction using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis. (a) Sum of the squared error to identify the markers with the highest variability among clusters. (b) TSNE plot showing the four different clusters. (c) The distributions of the different markers in relation to the four identified clusters. Page 25/27 Figure 7 Figure 7 Figure 7 Overall Percentage of cell clusters per group, time points, and regions. Significance is calculated in reference to the Sham by Chi-square test followed by Bonferroni for multiple comparison post‐test. p < 0.05. 0.05. Page 26/27 0.05. Figure 8 Figure 8 Figure 8 Page 26/27 Page 26/27 The relation between spatial learning deficit and microglia activation. (a) Cumulative latency to platform showing spatial learning curve and deficit in severe TBI group. (b) Frequency of highly activated microglia in subcortical areas. (c) Pearson correlation between cumulative latency to the platform and activated microglia frequency. (d) Linear regression output showing the relationship between the different cell clusters and the cumulative latency to the platform (dependent variable). (*p < 0.05). y p ( p ) ( p ) Figure 9 Controlled cortical impact (CCI)-induced brain injury. Representative sections shown were stained with hematoxylin and eosin. The data represent the percentage lesion volume as assessed two days following the impact. The mean lesion volume of the animals with severe injury (n = 3; 8.31% ± 3.35) was more significant than the animals with mild injury (n = 2; 3.58% ± 0.33). Figure 9 Figure 9 Controlled cortical impact (CCI)-induced brain injury. Representative sections shown were stained with hematoxylin and eosin. The data represent the percentage lesion volume as assessed two days following the impact. The mean lesion volume of the animals with severe injury (n = 3; 8.31% ± 3.35) was more significant than the animals with mild injury (n = 2; 3.58% ± 0.33). Page 27/27
https://openalex.org/W3032111945	https://repository.gsi.de/record/184272/files/jpconf16_665_011001.pdf	English	null	Nuclear Physics in Astrophysics VI (NPA6)	Journal of physics. Conference series	2,016	cc-by	595	Preface The Nuclear Physics in Astrophysics VI conference was the 6th event of the NPA biannual conference series. Previous events of this series were held at the Institute of Nuclear Research of the Hungarian Academy of Sciences (ATOMKI), Debrecen, Hungary, in 2002 and 2005; at the Forschungszentrum Dresden-Rossendorf, Dresden, Germany, in 2007; at the Laboratori Nazionali del Gran Sasso (LNGS), L’Aquila, Italy, in 2009; and in Eilat, Israel, in 2011. This edition of the NPA conference series was organized by the European Physical Society (EPS) through its Nuclear Physics Division, corresponding to the 26th edition of the Topical Conferences of the EPS. As in previous editions, the goal of the NPA conference was to bring together the specialists in the fields of Nuclear Physics (theory and experiment) and Nuclear Astrophysics (theory and observation), providing the appropriate forum for review and discussion of the status and prospects of the field of Nuclear Astrophysics. During the discussions, special attention was given to the Nuclear Physics aspects that have an impact in Astrophysics. The list of topics discussed during the conference was: • Big Bang Nucleosynthesis • Stellar Nucleosynthesis and Evolution • Experimental Facilities for Nuclear Astrophysics (Underground Lab • Experimental Facilities for Nuclear Astrophysics (Recoil Mass Sepa • The s-process • Explosive Nucleosynthesis I (r- and vp-process) • Explosive Nucleosynthesis II (p and rp-process) • Astrophysics of Compact Stars and Supernovae • Neutron Stars and Equation of State (EoS) • Isotope Detection Technologies for Nuclear Astrophysics • Solar Standard Model • Nuclear Physics of Exotic Nuclei: Experiments with RIB • Nuclear Physics of Exotic Nuclei: Experiments with RIB The program consisted of keynote talks, providing a review on the recent advances in the field; invited talks and selected oral contributions that focused on specific topics; and poster contributions that had the opportunity to present the work to the audience via one minute oral contributions. In total 78 oral contributions and 64 posters were presented. A total of 146 delegates participated in lively discussions during the oral presentations, the poster session scheduled during the second day of the conference, the coffee breaks and all programmed events of a social character. Regarding the participants, 41 were PhD candidates, 33 postdoctoral researchers and 72 senior scientists. The participation rate of women at the NPA-VI was 1/3. Nuclear Physics in Astrophysics VI (NPA6) Journal of Physics: Conference Series 665 (2016) 011001 Nuclear Physics in Astrophysics VI (NPA6) Journal of Physics: Conference Series 665 (2016) 011001 Nuclear Physics in Astrophysics VI (NPA6) Journal of Physics: Conference Series 665 (2016) 011001 Nuclear Physics in Astrophysics VI (NPA6) Journal of Physics: Conference Series 665 (2016) 011001 IOP Publishing doi:10.1088/1742-6596/665/1/011001 IOP Publishing doi:10.1088/1742-6596/665/1/011001 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 Preface Fellowships to students and selected participants were supported by the Nuclear Astrophysics Virtual Institute (NAVI), the Joint Institute for Nuclear Astrophysics (JINA), the Network Activity ATHENA, and the ExtreMe Matter Institute (EMMI). This support was crucial to warrant the high participation of young researchers at the conference. The excellent scientific atmosphere we experienced during the conference promoted vivid discussions, strengthened existing collaborations and motivated the continuation of the work in the interdisciplinary field of Nuclear Astrophysics, keeping the different communities on a joint working perspective. Nuclear Physics in Astrophysics VI (NPA6) IOP Publishing doi:10.1088/1742-6596/665/1/011001 doi:10.1088/1742-6596/665/1/011001 Journal of Physics: Conference Series 665 (2016) 011001 2
https://openalex.org/W2526047447	https://www.annualreviews.org/doi/pdf/10.1146/annurev-publhealth-082516-012750	English	null	Toward Greater Implementation of the Exposome Research Paradigm within Environmental Epidemiology	Annual review of public health	2,017	cc-by-sa	7,939	Keywords First published online as a Review in Advance on January 6, 2017 First published online as a Review in Advance on January 6, 2017 exposome, environmental epidemiology, life-course epidemiology, multiple exposures The Annual Review of Public Health is online at publhealth.annualreviews.org The Annual Review of Public Health is online at publhealth.annualreviews.org The Annual Review of Public Health is online at publhealth.annualreviews.org Jeanette A. Stingone,1 Germaine M. Buck Louis,2 Shoji F. Nakayama,3 Roel C.H. Vermeulen,4 Richard K. Kwok,5 Yuxia Cui,6 David M. Balshaw,6 and Susan L. Teitelbaum1 1Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, New York, NY 10029; email: jeanette.stingone@mssm.edu, susan.teitelbaum@mssm.edu 2Division of Intramural Population Health Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20817; email: louisg@mail.nih.gov 2Division of Intramural Population Health Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20817; email: louisg@mail.nih.gov 3National Institute for Environmental Studies, Tsukuba 305-0053, Japan; email: fabre@nies.go.jp 4Institute for Risk Assessment Sciences, Environmental Epidemiology Division, Utrecht University, Utrecht 3584 CM, Netherlands; email: R.C.H.Vermeulen@uu.nl 4Institute for Risk Assessment Sciences, Environmental Epidemiology Division, Utrecht University, Utrecht 3584 CM, Netherlands; email: R.C.H.Vermeulen@uu.nl 5Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; email: richard.kwok@nih.gov 5Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; email: richard.kwok@nih.gov 6Exposure, Response, and Technology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; email: yuxia.cui@nih.gov, balshaw@niehs.nih.gov Annu. Rev. Public Health 2017. 38:315–27 Toward Greater Implementation of the Exposome Research Paradigm within Environmental Epidemiology Click here to view this article's online features: • Download ﬁgures as PPT slides • Navigate linked references • Download citations • Explore related articles • Search keywords ANNUAL REVIEWS Further Click here to view this article's online features: • Download ﬁgures as PPT slides • Navigate linked references • Download citations • Explore related articles • Search keywords ANNUAL REVIEWS Further Jeanette A. Stingone,1 Germaine M. Buck Louis,2 Shoji F. Nakayama,3 Roel C.H. Vermeulen,4 Richard K. Kwok,5 Yuxia Cui,6 David M. Balshaw,6 and Susan L. Teitelbaum1 Abstract https://doi.org/10.1146/annurev-publhealth- 082516-012750 INTRODUCTION AND OBJECTIVES In 2005, Christopher Wild championed the need for a comprehensive environmental exposure complement to the genome to reﬁne epidemiologic ﬁndings and public health research (49). He referred to this new research paradigm as the “exposome” and deﬁned it as that which “encom- passes life-course environmental exposure (including lifestyle factors) from the prenatal period onwards” (49, p. 1848). Multiple researchers in ﬁelds ranging from perinatal and chronic disease epidemiology to exposure science have written commentaries and reviews that highlight the po- tential beneﬁts of characterizing the exposome and promoting its integration into future studies (2, 6, 10, 24–26, 36, 38, 39, 45, 47, 49). The exposome research paradigm is distinguished from traditional epidemiologic approaches by three characteristics: expanded and dynamic exposure assessment across multiple exposure do- mains (50); the integration of data on exposure and response across multiple scales of variation, including across populations, as well as over time and space; and the use of the resulting high- dimensional information on multiple exposure–response relationships for data-driven discovery (9). Researchers’ views on the exposome are evolving, ranging from skepticism to enthusiasm de- spite challenges (10, 33, 36, 40). Additionally, researchers have differing views of the domains that should be included within the exposome research paradigm. Wild reﬁned his original deﬁnition to explicitly include three domains of the exposome: internal (e.g., endogenous factors such as metabolism), speciﬁc external (e.g., environmental pollutants, chemical exposures, occupation), and general external (e.g., broader social, economic, and/or psychological factors such as socioeco- nomic status, mental stress, and climate) (50). Some have advocated for at least an initial focus on the internal exposome (37), whereas others have expanded the Wild deﬁnition to include measures of biological response to the environment (26). This article adopts the view that there is merit to incorporating the exposome into environmen- tal health research and applies a broad deﬁnition of the exposome that includes multiple domains of exposure as well as biological responses to those exposures. This deﬁnition includes multilevel exposures, for example the built environment of a neighborhood and the individual physical activ- ity of its residents, biological markers of exposure and response, and exogenous stressors that may or may not have an associated biomarker. This view allows for ﬂexibility in the implementation of the exposome within environmental health research because individual investigators can approach this paradigm from different perspectives in light of their targeted research goals. https://doi.org/10.1146/annurev-publhealth- 082516-012750 Investigating a single environmental exposure in isolation does not reﬂect the actual human exposure circumstance nor does it capture the multifacto- rial etiology of health and disease. The exposome, deﬁned as the totality of environmental exposures from conception onward, may advance our under- standing of environmental contributors to disease by more fully assessing the multitude of human exposures across the life course. Implementation into studies of human health has been limited, in part owing to theoret- ical and practical challenges including a lack of infrastructure to support Copyright c⃝2017 Annual Reviews. This work is licensed under a Creative Commons Attribution- ShareAlike 4.0 (CC-BY-SA) International License, which permits unrestricted use, distribution, and reproduction in any medium and any derivative work is made available under the same, similar, or a compatible license. See credit lines of images or other third-party material in this article for license information. 315 comprehensive exposure assessment, difﬁculty in differentiating physiologic variation from envi- ronmentally induced changes, and the need for study designs and analytic methods that accom- modate speciﬁc aspects of the exposome, such as high-dimensional exposure data and multiple windows of susceptibility. Recommendations for greater data sharing and coordination, methods development, and acknowledgment and minimization of multiple types of measurement error are offered to encourage researchers to embark on exposome research to promote the environmental health and well-being of all populations. xposome research to pr S ed for a comprehensive ogic ﬁndings and public exposome” and deﬁned uding lifestyle factors) ﬁelds ranging from peri commentaries and revie and promoting its integ ished from traditional e exposure assessment acr and response across mu me and space; and the u response relationships lving, ranging from skep searchers have differing ch paradigm. Wild reﬁn some: internal (e.g., en ntal pollutants, chemica mic, and/or psychologica ome have advocated for expanded the Wild deﬁn 316 Stingone et al. Rationale for the Exposome Research Paradigm Rationale for the Exposome Research Paradigm The exposome paradigm marks a signiﬁcant departure from traditional approaches to environ- mental health research by promoting a more holistic view of environmental effects on human health. Traditional epidemiologic approaches often focus on a single exposure, or a class of expo- sures, and a single outcome or closely related outcomes. Interactions among multiple exposures are often investigated with a focus on an individual pollutant or a related class of toxicants and how its effects may be modiﬁed by other environmental, social, and/or genetic factors. The expo- sure and environmental health communities have already begun to acknowledge the limitations of a one-chemical-at-a-time approach and explore approaches to quantify the health effects of chemical mixtures (3). The exposome adds another level of complexity and is more similar to a mixture of mixtures because, by deﬁnition, it encompasses the totality of exposures (49). Owing to its expansive nature, characterizing the exposome requires multiple exposure assessment method- ologies, including biological sample analyses and measurement of one’s personal environment using sensors, wipes, simulation modeling, and interviews/questionnaires. As a result of this more holistic view, the exposome paradigm is more consistent with the dynamic nature of exposures experienced by individuals across their life course. The exposome encompasses exposure from be- fore conception onward and through its application researchers can begin to investigate acute and cumulative exposures and their effects as well as to identify windows of susceptibility throughout the life course. In addition to assessing multiple exposures (and multiple outcomes), the holistic view of the exposome promotes the inclusion of both longitudinal and hierarchical exposure measures within epidemiologic approaches. Hierarchical data structures arise when exposure measures are nested within each other. Examples include spatial hierarchy, where exposures can be measured at the individual, local, and community level, and familial hierarchy, where both parental and fetal mea- sures are assessed. Hierarchical data can also arise from within individuals, such as when both exogenous (external) and endogenous (internal) measures of an exposure are assessed. There are existing epidemiologic methods to assess these types of multilevel exposures (10). However, expanded exposure assessment across the multiple domains (i.e., speciﬁc external, general exter- nal, and internal) of exposure that are the hallmarks of the exposome would greatly facilitate the investigation of multilevel or hierarchical data structures (38). INTRODUCTION AND OBJECTIVES This broad view is inclusive of researchers who use the exposome paradigm to understand the exogenous expo- sures, including policy and social environments, that contribute to health disparities (19), and includes those who utilize untargeted approaches to identify metabolic proﬁles associated with known environmental exposures (13). This review addresses the potential beneﬁts and overarching challenges surrounding the incor- poration of the exposome into epidemiologic research. Although it does not provide step-by-step 316 Stingone et al. instructions for conducting exposome research, it summarizes the current approaches and initia- tives aimed at implementing the exposome research paradigm within epidemiologic studies. In addition, it provides recommendations to address both scientiﬁc and practical barriers in order to move the ﬁeld forward and encourage researchers to integrate the exposome within their own work. www.annualreviews.org • Epidemiology and the Exposome Rationale for the Exposome Research Paradigm Although the exposome does not necessarily possess an inherent hierarchical structure, the ability to incorporate hierarchical exposures within the exposome facilitates the statistical analysis of upstream contributors to envi- ronmental exposures such as climate change and allows for the investigation of exposures that lack internal biomarkers. It also allows researchers to investigate the interplay between the parental and offspring exposome during fetal and early childhood development. Within this holistic framework, there is active discussion about how best to conceptualize and consider confounding in light of the totality of exposures being considered. If all environmental factors are coexposures within the exposome paradigm, then nonenvironmental factors remain www.annualreviews.org • Epidemiology and the Exposome 317 as potential confounders when assessing the combined effect of the totality of the environmental exposures (i.e., the exposome). The deﬁnition of environment used will then dictate which factors are nonenvironmental and thus potential confounders. From a practical standpoint, the statistical methodologies to estimate these combined effects are limited (40). Many researchers will continue to estimate effects between a single causative agent and outcomes within the exposome paradigm, which will require multiple confounding sets depending on the individual exposure of interest (38). In this case, the holistic nature of the exposome can improve researchers’ ability to control for confounders and reduce unmeasured confounding by promoting expanded and improved data collection for a variety of environmental (i.e., nongenetic) coexposures (40). The exposome affords epidemiologists an opportunity to utilize well-established epidemio- logic methods aimed at minimizing measurement error and reducing bias, while looking for new associations between environmental exposures and health or disease end points. The complexity of the exposome underscores the need for continued vigilance and adherence to epidemiologic principles such as accurate assessment and adjustment for confounders, consideration of selection biases when reexamining existing populations or recruiting new study populations, and acknowl- edgment of measurement error and the potential impacts that such errors can have on analytic results. These can help ensure an accurate and reliable interpretation of the data that are emerging from exposome research. Findings from exposome-related analyses have the potential to generate signals that need to be interpreted and followed up using bioinformatics and in vivo and in vitro ex- periments. Promising signals or pathways will need subsequent validation in independent studies, similar to validation of genome-wide association study ﬁndings (18). Rationale for the Exposome Research Paradigm Thus, the exposome repre- sents a complement to, not a replacement for, the hypothesis-driven research that has successfully advanced the ﬁeld of environmental health. Stingone et al. Current Approaches to Implement the Epidemiologic Method Relative to the Exposome The Health and Exposome Re- search Center: Understanding Lifetime Exposures (HERCULES; http://emoryhercules.com) was funded to develop the expertise and necessary tools and methodologies to provide a more comprehensive assessment of exposures and to advance exposome-related research. More re- cently, the NIEHS has established an infrastructure to promote the incorporation of the exposome within child health research studies. The Children’s Health Exposure Analysis Resource (CHEAR; https://www.niehs.nih.gov/research/supported/exposure/chear/) includes a network of labo- ratories with extensive analytic abilities for exposure assessment and measures of biologic response in a variety of biological samples. CHEAR also supports a data center that serves as a central data repository and provides methodological support for analysis of multiple environmental exposures, as well as develops novel statistical approaches for combining data across studies and analysis of high-dimensional exposure data. Whereas these previous examples represent coordinated efforts to advance the incorporation of the exposome into epidemiologic research, other examples of current epidemiologic studies have integrated measures of the exposome within their initial study designs. One large-scale initiative is the Japan Environment and Children’s Study ( JECS) (23). The JECS has enrolled more than 100,000 expectant mothers and utilizes multiple exposure measurement methodologies, including frequent questionnaire application, extensive biospecimen analyses, simulation models, and ambient environmental measurements to prospectively follow children through age 13 years. Having data on multiple exposures within all the exposome exposure domains across time for a large sample of children will allow researchers to characterize the early-life and childhood exposomes within this population. The US National Institute of Child Health and Human Development (NICHD) Longitudinal Investigation of Fertility and the Environment (LIFE) study focuses on the preconception period and aims to characterize the maternal and paternal exposomes and assess their effects on fertility and birth outcomes (5). A recent publication from this study identiﬁed reductions in couples’ fecundity associated with a number of paternal environmental exposures, including lead, selected phthalates, and polychlorinated biphenyls (4). Although these ﬁndings still reﬂect a chemical-class approach to analysis, the study’s data availability on multiple parental exposures facilitates a later, agnostic-based, exposome approach to understanding fertility and time to pregnancy. One example of an approach that examines the breadth of environmental exposures at a single time point related to disease is a data-driven analysis known as environment-wide association stud- ies (EWAS). Current Approaches to Implement the Epidemiologic Method Relative to the Exposome Successful implementation of the epidemiologic method relative to the exposome requires two components: the ability to accurately characterize the exposome and the ability to relate the com- plexity of the exposome to health-related end points. Many, but not all, of the current approaches seeking to examine health-related end points focus speciﬁcally on fetal and early childhood devel- opment owing to the close temporal association between exposure and effect within this window. Although the examples detailed below often examine the exposome within similar time windows, they illustrate the multiple, diverse avenues that researchers are taking to develop methods and incorporate the exposome into epidemiologic research. The three projects funded by the European Union’s Framework Program 7 (22) rep- resent a coordinated effort to advance this ﬁeld through characterization of the exposome within multiple time windows, development of exposure assessment and analytic methods, and examination of exposome–response relationships related to a number of health end points. The Health and Environment-wide Associations based on Large population Surveys (HEALS; http:/heals-eu.eu/) will develop an integrated methodology for compiling and organizing expo- sure data across multiple domains and will relate these factors to predict health outcomes. These methods can then be applied to conduct European-wide health assessments related to the expo- some, with the eventual goal of improved risk assessment. The EXPOsOMICS project leverages existing long-term European cohorts, and their stored biospecimens, to integrate external expo- sures from personal exposure monitoring technologies and population-based measures of exposure with internal measures resulting from -omics technologies (46). With a focus on air pollution and water contaminants, EXPOsOMICS will examine this segment of the exposome across studies 18 Stingone et al. 318 of populations at various points in the life course (46). Finally, the Human Early-Life Exposome (HELIX) project supplements existing data for 32,000 mother–child pairs from 6 European birth cohorts with new internal measures of exposure and biological response on a smaller subsam- ple of 1,200 mother–child pairs (48). This approach effectively capitalizes on the existing study infrastructure and past data collection to facilitate the newer, exposome-related measurements, including personal exposure monitoring and analysis of molecular signatures within stored and new biological samples. Within the United States, the National Institute of Environmental Health Sciences (NIEHS) has supported efforts to advance the technology, capacity, and infrastructure needed to imple- ment the exposome within environmental health research (9). www.annualreviews.org • Epidemiology and the Exposome 3 CHALLENGES TO INCORPORATING THE EXPOSOME INTO EPIDEMIOLOGIC RESEARCH Investigators have encountered three primary challenges to incorporating the exposome paradigm into epidemiologic research on a larger scale: (a) the lack of infrastructure to support the compre- hensive exposure assessment activities that are critical to exposome research; (b) the difﬁculty in differentiating normal physiologic variation from environmentally induced changes in measures of biological response (33); and (c) the lack of analytical, bioinformatics, and statistical method- ologies to process, integrate, and analyze high-dimensional data and the corresponding lack of cross-trained scientiﬁc investigators who can develop and implement these approaches. First, owing to their complexity and the requirement for a large commitment of resources, exposome studies are well served by centralized support and coordination to ensure that potential exposure assessment strategies are rigorously evaluated. This approach promotes valid assessment of exposure and facilitates data sharing, replication, and validation of ﬁndings across studies. En- couraging the increased availability of tools and methodologies could especially beneﬁt smaller efforts driven by investigators who could then apply the tools and approaches developed by larger- scale initiatives within their ﬁelds of research. Existing coordinated approaches of the European Initiative and the NIEHS CHEAR centers have already produced some discussion and develop- ment of analytic methods for exposomic data (1, 15). Similar investments in infrastructure and cross-study collaboration are necessary for life stages beyond pregnancy and birth, including sup- port for linking the early-life exposome to adult disease, as suggested by the developmental origins of health and disease hypothesis (17). Second, as ﬁrst suggested by Wild, the exposome is dynamic within and over time; thus, inves- tigators must differentiate periods of random variation from periods of induced biologic response (50). This observation not only translates into a need for accurate and precise measurements for various populations in order to characterize typical biological variation, but also stresses the importance of capturing the temporal variation in the exposome that occurs throughout an indi- vidual’s life course. Understanding this temporal variation is especially critical for time periods, such as development, puberty, and pregnancy, which are hypothesized to have speciﬁc patterns of greater biological variation and may be more susceptible to disruption by environmental expo- sures (8). Constructing a reference exposome is not feasible due to the dynamic nature of exposure across space and time. However, we can begin to characterize the temporal interrelatedness and variability of both exposures and responses throughout the life course. Current Approaches to Implement the Epidemiologic Method Relative to the Exposome Using cross-sectional data from the National Health and Nutrition Examination Sur- vey (NHANES), researchers examined 266 environmental factors collected at a single time point; they aimed to discover nongenetic contributors to type 2 diabetes by examining each factor within a statistical model and then simultaneously assessing the results across all models (30). The EWAS approach has since been applied to studies of preterm birth (32), communication impairments in nine-year-old children (42), and leukocyte telomere length (31). Individual studies have also fo- cused on providing proof-of-principle data using agnostic techniques to identify environmental www.annualreviews.org • Epidemiology and the Exposome 319 signals. These studies often utilize well-deﬁned exposure scenarios such as smoking to investigate whether speciﬁc biological signals can be traced in the biological system (16). These types of studies willresultinbestpracticesthatcanbeimplementedinfutureepidemiologicstudies.Togetherthese examples, from prospective cohorts to secondary analyses of cross-sectional data, illustrate that the exposome research paradigm can be implemented at different scales of epidemiologic research, de- pending on the research question and the availability and access to data and biospecimen resources. Stingone et al. CHALLENGES TO INCORPORATING THE EXPOSOME INTO EPIDEMIOLOGIC RESEARCH Finally, as discussed above, investigations of the exposome are considerably different from tra- ditional epidemiologic approaches. Practically, they require more data storage and more complex data management than do traditional studies. Analytically, statistical and other computational models are needed that can simultaneously accommodate heterogeneity from merging data across populations, longitudinal measurements, and large numbers of correlated exposures. Although genomic research also encounters a multitude of possible features coupled with their potential interactions, the longitudinal nature of environmental exposures is an added point of complexity for exposome research. This unique feature of the exposome needs to be considered in all phases 320 Stingone et al. of research and will stimulate the development of analytic methods that are sensitive to these fea- tures. To develop and implement these novel approaches, current and upcoming epidemiologists will require training in complementary disciplines such as computer science, bioinformatics, and advanced biostatistics (27). Catalog Existing Tools and Methodologies to Facilitate Exposome Research Catalog Existing Tools and Methodologies to Facilitate Exposome Rese Catalog Existing Tools and Methodologies to Facilitate Exposome Research As discussed above, researchers require easily accessible tools and approaches that can be used to characterize both exogenous and endogenous measures of exposure, including those exposures that do not have known corresponding biomarkers. The creation of a database to catalog and curate exposure assessment methods, ranging from questionnaires to assay protocols, along with their performance characteristics, would allow for wide implementation among the research com- munity. This step would ensure that epidemiologists have access to the most appropriate exposure assessment tools, while fostering the development and testing of methods using collected data. Having an easily accessible inventory of exposure assessment tools would also facilitate the sharing of methods and data across studies. As part of the NIEHS exposome workshop in January 2015 (9), working groups began the process of cataloging available tools for assessing environmental exposures and associated biological responses within their respective ﬁelds (11, 12, 44). Thus, this recommendation can likely be implemented soon. Similarly, establishing a standard methodology to examine and report ﬁndings from exposome- focused investigations would serve as a resource to investigators who are planning studies, as well as those who are at the publication stage. This research framework could emulate the STrengthening the Reporting of OBservational studies in Epidemiology–Molecular Epidemiology (STROBE- ME) guidelines (14). It could include guidance for describing biological specimen collection and analysis, as well as for incorporating guidelines for presenting agreement (or lack thereof ) between external and internal measures of exposure and potential sources of bias and error throughout the exposure assessment process. Again, this recommendation can be initiated in the short term, and the research framework can continue to evolve as research within this ﬁeld progresses. www.annualreviews.org • Epidemiology and the Exposome 32 Develop New Tools and Methods to Account for the Unique Aspects of the Exposome As presented above, one of the primary challenges to interpreting exposome research is differen- tiating between periods of natural variation and environmentally induced variation in measures of the exposome and then relating these measurements to health outcomes. The key to solving this challenge is two-pronged: First, deﬁne patterns of variation for exposures, particularly during critical and sensitive windows of human reproduction and development, and then design studies that examine the effect of changes in the exposome throughout the life course. Similar to how we use NHANES to understand background levels of contaminants, deﬁning variation in critical and sensitive windows is an important step in allowing researchers to place into its appropriate context the variation observed as a result of environmental exposures. The second step is to create study designs that facilitate comparisons in the exposome over time and in response to environmental exposures. Transgenerational, prospective life-course studies with repeated follow-up, including multiple periods of biological sampling, are one way to explore changes in the exposome that occur from preconception on throughout the life course. However, www.annualreviews.org • Epidemiology and the Exposome 321 Exposure -omics Health outcome Young children lth me Exposure -omics Health outcome Children Exposure Hea outco alth me a m Exposure -omics Health outcome Young adults -omics Exposure -omics Hea outco Exposure Ex -omics o Health outcome outcome a o Exposure -omics Health outcome Adults Age integration Figure 1 Framework for methodologies that integrate across individual studies to assess the exposome. Each square represents a cohort at a speciﬁc life stage. Black arrows illustrate the potential bidirectional relationships between measures of exposure assessment, -omics technologies, and health outcomes assessed within each cohort. Integrating across the ages represented by the different life-stage cohorts will allow investigators to explore the exposome across the life course. Framework for methodologies that integrate across individual studies to assess the exposome. Each square represents a cohort at a speciﬁc life stage. Black arrows illustrate the potential bidirectional relationships between measures of exposure assessment, -omics technologies, and health outcomes assessed within each cohort. Integrating across the ages represented by the different life-stage cohorts will allow investigators to explore the exposome across the life course. they require lifetimes for completion and an extensive commitment of resources, even when build- ing on existing studies. Additionally, relevant exposures may change during these expansive periods of time. Stingone et al. Develop New Tools and Methods to Account for the Unique Aspects of the Exposome As an alternative, we recommend the development of methods that account for timing of exposure within multiple critical and sensitive windows by merging across different cohorts that have focused on shorter windows. This approach is being investigated by the EXPOsOMICS research group (46). As shown in Figure 1, exposure is linked to health end points using advanced exposure science technologies including -omics within each critical life stage, and then integrated across life stages for a life-course view. These methods would require the ability to account for the heterogeneity in study populations as well as the potential differences in exposure assessment and the presence of coexposures. Developing these methods will require an investment of resources and a commitment to increase data sharing and consortium building. In addition to developing new epidemiologic study designs and approaches for the exposome, the ﬁelds of exposure science and biostatistics will also need to develop approaches that can account for the increased complexity of the exposome. New technologies such as sensors that enable individualized measures of external exposure and advanced modeling techniques that better estimate population-level exposures are two examples of the types of improvements in exposure science that will beneﬁt exposome research and minimize measurement error. The Pediatric Research using Integrated Sensor Monitoring Systems (PRISMS; https://www. nibib.nih.gov/research-funding/prisms) is a program to develop technologies such as smart phone applications and wearable and home-based sensors to improve exposure assessment in Stingone et al. 322 studies of childhood asthma. It serves as a model of the types of initiatives that are needed for other diseases and stages of life. New statistical methods are needed speciﬁcally to handle the high-dimensional data that will be produced by exposome studies. These methods need to accommodate the increased levels of heterogeneity from combining data from potentially different study populations, as well as the greater amount of uncertainty that arises from examining multiple exposures. However, these methods must continue to account for confounding factors and provide interpretable estimates of disease risk that can be used in risk assessment and health communication. Similarly, we will require better data management tools and increased available storage for the products of exposome-related research (27). All these new tools and methodologies, whether they are exposure assessment techniques, bioas- says, or new approaches to statistical modeling, need to be rigorously assessed in populations with gender, racial, socioeconomic, and geographic diversity. Develop New Tools and Methods to Account for the Unique Aspects of the Exposome Researchers have suggested that popu- lations with greater susceptibility or higher levels of environmental exposures, such as migrants and low socioeconomic groups, should be a priority for exposome research (41). The exposome has great potential to improve our understanding of health disparities but only if measures and methods are assessed and adapted for various health states in diverse populations. Doing so may require additional outreach to communities that are typically underrepresented in epidemiologic research to both improve recruitment into prospective studies and identify existing biospecimen repositories that represent diverse populations. www.annualreviews.org • Epidemiology and the Exposome Promote Data Sharing and Cross-Discipline Collaboration To develop the methodologies needed to advance exposomic research, exposure scientists, statisti- cians, and data scientists need access to epidemiologic data, particularly stored biological specimen resources for diverse populations. The National Institutes of Health (NIH) has a long-standing policy to promote data sharing among its intramural (29) and extramural research (28), although there are obstacles and considerations, particularly when using data derived from human subjects. In 2014, the Roundtable on Environmental Health Sciences of the National Academies of Sci- ences, Engineering, and Medicine held a workshop on the principles of and obstacles to sharing data from environmental research. As part of this work, researchers discussed ways to increase data sharing, including conducting quality control prior to sharing, developing common language and standards, and providing incentives to share beyond the minimal amount of data (34). Ef- forts aimed at improving data sharing in the environmental health community would facilitate the development of tools and methods needed to advance exposome research. In addition, epi- demiologic researchers both within and external to the environmental health community need to collaborate with researchers who are engaged in exposome methods development to ensure that these approaches are consistent with core epidemiologic principles of study design and analysis. Consider Causal Inference in the Context of the Exposome Just as statistical approaches need to be adapted to accommodate unique aspects of the exposome, the approach to causal inference may change with the increased complexity of the exposome. Epi- demiology draws on the replication of results and/or the use of meta-analyses to create a consensus out of individual observational research studies (43). The question arises about whether replica- tion is possible if the exposome varies in composition across different populations. Additionally, the exposome is composed of highly correlated exposures, both known and unknown. Within this context, the causal agent and the avenues for intervention may not be clear. There are examples in public health, such as the use of tobacco and exposure to diesel engine exhaust, where an in- tervention aimed at prevention was implemented without isolating a single chemical responsible for the adverse health effect (21, 35). However, it is possible that targeted studies of individual chemicals/exposures, which incorporate knowledge of sources, toxicology, and other scientiﬁc information, can inform public health in a way that might not be possible with the untargeted approaches of the exposome. As causal analysis methods applicable to observational research continue to advance, investiga- tors incorporating the multiple exposure domains of the exposome will have both the data and the tools for more comprehensive analysis and interpretation of ﬁndings from a causal perspective. For example, mediation analyses, which can increase our understanding of the mechanistic path from exposure to disease, will be greatly facilitated by having comprehensive data on exposures, includ- ing those with and without biomarkers and associated biological responses for study populations. We may be able to rely more on these investigations of mechanistic pathways to infer causality once these diverse data are more widely available as the exposome research paradigm is implemented. Address the Multiple Sources and Types of Uncertainty Present within Exposome Research The multiple exposures and responses within exposome research can be measured by a variety of methods, ranging from self-administered questionnaires to laboratory assays. Each approach has its own potential for error or misclassiﬁcation, which when considered in the context of the exposome contributes to the overall uncertainty of the research study. Although uncertainty in the characterization of the exposome should not stop progress, its implications on the obtained results should be thoughtfully considered and reported by investigators. A growing body of empirical research has focused on uncertainty and measurement error for single exposures in epidemiology (7). However, it is not a straightforward process to translate this work to multiple exposures and across the life course. We caution against using exposome research to rule out exposures of interest, owing to the potential for multiple sources of nondifferential error or misclassiﬁcation. However, even when exposure misclassiﬁcation is hypothesized to be nondifferential, additional conditions need to be met in order to conclude that the bias would be toward the null (20). Additionally, the potential for false discovery can be heightened in the context of highly correlated exposures within the exposome, particularly when these exposures are measured with different levels of error (51). As discussed by Zidek et al. (51), simulation studies have illustrated that the association between an outcome and a causal exposure with high measurement error can fail to be observed and in- stead manifest as an association between the outcome and a highly correlated surrogate of the causal exposure measured with less error. This situation is especially problematic when relying solely on statistical signiﬁcance to denote associations of interest. As a result, the roles of mea- surement error and collinearity need to be considered when positive associations are observed in exposome research. As in traditional epidemiologic studies, these ﬁndings need to be followed up with additional targeted studies. In the long term, we encourage the development of statistical methodologies that can combine measurement error across multiple sources within the exposome. www.annualreviews.org • Epidemiology and the Exposome 323 Stingone et al. ACKNOWLEDGMENTS This work began as part of a January 2015 workshop on the exposome hosted by the NIH and the National Institute of Environmental Health Sciences. This work was supported in part by NIH grants P30ES023515 and U2CES026555. J.A.S. was supported by the Elizabeth Mascia Schol- arship from the Mount Sinai Children’s Environmental Health Center. We thank M. Chadeau- Hyam for producing Figure 1. DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review. CONCLUSION The goal of incorporating the exposome into epidemiologic research is to improve our ability to uncover the environmental contributors to health and disease. The discovery science enabled by the exposome presents a complement to the hypothesis-driven investigations of modern epidemi- ology, and both are needed to move the ﬁeld of environmental epidemiology forward. As discussed in this review, many challenges have hindered the incorporation of the exposome on a larger scale 324 Stingone et al. Stingone et al. 324 within environmental health research. Infrastructure and coordinated support for the new tech- nologies and approaches needed to measure multiple domains of the exposome within populations have been limited. Characterizing the complexity of the exposome is a daunting task, and many epidemiologists have not been trained to manage, store, or analyze the high-dimensional data that result from exposome investigations. In addition, analytic methods that can account for the com- plexity of multiple, highly correlated exposures measured at multiple time points have been limited. Over the past ten years, however, the ﬁeld has experienced some progression from initial discus- sions of the paradigm to empirical examples of how the exposome could be implemented, including proof-of-principle studies showing that environmental signals can be traced within the body (16). Investigators have now initiated efforts, such as the HELIX (48) and JECS (23) studies, to design epidemiologic research that capitalizes on new technologies and seeks to characterize the early-life exposome and relate it to health end points. The infrastructure available to support these types of studies has also expanded. To continue this progression and successfully conduct research within the exposome paradigm, we need to adapt current methodologies and create new approaches to handle the unique requirements of assessing the exposome. These include the high dimensional- ity of data, the dynamic nature of exposure and response throughout the life course, the need for greater and broader data sharing, and the demand for transdisciplinary research teams composed of investigators with diverse skill sets. These recommendations are offered to encourage researchers to embark on exposome research and to further develop the needed approaches and analytic meth- ods in our collective efforts to promote the environmental health and well-being of all populations. www.annualreviews.org • Epidemiology and the Exposome LITERATURE CITED 1. Agier L, Portengen L, Chadeau-Hyam M, Basagana X, Giorgis-Allemand L, et al. 2016. A systematic comparison of linear regression-based statistical methods to assess exposome-health associations. Environ. Health Perspect. 124:1848–56 2. Andra SS, Austin C, Arora M. 2016. The tooth exposome in children’s health research. Curr. Opin. Pediatr. 28:221–27 3. 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https://openalex.org/W4283813442	https://ieeexplore.ieee.org/ielx7/9424/10064202/09795904.pdf	English	null	A Functional Architecture for 6G Special-Purpose Industrial IoT Networks	IEEE transactions on industrial informatics	2,023	cc-by	11,712	A Functional Architecture for 6G Special-Purpose Industrial IoT Networks Nurul Huda Mahmood , Gilberto Berardinelli , Senior Member, IEEE, Emil J. Khatib , Member, IEEE, Ramin Hashemi , Student Member, IEEE, Carlos De Lima, Member, IEEE, and Matti Latva-aho , Senior Member, IEEE 2020s is expected to be succeeded by the sixth generation (6G) wireless network around 2030. The International Telecommu- nications Union, which is responsible for deﬁning International Mobile Telecommunications (IMT) systems, has already started to examine future technology trends for “IMT towards 2030 and beyond”1; with the ﬁrst set of deﬁnitions expected to be available around mid-2024. The standardization body 3GPP also plans to initiate studies into 6G requirements from mid-2024 with the ﬁrst basic 6G standard anticipated to be deﬁned around 2027. Abstract—Future industrial applications will encompass compelling new use cases requiring stringent performance guarantees over multiple key performance indicators, such as reliability, dependability, latency, time synchronization, security, etc. Achieving such stringent and diverse service requirements necessitates the design of a special-purpose Industrial-Internet-of-Things (IIoT) network comprising a multitude of specialized functionalities and technological enablers. This article proposes an innovative architecture for such a special-purpose sixth generation (6G) IIoT net- work incorporating seven functional building blocks cate- gorized into special-purpose functionalities and enabling technologies. The former consists of Wireless Environment Control, Trafﬁc/Channel Prediction, Proactive Resource Management, and End-to-End Optimization functions, whereas the latter includes Synchronization and Coordi- nation, Machine Learning and Artiﬁcial Intelligence Algo- rithms, and Auxiliary Functions. The proposed architecture aims at providing a resource-efﬁcient and holistic solution for the complex and dynamically challenging requirements imposed by future 6G industrial use cases. Selected test scenarios are provided and assessed to illustrate cross- functional collaboration and demonstrate the applicability of the proposed architecture in a wireless IIoT network. Alongsideconventionalhuman-typecommunications,5GNR has incorporated two dedicated service classes to support ma- chine type communications (MTC), namely massive MTC and ultra-reliable low-latency communications (URLLC). MTC will play a dominant role in future beyond 5G/6G systems owing to its huge potential for business and technological innovations. From a business perspective, the ability to automate commu- nications between machines paves the way toward connectivity as a service business model, thereby enabling a wide range of novel applications and use cases [1], [2]. New technological innovations are also urgently needed to allow intelligent, scal- able, and energy efﬁcient solutions that can meet the challenging requirements of future MTC networks [3]. 2530 IEEE TRANSACTIONS ON INDUSTRIAL INFORMATICS, VOL. 19, NO. 3, MARCH 2023 A Functional Architecture for 6G Special-Purpose Industrial IoT Networks Index Terms—Artiﬁcial intelligence (AI), beyond ﬁfth gen- eration (5G), Industrial Internet of Things (IIoT), machine learning (ML), reconﬁgurable intelligent surfaces (RISs), sixth generation (6G), special-purpose networks, ultra- reliable low-latency communications. Industrial Internet of Things (IIoT) primarily caters to MTC applications with requirements from an industrial network such as a manufacturing setup and control systems for railways and energy management [3], [4]. IIoT converges information and communication technology with operational technology and is an integral component of the fourth industrial revolution (Indus- try 4.0), where cyber-physical systems utilize reliable and fast control loops between sensors and actuators to automate delicate control tasks [5]. Communications networks designed to provide seamless connectivity for various IIoT applications are realized using a wide range of, mostly proprietary, wired and wireless solutions [1]. In many cases, such solutions are customized to the local use case motivated by security considerations, technology limitations, and legacy design, which limit their scalability and adaptability to different situations [5]. The ﬂexibility and mass customization of production envisioned by Industry 4.0 requires agile and open connectivity solutions while maintaining the high performance guarantees accorded by existing customized solutions [6]. The introduction of URLLC service class in 5G 1Beyond 5G: What’s next for IMT? [Online]. Available: https: //www.itu.int/en/myitu/News/2021/02/02/09/20/Beyond-5G-IMT-2020- update-new-Recommendation This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ ) Gilberto Berardinelli is with the Department of Electronic Systems, Aalborg University, 9100 Aalborg, Denmark (e-mail: gb@es.aau.dk). ive Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ g y g ( g ) Emil J. Khatib is with the University of Málaga, 29016 Málaga, Spain (e-mail: emil@UMA.ES). II. KEY REQUIREMENTS OF 6G IIOT NETWORKS The notion of multiservice communications introduced in 5G is expected to expand further in 6G with the emergence of new use cases and service classes driven by advances in communications and other technologies such as sensing, imag- ing, and artiﬁcial intelligence (AI) [2], [13]. The potential use cases and expected key performance indicators (KPIs) of future 6G networks have recently been considered by various authors (e.g., [2], [8], [9], [12]). The diverse set of expected 6G use cases can be described in terms of a set of KPIs representing 1) ultra- reliability, 2) low-latency, 3) high data-rate, 4) massive access, 5) energy-efﬁciency, and 6) localization and sensing accuracy. However, all of these—sometimes conﬂicting—requirements are not needed to be met simultaneously in a single system. Ultra-reliability, low-latency, localization, and sensing accuracy, and to a certain extent high data-rate, will be the most important KPIs for future 6G IIoT networks, which is the focus application in this work. This section brieﬂy summarizes some use cases and requirements relevant to future 6G IIoT networks. We propose an architecture consisting of seven specialized functions grouped into special-purpose functionalities and key enabling technologies. Such an architecture will allow opti- mizing the implementation of the key functionalities through dedicated network components. We envision that the proposed 6G special-purpose IIoT network will be a part of the wider 6G ecosystem of Network of networks. The foundation for such networks dedicated to speciﬁc use cases like IIoT is rooted in the Non-public (i.e. private) networks (NPN) feature in the 3GPP Release-16 standard [3], [14], and the URLLC/IIoT en- hancement features to be ﬁnalized in the forthcoming Release 17. Although some of the special-purpose functionalities and enabling technologies presented in this article have already been discussed in several 6G related articles, to the best of our knowledge, thereisnoexistingliteraturediscussingaframework integrating all these functionalities into a system concept and highlighting the tight integration among them. Furthermore, a few of the functionalities presented in this article, such as high-accuracy positioning through integrated communications and sensing, are rather novel concepts that have started being discussed in the literature just recently [15]. The novel contri- butions of this work with respect to the state of the art can be summarized as follows. The key requirements and expected KPIs of a 6G IIoT special- purpose network stem from its various potential use cases. MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS 2531 NR is the ﬁrst step toward having an universal wireless standard to meet these needs. future industrial communications networks is detailed. We limit ourselves to a selected set of features relevant for IIoT applications since designing a complete system architecture requires multidisciplinary expertise and col- laboration that is beyond the scope of this work. Although the 5G NR URLLC solutions have made progress in enabling wireless connectivity for IIoT use cases, the true vision of replacing proprietary connectivity solutions with a universal wireless system in industrial networks is yet to be realized [7]. Moreover, emerging use cases in the coming decade will impose new requirements that were not considered in 5G NR. A robust, scalable, and efﬁcient 6G network is thus neces- sary to meet the diverse requirements of the upcoming decade. Research efforts toward deﬁning 6G have been pursued by academic researchers [2], [8]–[10], major international research projects [11], and key industrial players [12], [13]. Due to the diversity of use cases and application areas, 6G is expected to be a “network of networks” that will aggregate multiple types of resources connecting at different scales [11]. 3) We demonstrate the feasibility of the proposed functional architecture through numerical evaluations, thus moving beyond merely presenting the concepts in generic terms. The rest of the article begins with an overview of the key requirements of a 6G IIoT special-purpose network in Section II. Each of the functional blocks in the proposed architecture is then elaborated in Section III. The effectiveness of the proposed architecture in enhancing the network performance is demon- strated with an example use case in Section IV, describing how the different blocks interact for the sake of controlling the radio environment to improve IIoT connectivity. Finally, Section V concludes this article. This article contributes to the ongoing efforts in deﬁning 6G by proposing an integrated functional architecture for a 6G special-purposeIIoTnetwork.Inparticular,wefocusonwireless IIoT networks motivated by the demand for robust wireless communication technologies that can seamlessly replace wired connections in the IIoT domain [1]. A transmission failure in an IIoT network may lead to downtime, which is costly in terms of money and efforts. Hence, there is an enormous value—not only in terms of academic research, but also from a commercial perspective—in designing robust IIoT networks. I. INTRODUCTION W IRELESS network evolution follows a trend of having a new generation every decade. Following this trend, the ﬁfth generation (5G) New Radio (NR) introduced in the W the ﬁfth Manuscript received 30 November 2021; revised 27 April 2022; ac- cepted 31 May 2022. Date of publication 14 June 2022; date of current version 3 March 2023. This work was supported by the Academy of Finland within 6G Flagship program under Grant 346208. Paper no. TII- 21-5323. (Corresponding author: Nurul Huda Mahmood.) ( p g ) Nurul Huda Mahmood, Ramin Hashemi, and Matti Latva-aho are with 6G Flagship, Centre for Wireless Communications, University of Oulu, 90014 Oulu, Finland (e-mail: nurulhuda.mahmood@oulu.ﬁ; ramin.hashemi@oulu.ﬁ; matti.latva-aho@oulu.ﬁ). ( p g ) Nurul Huda Mahmood, Ramin Hashemi, and Matti Latva-aho are with 6G Flagship, Centre for Wireless Communications, University of Oulu, 90014 Oulu, Finland (e-mail: nurulhuda.mahmood@oulu.ﬁ; ramin.hashemi@oulu.ﬁ; matti.latva-aho@oulu.ﬁ). ( p g ) Nurul Huda Mahmood, Ramin Hashemi, and Matti Latva-aho are with 6G Flagship, Centre for Wireless Communications, University of Oulu, 90014 Oulu, Finland (e-mail: nurulhuda.mahmood@oulu.ﬁ; ramin.hashemi@oulu.ﬁ; matti.latva-aho@oulu.ﬁ). @ ; @ ) Gilberto Berardinelli is with the Department of Electronic Systems, Aalborg University, 9100 Aalborg, Denmark (e-mail: gb@es.aau.dk). g y g ( g @ ) Emil J. Khatib is with the University of Málaga, 29016 Málaga, Spain (e-mail: emil@UMA.ES). Carlos De Lima is with the Nokia Bell Labs, 90620 Oulu, Finland (e-mail: carlos.de_lima@nokia.com). Color versions of one or more ﬁgures in this article are available at https://doi.org/10.1109/TII.2022.3182988. p g Digital Object Identiﬁer 10.1109/TII.2022.3182988 p g Digital Object Identiﬁer 10.1109/TII.2022.3182988 p g Digital Object Identiﬁer 10.1109/TII.2022.3182988 ct Identiﬁer 10.1109/TII.2022.3182988 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ reative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creat II. KEY REQUIREMENTS OF 6G IIOT NETWORKS For instance, the application of digital twins (DT), which provide a real-time representation of physical objects in the virtual world, will be further extended to include digital representation of the (wireless propagation) environment and assets beyond manufacturing—leading to the massive DT use case [11]. This will require a new class of URLLC with high data rates, thereby imposing novel design challenges. As another exam- ple, wirelessly connected multirobot systems in smart factories with highly reliable driving systems (i.e. navigation, collision avoidance, sensing, etc.) will increase the demand on the scale, complexity, and quality of service (QoS) of the connectivity requirement [6], [16]. 6G will bring further advances in pri- vate networks [3], [17], where the ownership of the network infrastructure and data belongs to the entity that is using it to support its own applications. Special-purpose networks often go hand in hand with private networks. IIoT is a typical example where special-purpose networks are used, since it is often a good 1) We take a clean slate approach and present a functional architecture for 6G special-purpose IIoT networks that can efﬁciently meet the stringent and diverse design goals. We believe that the distribution of different enabling technology components across different functionalities within the proposed architecture make its management and integration agile and seamless. We believe that the distribution of different enabling technology components across different functionalities within the proposed architecture make its management and integration agile and seamless. 2) The tight integration among the presented functionalities enabling them to meet the stringent requirements of the IEEE TRANSACTIONS ON INDUSTRIAL INFORMATICS, VOL. 19, NO. 3, MARCH 2023 2532 Fig. 1. Proposed functional architecture for a 6G special-purpose IIoT network. Fig. 1. Proposed functional architecture for a 6G special-purpose IIoT network. Fig. 1. Proposed functional architecture for a 6G special-purpose IIoT network. TABLE I COMPARISON OF SELECTED MTC KPIS IN 5G AND 6G prospective IIoT relevant KPIs in 6G and their comparison with 5G KPIs is presented in Table I. COMPARISON OF SELECTED MTC KPIS IN 5G AND 6G III. KEY FUNCTIONAL BLOCKS A summary of the MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS 2533 networkmanagementfunctionalitiesarebeyondthescopeofthis work. Proactive Resource Management and E2E Optimization func- tionalities, whereas itself is enabled by the Auxiliary Functions (e.g., localization) and, to a certain extend, ML and AI Algo- rithms. The proposed special-purpose 6G IIoT network is envisioned to be a private network deployed in a industrial setting. For instance, it can be a completely private network deployed, owned, and operated by the factory owner; a private network deployedand(partially)operatedbyanetworkoperator,network equipment vendor, or a third party at the premise of, and in col- laboration with, the factory owner; or a virtual private network operating on a sliced portion of a public commercial cellular network [1]. Such a special-purpose 6G IIoT network will be supported by the envisaged spectrum usage scenarios for future 6G networks, where spectrum may be allocated to a conﬁned area in line with the Network of networks concept [17]. B. Trafﬁc/Channel Prediction a) Functionality deﬁnition: IIoT devices vary widely in their functionalities, leading to diverse trafﬁc types in the industrial network. The trafﬁc generated by a given node can be tempo- rally and/or spatially correlated. A detailed modeling of such spatio-temporal trafﬁc correlation, and possibly collating it with other contextual information, can be utilized to better predict the arrival of the source trafﬁc. This in turn leads to more efﬁcient access to the shared wireless media, better resource utilization, lower latency, and higher reliability. y g y b) Research challenges and enabling techniques: Trafﬁc in IIoT networks can be broadly categorized as periodic and event- triggered. Predicting periodic trafﬁc arrival requires identifying the exact time instance when the trafﬁc will be generated and its size. There is a rich body of literature investigating math- ematical models for such trafﬁc behavior, such as the nonho- mogeneous Poisson process model and Markovian processes and frequency-based analysis [27] for long-term correlation modeling. However, such linear prediction models are not well suited to predict event-driven IIoT trafﬁc. ML-based prediction methods have recently emerged as viable solutions to capture the complex and nonlinear spatiotemporal dependence structure of the event-driven trafﬁc generation. The list of the proposed solutions include deep belief network-based prediction method, applying a long short-term memory structure [28], and applying deep convolutional neural networks to model the spatial and temporal trafﬁc dependence [29]. Despite their demonstrated performance under speciﬁc scenarios, most ML-based solutions are very much scenario-speciﬁc, dependent on large training data, and not easily amenable to interpretation. To address this, the main operational goal for the proposed prediction func- tionality is to integrate learning-based prediction with accurate modeling of the spatial–temporal trafﬁc correlation considering the available domain knowledge and contextual information. Such an approach in 6G will likely lead to more accurate and explainable prediction models in 6G. b) Research challenges and enabling techniques: Trafﬁc in IIoT networks can be broadly categorized as periodic and event- triggered. Predicting periodic trafﬁc arrival requires identifying the exact time instance when the trafﬁc will be generated and its size. There is a rich body of literature investigating math- ematical models for such trafﬁc behavior, such as the nonho- mogeneous Poisson process model and Markovian processes and frequency-based analysis [27] for long-term correlation modeling. However, such linear prediction models are not well suited to predict event-driven IIoT trafﬁc. A. Wireless Environment Control Similarly, preceding RF signals and depth-images can be exploited for received power prediction in wireless channels, so that resulting outages due to deep fades or channel blockages can be predicted beforehand and managed proactively. Recently, reconﬁgurable intelligent surface (RIS)-aided wireless communication is gain- ing widespread attention as a means to engineer the propagation environment [25]. An RIS is made of meta-materials with ad- justable phase shift and amplitude response. By adjusting these, anRIScanpassivelytransformtherandomwirelessenvironment into a controllable channel, thereby rendering the signal propa- gation more deterministic. Additionally, centralized information about the activities of different transmitters can lead to better information about the interference generation and its variation across time and space. Advanced ML and AI algorithms can be applied to reliably track the interference variation and predict its future values. Thus, guarantees on the interference experienced by different receivers can be provided with high accuracy, which leads to better performance guarantees and efﬁciency [26]. c) Interaction with other functionalities: We envision this functional block to be receiving data from multiple UEs, RAN elements such as access points, and the CN interfaces, while interacting with the Auxiliary Functions and the ML and AI Algorithms as the key enablers. The trafﬁc patterns predicted by this functionality will serve as inputs to the Proactive Resource Management and E2E Optimization functionalities. C. Proactive Resource Management III. KEY FUNCTIONAL BLOCKS This section presents the proposed functional architecture for a 6G IIoT network, comprising of seven specialized functional blocks, as illustrated in Fig. 1. The 6G network is divided into four sections [user equipment (UE), radio access network (RAN), core network (CN), and external services]. The pro- posed architecture is categorized into special-purpose function- alities and enabling technologies. The former includes Wire- less Environment Control, Trafﬁc/Channel Prediction, Proactive Resource Management, and End-to-End (E2E) Optimization. The Wireless Environment Control functionality deals mainly with the air interface, that is, the part of the UE and RAN directly related to radio communications. The Trafﬁc/Channel Prediction functionality analyzes the trafﬁc occurring both in the air interface and internal interfaces within the network, which is closely related to the services provided over the network. The Proactive Resource Management functionality orchestrates the resources in the 6G network, such as the scarce radio resources and computing nodes in the RAN and the CN. The E2E Optimization functionality monitors and improves all the elements in all the sections of the network in order to provide service-speciﬁc optimizations and increase the E2E QoS. On the other hand, the special-purpose functionalities rely on a set of enabling technologies that provide the computational tools [Machine Learning (ML) and AI Algorithms] and services (Auxiliary Functions) that compose their operation, as well as a Synchronization and Coordination functionality that orches- trates their work, overviews their effects, and prevents conﬂicts. The proposed architecture focuses on the lower layers of the open systems interconnect (OSI) protocol layer, and higher layer practice, both in technical and managerial terms to separate the critical business resources from the rest. The diverse set of 6G KPIs will either evolve from the main 5G KPIs or are novel ones introduced in 6G. Important 5G KPIs such as reliability and latency will be further extended in 6G to consider the broader concept of dependability, whose main attributes are availability, reliability, safety, integrity, and security [18], [19]. Similarly, emerging applications like con- nectivity for multirobot systems including unmanned aerial objects will render connectivity requirements to be measured in volumetric unit. In addition, 6G will witness a number of novel KPIs coming to the fore. Many emerging applications requiring precise localization will render localization accuracy an important KPI [15]. Alongside, sophisticated industrial con- trol applications will require tight synchronization, low jitter, and time-sensitive communications [3], [20]. A. Wireless Environment Control a) Functionality deﬁnition: The stochastic nature of the wire- less environment and random interference are among the key challenges when ensuring high reliability in wireless communi- cations. We envision a dedicated functionality addressing these challenges. Generally, ﬂuctuation of the received signal power due to fast fading is tackled through diversity techniques, such as multiconnectivity [23]. However, not all forms of diversity solutions are applicable in industrial scenarios. In order to tame the random fading in wireless channels, novel approaches that can track, predict, and even favorably shape the propagation condition are anticipated toward 6G. b) Research directions and enabling techniques: Modeling the wireless environment through a DT and/or with the as- sistance radio frequency (RF; e.g., ray-tracing), non-RF tech- nologies (e.g., color and depth images) and integrated sensing and communications techniques can be used to better track and predict the channel behavior [15]. An accurate wireless map can provide deterministic knowledge of the path loss and also allow better prediction of the channel fading [24]. Similarly, preceding RF signals and depth-images can be exploited for received power prediction in wireless channels, so that resulting outages due to deep fades or channel blockages can be predicted beforehand and managed proactively. Recently, reconﬁgurable intelligent surface (RIS)-aided wireless communication is gain- ing widespread attention as a means to engineer the propagation environment [25]. An RIS is made of meta-materials with ad- justable phase shift and amplitude response. By adjusting these, anRIScanpassivelytransformtherandomwirelessenvironment into a controllable channel, thereby rendering the signal propa- gation more deterministic. Additionally, centralized information about the activities of different transmitters can lead to better information about the interference generation and its variation across time and space. Advanced ML and AI algorithms can be applied to reliably track the interference variation and predict its future values. Thus, guarantees on the interference experienced by different receivers can be provided with high accuracy, which leads to better performance guarantees and efﬁciency [26]. b) Research directions and enabling techniques: Modeling the wireless environment through a DT and/or with the as- sistance radio frequency (RF; e.g., ray-tracing), non-RF tech- nologies (e.g., color and depth images) and integrated sensing and communications techniques can be used to better track and predict the channel behavior [15]. An accurate wireless map can provide deterministic knowledge of the path loss and also allow better prediction of the channel fading [24]. B. Trafﬁc/Channel Prediction ML-based prediction methods have recently emerged as viable solutions to capture the complex and nonlinear spatiotemporal dependence structure of the event-driven trafﬁc generation. The list of the proposed solutions include deep belief network-based prediction method, applying a long short-term memory structure [28], and applying deep convolutional neural networks to model the spatial and temporal trafﬁc dependence [29]. Despite their demonstrated performance under speciﬁc scenarios, most ML-based solutions are very much scenario-speciﬁc, dependent on large training data, and not easily amenable to interpretation. To address this, the main operational goal for the proposed prediction func- tionality is to integrate learning-based prediction with accurate modeling of the spatial–temporal trafﬁc correlation considering the available domain knowledge and contextual information. Such an approach in 6G will likely lead to more accurate and explainable prediction models in 6G. E. Synchronization and Coordination Traditionally, wireless communication systems distribute time reference among BSs using the global navigation satel- lite system (GNSS) infrastructure, which is enough to meet present-day time requirements [35]. 5G NR implements two main procedures to propagate time reference across communi- cating devices, namely the typical GNSS-based approach and over-the-air synchronization. 5G NR synchronization require- ments are not necessarily more restrictive, though high-accuracy time synchronization has become a critical aspect owing to the frame structure and advanced radio resource algorithms. GNSS/global positioning system time synchronization provides areasonabletimereference,thoughtheperformancedeteriorates in canyon or indoor deployment scenarios. In this regard, trans- port network over-the-air synchronization becomes a promising alternative. Moreover, 5G NR coordinated RAN features such c) Interaction with other functionalities: The Proactive Re- source Management functionality logically lies at the center of the proposed functional architecture. On one hand, it is driven by system requirements including E2E optimization constraints and the network trafﬁc proﬁles. On the other hand, it relies upon the Synchronization and Coordination, Wireless Environment Control, and Auxiliary Functions to implement the outputs of its ML and AI-powered algorithms. C. Proactive Resource Management In- stead, intelligent mechanisms that proactively allocate resources by predicting the trafﬁc demand and the channel conditions beforehand are needed [3]. Scheduling and assignment of trans- mission resources, e.g., transmit power, bandwidth, etc., are the two main aspects of resource management at the physical and the access layers. A mismatch between the trafﬁc arrival and the allocated slots for existing proactive scheduling techniques like semipersistent scheduling leads to underutilized resources or additional access latency. Such concerns can be addressed by dynamically grouping users based on their predicted trafﬁc proﬁle and allocating resources accordingly. For example, users with low-trafﬁc correlation may access the same channel using grant-free schemes, leading to signiﬁcantly reduced collision probability. b) Research challenges and enabling techniques: The lat- est network optimization trends include an E2E perspective, where the monitored performance indicators cover speciﬁc data sessions or application classes, and represent the QoS at the application layer. At the same time, the optimized parameters affect all the network elements that serve the corresponding application [33]. E2E optimization also gains relevance with functions such as Multiaccess Edge Computing [34], where network computing resources are moved “closer” to the end- users to reduce the response time of certain services. With the help of ML/AI functions running on Big Data resources of the block described in Section III-B2, these functions can be implemented, replicated for diverse applications, and reused for novel optimization loops. b) Research challenges and enabling techniques: The lat- est network optimization trends include an E2E perspective, where the monitored performance indicators cover speciﬁc data sessions or application classes, and represent the QoS at the application layer. At the same time, the optimized parameters affect all the network elements that serve the corresponding application [33]. E2E optimization also gains relevance with functions such as Multiaccess Edge Computing [34], where network computing resources are moved “closer” to the end- users to reduce the response time of certain services. With the help of ML/AI functions running on Big Data resources of the block described in Section III-B2, these functions can be implemented, replicated for diverse applications, and reused for novel optimization loops. Current E2E optimization functions cover the communication channels between the endpoints of a data session. Nevertheless, the problems and suboptimal behavior may come from the conﬁguration of the end devices. C. Proactive Resource Management a) Functionality deﬁnition: The preceding two blocks discuss methods to predict the trafﬁc arrival, and track the channel and interference variation. The Proactive Resource Management block explores tools and techniques to utilize the predicted trafﬁc and interference information in designing scheduling and interference management techniques. The main goals are to reduce the access and transmission latency through prompt c) Interaction with other functionalities: This functional block closely interacts with and aids the implementation of IEEE TRANSACTIONS ON INDUSTRIAL INFORMATICS, VOL. 19, NO. 3, MARCH 2023 2534 scheduling, ensure high reliability through intelligent interfer- ence management schemes and guarantee efﬁcient utilization of scarce resources. Toward this end, network intelligence tech- nologies running at different elements across the network that fully automate the network operations will become the standard in 6G [3], [30]. having numerous conﬁguration parameters, whose optimization is a multidimensional problem. Once an optimal conﬁguration point is achieved, it must be actively maintained, since the envi- ronmental conditions of the network change over time and some elements may malfunction. Optimization involves iteratively adjusting the parameters of interest until reaching a desired performance. This scheme can target functionalities at different levels, for instance, at the RAN, the CN, or the radio interface level. b) Research challenges and enabling techniques: Conven- tional reactive resource management principles, where trafﬁc is served on-demand and transmission failures are addressed through retransmissions, are not well suited to meet the stringent latency and reliability requirements of many IIoT use cases. In- stead, intelligent mechanisms that proactively allocate resources by predicting the trafﬁc demand and the channel conditions beforehand are needed [3]. Scheduling and assignment of trans- mission resources, e.g., transmit power, bandwidth, etc., are the two main aspects of resource management at the physical and the access layers. A mismatch between the trafﬁc arrival and the allocated slots for existing proactive scheduling techniques like semipersistent scheduling leads to underutilized resources or additional access latency. Such concerns can be addressed by dynamically grouping users based on their predicted trafﬁc proﬁle and allocating resources accordingly. For example, users with low-trafﬁc correlation may access the same channel using grant-free schemes, leading to signiﬁcantly reduced collision probability. b) Research challenges and enabling techniques: Conven- tional reactive resource management principles, where trafﬁc is served on-demand and transmission failures are addressed through retransmissions, are not well suited to meet the stringent latency and reliability requirements of many IIoT use cases. C. Proactive Resource Management The detection of such issues can be very important to discard nonexisting network problems that may reﬂect in certain performance indicators (such as the data throughput). In the future, standardized interfaces may even allow the network to propose conﬁguration improvements to the endpoints, such as buffer sizes or priority queue parameters. The full-scale E2E connectivity landscape will also include integra- tion of wired and wireless networks to support the multitude of challenging QoS requirements [1]. Predictive resource allocation is more challenging than scheduling, especially in the case of an interferred network. Pre- dicting the received signal and interference strength can result in a more accurate prediction of the future signal-to-interference plus noise ratio (SINR), which in turn leads to improved link adaptation [26]. In the case of centralized resource allocation or coordination between base stations (BS), the predicted strongly interfering BS can be proactively requested to manage the inter- ference through muting or silencing a speciﬁc resource block. The proactive resource allocation paradigm extends beyond the lower layers to include proactive allocation of network slicing and edge-computing resources. Resource allocation for network slicing is particularly challenging since the physical network and edge-computing resources can be particularly scarce, and due to the limitations imposed by the mobility requirements of the end-user [30], [31]. In this context, proactive resource allocation by forecasting the end-user demand and dynamically matching it with the available resources leads to better QoS, and fairer and more efﬁcient allocation of the scarce resources [3], [32]. c) Interaction with other functionalities: Since the E2E com- munication link spans across the entire network, the E2E Opti- mization functionality has to interact with all other functionali- ties, including the enabling technologies. c) Interaction with other functionalities: Since the E2E com- munication link spans across the entire network, the E2E Opti- mization functionality has to interact with all other functionali- ties, including the enabling technologies. F. ML and AI Algorithms The ML and AI Algorithms functional block perform ac- tions to solve a problem based on information extracted from an environment that is either too complex or has too many variables to be efﬁciently solved by conventional optimization algorithms. This is precisely the case for many problems in the 6G network, where management functionality is bound to have both complex and large datasets given the great amount of users, service transactions, and infrastructure components [3]. In ML algorithms, the operation can be separated in a learning and an exploitation phase. The learning phase is usually slow, requires large datasets and is computationally costly, whereas the exploitation, that is, using the model to obtain new outputs based on unobserved inputs, is usually based on simple and fast operations. For the learning phase, there are three main possible locations: 1) distributed (i.e., running in the terminals, using only the dataset visible to the computing node), 2) federated (i.e., the algorithm runs in the devices and no data are shared among the nodes, but models are shared, improving the information available at each node), or 3) centralized (data are sent to the cloud, where the ML algorithms run). For the exploitation, that is, the use of the learned model to obtain new estimations, the algorithm can either run in the device, where it will produce fast results at relatively low computational cost, or in the 6G CN or network edge [6]. As depicted in the functional architecture in Fig. 1, this block will gather relevant information and then implement such auxiliary functions. In this context, new ML/AI solutions have the potential to leverage the availability of sensing information to streamline operational procedures and optimize the overall performance. Thus, aiming to support very high reliability and extreme low latency, real-time and accurate sensing becomes a crucial component. In this aspect, the research challenges are related to tradeoffs between sensing and communications, dual- functional (sensing and communications) waveform design, and signal-processingalgorithmstoaddressmutualinterferencecan- cellation [15]. Similarly, ML/AI-enabled intelligent spectrum management has the potential to not only cope with spectrum scarcity and harsh interference, but also to signiﬁcantly improve spectrum allocation, especially in the context of NPNs and licensed shared access [1]. In such deployments, collaborative- and edge-computing will become increasingly indispensable thus raising concern about the proper implementation of privacy, security, and trust. G. Auxiliary Functions 5G NR offers very promising features such as large band- width, very high carrier frequency, massive antenna arrays, and densiﬁcation. B5G/6G systems are expected to continue this trend and further develop them. This will be enabled by ad- vanced RAN infrastructure and auxiliary network functionalities beyond communication technologies. Future wireless networks will thus increasingly rely on auxiliary side-information for their overall operation and performance optimization. These include high-accuracy localization and high-resolution environ- ment mapping through integrated sensing and communications to support adaptive resource allocation [15], dynamic/intelligent spectrum management, as well as security enablers at the lower layers of the protocol stack. D. E2E Optimization a) Functionality deﬁnition: A cellular network is a very com- plex system with each deployed hardware and software element MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS 2535 as coordinated multipoint transmission, beamspace processing, and time-of-ﬂight-based positioning demand much tighter time synchronization. As time has progressed, ML has been adopted in a wider set of applicationsinmanydiverseﬁelds.Thishasledtotheemergence of a need for trustworthiness. While classical ML algorithms act as a black box, which take datasets as input and produce a model as an output, there is a growing demand for explainable ML [37], i.e., that the produced model is somehow justiﬁed. There are two approaches for achieving this; either using glass box (or white box) ML algorithms, that is, algorithms that are self-explanatory,orusingalgorithmsthat,attachedtoablackbox ML algorithm can obtain a human-understandable explanation. The development and use of these systems in 6G (and, therefore, in the present block) are key for a widespread adoption, based on the trust of operators and end-users. In order to provide reliable data transport, as well as seam- less connectivity of sensors, actuators, and controllers, special- purpose IIoT networks demand accurate time and clock synchro- nization so as to harmonize time among often independent and distributed clocks. For example, programable logical controllers are typically employed in sequential processes and thus need very tight clock synchronization with time variation limited to at most 1 μs. Legacy industrial settings are typically deployed through wired networks implementing time-sensitive network- ing (TSN) standards [1]. Hence, the proper integration of 5G and TSN requires E2E time synchronization to support time-critical industrial applications, which can be implemented via generic precision time protocol [35]. F. ML and AI Algorithms In particular, the network need to be made more resilient to security vulnerabilities at the CN-edge-RAN communication links, privacy issues in private networks (espe- cially with shared network infrastructure), and advanced attacks employing ML/AI techniques [3], [38]. The objective of this block is to simplify the development and deployment of novel ML-based applications. Centralizing the learning phase within the 6G network will have several advantages; ﬁrst, the management of learning datasets is easier andenablesthecreationofnovelfunctionsquickly.Furthermore, it allows access to a larger dataset compared to a decentralized implementation. Second, the possibility of creating libraries and reusing code increases the productivity of future develop- ment. Finally, centralization allows for more efﬁcient usage of computingresources,includingBigDatatechnologies[36],such as Cloud Computing, schema-free databases, etc. The exploita- tion phase can be done, as earlier indicated, in the network edge or even in the terminals, so response times of ML-based services are not compromised [30]. DEPLOYMENT SCENARIOS AND IMPLEMENTATION COSTS One of the main advantages of the proposed functional archi- tecture is that it is modular, i.e., each block can be implemented independently while still being interconnected with the other IEEE TRANSACTIONS ON INDUSTRIAL INFORMATICS, VOL. 19, NO. 3, MARCH 2023 2536 Fig. 2. Considered system model where a BS serves multiple actua- tors in a wireless industrial environment. blocks. Hence, the proposed functionalities can be placed cen- trally or distributed across the network. The blocks that require more prompt actions, such as wireless environment control, can be located in close proximity to the end devices, e.g. on a edge cloud. On the other hand, functionalities operating over a more relaxed time scale can eventually run in a central cloud, therefore beneﬁting from higher computational capacity, besides ofﬂoading the edge cloud. As many of the functionalities may rely on ML and AI algorithms, the models can eventually be trained in the central cloud and then transferred to the edge cloud for their prompt execution. The models can be then further optimized online by relying on persistent connections between theedgeandthecentralcloud.Byofﬂoadingcomplexcomputing tasks and data storage from the devices to the edge and/or the cloud, edge computing further enhances the operation of limited terminals not only as a way to reduce latency, but also to save energy and carry out highly demanding computational procedures. There are, of course, several tradeoffs in exchange of this advantage, which are as follows. Fig. 2. Considered system model where a BS serves multiple actua- tors in a wireless industrial environment. Fig. 3. SNR cdf for the phase optimized RIS as well as when the RIS acts as a relay, with and without the direct link f(t), N = 512. 1) The proposed architecture will not be readily applicable in other scenarios, e.g., massive IoT networks. Different conﬁgurations and functionalities of 6G will have to be adapted to those uses. 2) There is an added communication overhead, especially when implemented as a distributed architecture. This overhead affects both the devices (that will have more functionalities available, but also will need to rely more on the network connectivity), and the network elements (edge computing nodes, remote servers, etc.). Fig. 3. SNR cdf for the phase optimized RIS as well as when the RIS acts as a relay, with and without the direct link f(t), N = 512. IV. EXAMPLE OF USAGE OF PROPOSED ARCHITECTURE Novel technologies like the use of RIS are introducing a paradigm shift by allowing to shape the propagation characteris- tics favorably and hence are poised to be an integral part of future 6G systems [25], [39]. An RIS-assisted wireless link allows the components of the received signal at a target receiver to add constructively resulting in boosting the received signal-to-noise ratio (SNR). In this section, we illustrate how the proposed architecture works for the usage and optimization of phase shifts of an RIS to improve the connectivity quality of an IIoT network. We consider an industrial scenario where a BS serves one or more actuators. Due to the presence of signiﬁcant noise and interference caused by large machinery and heavy multipath propagation effects induced by highly reﬂective structures, the wireless channel in an industrial environment is much different compared to conventional wireless propagation scenarios [40]. DEPLOYMENT SCENARIOS AND IMPLEMENTATION COSTS 3) The computational cost for the network operator will be higher and the operator has to dedicate more resources to automatic functions that allow for deploying, maintain- ing, and optimizing a much more complex network than prior generations. We investigate the use of RIS to control the wireless environ- ment with the objective of mitigating the adverse propagation effects considering the deployment scenario presented in Fig. 2. RIS represents an instance of the Wireless Environment Control functionality. In this example, we show the interconnection of Wireless Environment Control block with the Trafﬁc/Channel Prediction block, which has the scope here of predicting channel state information (CSI) as this is fundamentally important for leveraging RIS, as well as with the ML and AI Algorithms block, that has the task of optimizing the phase response of the RIS. Nevertheless, these tradeoffs allow devices to have more func- tionalities available from the network and efﬁcient development of new services. Hence, the computational and communication costs of the proposed architecture can be very efﬁciently man- aged while reaping its advantages. B. Application of the Trafﬁc/Channel Prediction Block Fig. 4. Normalized gain of the BS–RIS–Rx link for different values of maximum phase errors due to channel estimation error. The results presented in Section IV-A consider an ideal sce- nario and give an indication of the general performance trends. In order to evaluate the beneﬁts of controlling the wireless environment using RIS in realistic scenarios, we evaluate the performance under nonideal assumptions in this section. The perfect CSI assumption is relaxed. In practice, an RIS contains a large number of elements that increases the number of links whose CSI are to be estimated, resulting in an unacceptably large pilot overhead. In addition, the RIS itself is a passive component, and hence, the channel can only be sensed at a receiver by sounding the channel from a transmitter. This implies the need to estimate the channels g and h in (1) from the observation of the cascaded effect of gHΘh [42]. strong assumptions allow assessing the full potential of RIS and the corresponding results serve as performance upper bounds. In the case of single-antenna BS and actuator, the received narrowband signal at the actuator in the presence of an RIS with N elements at time instant t is given by y(t) = f(t) + g(t)HΘ(t)h(t) s(t) + n(t) (1) (1) where f(t), g(t) ∈CN×1, and h(t) ∈CN×1 represent the combined effects of the small-scale fading and the distance- dependent path loss of the BS–actuator, RIS–actuator, and BS– RIS channels, respectively. The N-dimensional square matrix Θ(t) = diag(β1eiθ1, β2eiθ2, . . . , βNeiθN ) represents the RIS el- ements with βn ∈[0, 1] being the amplitude attenuation and θn ∈(0, 2π] being the phase shifts, ∀n ∈{1, 2, . . . , N}. The overhead and complexity of channel estimation in an RIS-assisted wireless systems can be reduced by implementing speciﬁc algorithms in the Trafﬁc/Channel Prediction block. The unique properties of the RIS channel can be exploited for this purpose. For instance, given the ﬁxed locations of the BS and RIS, the BS–RIS channel g usually remains unchanged over a longer period and hence can be estimated less often compared to the RIS–actuator channel h [42]. In the case of multiple actuators, the fact that the BS–RIS channel is the same for all users results in the cascaded channels associated with different users having some correlation among them, which can be ex- ploited to reduce the estimation overhead. A. Application of the Wireless Environment Control Block In the ﬁrst illustrative example, we demonstrate how an RIS can be used to control the wireless environment. We assume an ideal scenario where the BS, which controls the RIS elements, has full CSI of the BS–RIS and the RIS–actuator channels. The BS optimizes the phase shifts of each of the N RIS elements such that the phases of complex baseband received signals coming from the BS–RIS and the RIS–actuator channels add construc- tively at the receiver end. We further consider the possibility of optimizing the reﬂection phases without any discretization error, i.e., the phases of the RIS elements are able to take any value in the continuous range (0, 2π] and the absence of any loss due to absorption or coupling between the RIS elements [41]. Such MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS 2537 TABLE II MEDIAN AND THE RANGE OF THE SNR, IN DECIBEL, FOR THE FOUR DIFFERENT CASES PRESENTED IN FIG. 3 Fig. 4. Normalized gain of the BS–RIS–Rx link for different values of maximum phase errors due to channel estimation error. TABLE II MEDIAN AND THE RANGE OF THE SNR, IN DECIBEL, FOR THE FOUR DIFFERENT CASES PRESENTED IN FIG. 3 elements’ phase shifts results in a signiﬁcant improvement in the SNR,withagainofover25dBinthemedianvalue.Furthermore, the variation in the SNR is greatly reduced, as indicated by the steep slope of the cdf plot and the range values in Table II. The lesser is the variation in the received SNR through a given channel, the more deterministic is that channel—which implies higher dependability. Hence, the use of RIS in controlling the wireless environment has important positive implications for industrial scenarios requiring dependable communications. The gains from employing RIS are less pronounced in the presence of the direct source–destination link with a modest gain of less than 4 dB in the median value. This is because the compound source– RIS–destination channel is reported to decay with the product of the source–RIS and RIS–destination distances, which leads to a much higher path loss compared to the direct channel [41]. Thus, the SNR gain accorded by the RIS is masked by the signiﬁcantly stronger source–destination path. Nonetheless, the range of the SNR distribution is still signiﬁcantly reduced by around 20 dB, thus making the channel more deterministic. B. Application of the Trafﬁc/Channel Prediction Block Similarly, the channel through the RIS can be transformed and tested for sparsity in the angular domain, which may arise in scenarios with limited propagation paths. In the case of such sparsity, the channel estimationproblemcanbeformulatedas asparsesignal recovery problem and subsequently solved using the rich body of knowl- edge available on solving compressed sensing problems [43]. The application of ML-based approaches to directly observe the impact of the RIS elements phase shifts on the achievable rate (or other performance metrics) without estimating the channel itself has also been proposed as an alternative solution [44], which is further investigated in Section IV-C. n ( , ] g p , { , , , } Fig. 3 presents the cumulative distribution function (cdf) of received SNR at the actuator in the presence of an RIS. The number of elements in the RIS, N = 512, and the path loss in decibel is given by PL = 34.53 + 38 log10(d), where d is the transmitter–receiver separation distance in metres. We consider a scenario where the BS, the RIS, and the actuator are located at points (0, 0), (10, 10), and (100,0) on the (x, y) grid (in meters), respectively. Four different cases are presented in Fig. 3. The SNR distribution when the RIS merely acts as a relay with static phase shifts (θn = 0 ∀n) considering the absence (f(t) = 0) and presence (f(t) ̸= 0) of the direct link are shown by the dashed lines. The solid lines represent the SNR distribution through an RIS with optimum phase shift, θ∗ n = −(ξn + ζn) ∀n, where ξn and ζn are the phase shifts of the nth element of g(t) and h(t), respectively. The median and the range of the SNR, in decibel, for the four different cases presented in Fig. 3 are tabulated in Table II. By comparing the values corresponding to the cases without the direct link (black lines), we observe that optimizing the RIS IEEE TRANSACTIONS ON INDUSTRIAL INFORMATICS, VOL. 19, NO. 3, MARCH 2023 2538 Fig. 4 illustrates the power of the BS–RIS–receiver link (i.e., ∥g(t)HΘ(t)h(t)∥2) when there is a mismatch in the phase shifts of the RIS elements, Θ(t), due to channel estimation error. The power is normalized by the ideal case corresponding to perfect CSI estimation. The mismatch is modeled as a random variable uniformly distributed between 0 and the maximum phase mismatch. B. Application of the Trafﬁc/Channel Prediction Block The number of RIS elements N is 1024. The ﬁrst two bar plots, respectively, present the continuous phase and the case where the phase shifts of the RIS elements are assumed to take discrete values from a ﬁnite set, whose members are determined by the number of quantization bits. For instance, a 2-b quantization corresponds to four different phase shift values. In both cases, the error stems from estimating the cascaded effect of g and h collectively. The last two bar plots represent the cases where the estimation error only occurs at either of these two channels. It is observed from Fig. 4 that the loss due to CSI estimation error is minimal for smaller estimation error values; it is less than 10% for a phase mismatch of up to π/3. Furthermore, the impact of the CSI estimation error on the power gain of the RIS-assisted link will be further minimized when considered together with other non-idealities such as quantization error, absorption loss (i.e., βn < 1), and mutual coupling between the RIS elements. This is evident from the plots corresponding to “2-bphase,”whereweobservethatsomeofthephaseerrordueto channel estimation error is positively offset by the quantization error resulting in a slightly better performance compared to the “continuous phase” case. Although not reported here, a similar trend is observed for other values of N. Fig. 5. Average and the standard deviation of the sum Shannon and FBL rate of four actuators in an RIS-assisted scenario with ideal and nonideal RIS. The RIS phase shifts are optimized using the TD3 RL algorithm. Fig. 5. Average and the standard deviation of the sum Shannon and FBL rate of four actuators in an RIS-assisted scenario with ideal and nonideal RIS. The RIS phase shifts are optimized using the TD3 RL algorithm. function R. The Shannon rate and the ﬁnite blocklength (FBL) rate [47] for a blocklength of 20 channel uses are considered. The latter is a more accurate representation of the achievable rate in factory automation use cases transmitting short but critical status update messages. The application of ML and AI Algorithms block to solve the complex problem of optimizing the RIS elements’ phase shifts also absolves the BS from needing to estimate the CSI of the involved channels. Fig. 5 presents the moving average (averaged over the past 50 samples) of the sum rate at the four actuators as a function of the training episodes. C. Application of the ML and AI Algorithms Block In short, the Twin Delayed Deep Deterministic Policy Gradient (TD3) deep reinforcement learning algorithm [46] is applied where the set of RIS phase shift values form the action space A, and the sum rate considering the outage probability constraint is the reward B. Application of the Trafﬁc/Channel Prediction Block The Shannon rate and the FBL rate considering an ideal RIS (i.e., continuous phase shifts), as well as nonideal RIS with βmin = 0.8 and 2-b quantized phase shifts, are shown by the blue and the black curves, respectively. The shadow represents the variation in the rate given by one stan- dard deviation of the averaging samples. The limited number of samples to average over for Episodes < 50 results in the discontinuity observed around Episode = 50. We also see that there is a nonnegligible loss in the sum rate of less than 10% in the converged state resulting from the nonideal assumptions, though the performance trend is the same. A signiﬁcant gain in terms of the average sum rate is attained by optimizing the RIS elements’ phase shifts through the ML and AI algorithms block. Approximate Shannon (FBL) rate gains of up to 50% (90%) and 75% (130%) are observed for ideal and nonideal RIS elements, respectively. However, it must be noted that such gains are obtained when the ML algorithms converge over hundreds of episodes, which incurs nonnegligible computational latency. Applications requiring low latency will impose constraints on the maximum number of episodes, and hence, the obtained phase shift values may lead to more modest performance gains. C. Application of the ML and AI Algorithms Block In this last section, we extend the investigation scenario to multiple actuators and consider nonideal RIS with absorption and mutual coupling loss and RIS phase shifts quantized into four levels, i.e., represented by 2 b. The amplitude of the RIS phase response is modeled following the empirical model pre- sented in [45]. The amplitude βn, which is a function of θn, is given as [45, (5)] βn(θn) = (1 −βmin) sin(θn −φ) + 1 2 α + βmin (2) (2) where φ and α are constants related to the speciﬁc circuit imple- mentation. We have set βmin = 0.8. A multiantenna BS concur- rently serves four single-antenna actuators placed in an indoor factory area at the points (135, 105), (105, 135), (120, 90), and (90, 120), respectively. The BS is positioned at (75, 75) and the RIS is located at the edge at (150, 150). The (quantized) phase shifts of the RIS elements are chosen such that the sum rate at the four actuators is optimized, subject to a stringent outage probability constraint of 10−6 at each of the actuators. The ensuing optimization problem is not solvable using conventional methods due to the mutual interference coupling among the different actuators. Given the complexity of the optimization problem, we apply ML algorithms to solve it. In short, the Twin Delayed Deep Deterministic Policy Gradient (TD3) deep reinforcement learning algorithm [46] is applied where the set of RIS phase shift values form the action space A, and the sum rate considering the outage probability constraint is the reward where φ and α are constants related to the speciﬁc circuit imple- mentation. We have set βmin = 0.8. A multiantenna BS concur- rently serves four single-antenna actuators placed in an indoor factory area at the points (135, 105), (105, 135), (120, 90), and (90, 120), respectively. The BS is positioned at (75, 75) and the RIS is located at the edge at (150, 150). The (quantized) phase shifts of the RIS elements are chosen such that the sum rate at the four actuators is optimized, subject to a stringent outage probability constraint of 10−6 at each of the actuators. The ensuing optimization problem is not solvable using conventional methods due to the mutual interference coupling among the different actuators. Given the complexity of the optimization problem, we apply ML algorithms to solve it. V. CONCLUSION [15] F. Liu et al., “Integrated sensing and communications: Towards dual- functional wireless networks for 6G and beyond,” IEEE J. Sel. Areas Commun., vol. 40, no. 6, pp. 1728–1767, Jun. 2022. This article proposed an innovative functional architecture for 6G special-purpose IIoT networks comprising four distinct special-purpose functionalities and three enabling technologies. The special-purpose functionalities are made up of RAN func- tions such as Wireless Environment Control and Trafﬁc/Channel Prediction; and cross-layer optimized functionalities such as Proactive Resource Management and E2E Optimization. The enabling technologies for these special-purpose functionalities include Synchronization and Coordination, ML and AI Algo- rithms, and Auxiliary Functions. The proposed architecture was designed to enable resource-efﬁcient solutions to the complex and dynamically changing requirement of emerging IIoT appli- cations. All these functionalities come at the cost of manage- able communications overhead and a higher computational cost from the side of the network, which, thanks to cloud and edge computing technologies, can be very efﬁciently managed. The applicability of the proposed architecture was demonstrated in an RIS-assisted wireless IIoT network where the phase shifts of the RIS elements residing in the Wireless Environment Control block were optimized with the help of the Trafﬁc/Channel Prediction and ML and AI blocks to improve the sum rate performance under a stringent outage constraint. The numerical results demonstrated a sum throughput gain of over 100% under realistic assumptions for the FBL rate. [16] E. J. Khatib and R. Barco, “Optimization of 5G networks for smart logistics,” Energies, vol. 14, no. 6, 2021, Art. no. 1758. [17] P. Ahokangas, M. Matinmikko-Blue, S. Yrjölä, and H. Hämmäinen, “Platform conﬁgurations for local and private 5G networks in complex industrial multi-stakeholder ecosystems,” Telecommun. Policy, vol. 45, no. 5, 2021, Art. no. 102128. [18] N. 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Mahmood et al., “On the resource utilization of multi-connectivity transmission for URLLC services in 5G new radio,” in Proc. IEEE Wireless Commun. Netw. Conf. Workshop, 2019, pp. 1–6. [24] Y. Zeng and X. Xu, “Toward environment-aware 6G communications via channel knowledge map,” IEEE Wireless Commun., vol. 28, no. 3, pp. 84–91, Jun. 2021. MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS 2539 RIS would need to interact with other blocks, namely the Traf- ﬁc/Channel Prediction and the ML and AI Algorithms blocks, to realize reliable and robust wireless links. Although not outlined here, the other blocks in the proposed functional architecture will be required to play a role in a more complex scenario. For instance, the Proactive Resource Management and the E2E Optimization blocks can introduce higher layer aspects, which are not considered in the physical layer analysis presented here. RIS would need to interact with other blocks, namely the Traf- ﬁc/Channel Prediction and the ML and AI Algorithms blocks, to realize reliable and robust wireless links. Although not outlined here, the other blocks in the proposed functional architecture will be required to play a role in a more complex scenario. For instance, the Proactive Resource Management and the E2E Optimization blocks can introduce higher layer aspects, which are not considered in the physical layer analysis presented here. [10] N. H. Mahmood et al., “Machine type communications: Key drivers and enablers towards the 6G era,” EURASIP J. Wireless Commun. Netw., vol. 2021, no. 134, pp. 1–25, Jun. 2021. [11] M. A. Uusitalo et al., “6G vision, value, use cases and technologies from European 6G ﬂagship project Hexa-X,” IEEE Access, vol. 9, pp. 160004–160020, 2021. pp [12] H. Viswanathan and P. E. 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We have demonstrated how the Wireless Environment Control block represented by an MAHMOOD et al.: FUNCTIONAL ARCHITECTURE FOR 6G SPECIAL-PURPOSE INDUSTRIAL IoT NETWORKS REFERENCES He is currently a doctoral student with the CWC, University of Oulu, Oulu, Finland, in the 6G Flagship project. His research interests in- clude resource allocation in wireless networks, ultra-reliable low-latency communications, re- conﬁgurable intelligent surface, multi-in multi-out wireless communica- tions, beyond 5G, and ﬁber-optic communications. Ramin Hashemi (Student Member, IEEE) was born in Ardabil, Iran. He received the B.Sc. and M.Sc. (with hons.) degrees in wireless commu- nication from the Amirkabir University of Tech- nology Tehran Polytechnic, Tehran, Iran, in 2016 and 2018, respectively. [39] R. Hashemi, S. Ali, N. H. Mahmood, and M. Latva-aho, “Joint sum rate and blocklength optimization in RIS-aided short packet URLLC systems,” IEEE Commun. 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Nurul Huda Mahmood received the Ph.D. de- gree in communications theory from Norwegian University of Science and Technology, Trond- heim, Norway, in 2013, and the Habilitation de- gree in critical MTC from the University of Oulu, Oulu, Finland, in 2021. He is currently a Senior Researcher and an Adjunct Professor with the Center for Wireless Communications, University of Oulu, where he is also the coordinator for wireless connectivity research area and leads critical machine type communications research within the 6G ﬂagship research program. His current research focuses on the resilient communications for the 6G era. Matti Latva-aho (Senior Member, IEEE) re- ceived the M.Sc., Lic.Tech., and Dr.Tech. (hons.) degrees in electrical engineering from the Uni- versity of Oulu, Oulu, Finland, in 1992, 1996, and 1998, respectively. Matti Latva-aho (Senior Member, IEEE) re- ceived the M.Sc., Lic.Tech., and Dr.Tech. (hons.) degrees in electrical engineering from the Uni- versity of Oulu, Oulu, Finland, in 1992, 1996, and 1998, respectively. p y From 1992 to 1993, he was a Research En- gineer with Nokia Mobile Phones, Oulu, after which he joined CWC, University of Oulu. He was the Director of CWC during 1998–2006 and the Head of the Department for Communication Engineering until August 2014. He is currently a Professor with the University of Oulu on wireless communications and Director for National 6G Flagship Programme. He is also a Global Research Fellow with Tokyo University. Emil J. Khatib (Member, IEEE) received the Ph.D. degree in mobile networks from the Uni- versity of Malaga, Malaga, Spain, in 2017. He is currently a Postdoctoral Researcher with the University of Málaga. He has par- ticipated in several national and international projects related to Industry 4.0 projects. He is currently working on the topic of security, energy efﬁciency, and localization in B5G/6G networks. REFERENCES E. 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Khatib (Member, IEEE) received the Ph.D. degree in mobile networks from the Uni- versity of Malaga, Malaga, Spain, in 2017. He is currently a Postdoctoral Researcher with the University of Málaga. He has par- ticipated in several national and international projects related to Industry 4.0 projects. He is currently working on the topic of security, energy efﬁciency, and localization in B5G/6G networks. [34] N. Abbas, Y. Zhang, A. Taherkordi, and T. Skeie, “Mobile edge computing: A survey,” IEEE Internet Things J., vol. 5, no. 1, pp. 450–465, Feb. 2018. [35] I.Godoretal.,“Alookinside5Gstandardstosupporttimesynchronization for smart manufacturing,” IEEE Commun. Standards Mag., vol. 4, no. 3, pp. 14–21, Sep. 2020. pp p [36] E. J. Khatib, R. Barco, P. Munoz, I. De La Bandera, and I. Serrano, “Self- healing in mobile networks with Big Data,” IEEE Commun. Mag., vol. 54, no. 1, pp. 114–120, 2016. pp [37] A. Rai, “Explainable AI: From black box to glass box,” J. Acad. Marketing Sci., vol. 48, no. 1, pp. 137–141, 2020. [38] M. Ylianttila et al., Eds., 6G White Paper: Research Challenges for Trust, Security and Privacy, Ser. 6G Research Visions, nr. 9. Oulu, Finland: Univ. Oulu, Jun. 2020. [Online]. Available: http://jultika.oulu.ﬁ/ﬁles/ isbn9789526226804.pdf Ramin Hashemi (Student Member, IEEE) was born in Ardabil, Iran. He received the B.Sc. and M.Sc. (with hons.) degrees in wireless commu- nication from the Amirkabir University of Tech- nology Tehran Polytechnic, Tehran, Iran, in 2016 and 2018, respectively. REFERENCES He has authored or coauthored more than 500 conference or journal papers in the ﬁeld of wireless com- munications. His group currently focuses on 6G systems research. His research interests include mobile broadband communication systems. Prof. Latva-aho was the recipient of the Nokia Foundation Award in 2015 for his achievements in mobile communications research. Gilberto Berardinelli (Senior Member, IEEE) received the ﬁrst- and second-level degrees (cum laude) in telecommunication engineering from the University of L’Aquila, L’Aquila, Italy, in 2003 and 2005, respectively, and the Ph.D. degree in wireless communication from Aalborg University, Aalborg, Denmark, in 2010. y, g, , He is currently an Associate Professor with the Wireless Communication Networks Section, Aalborg University, and also working in tight co- operation with Nokia Bell Labs, Oulu, Finland. He is the author or coauthor of more than 100 international publica- tions, including conference proceedings, journal contributions, and book chapters. His research interests include physical layer, medium access control, and radio resource management design for 5G systems and beyond.
https://openalex.org/W4367051719	https://nottingham-repository.worktribe.com/preview/20834652/Sliding%20mode%20corrector%20for%20jet%20UAV%20control.pdf	English	null	Sliding mode corrector for jet UAV control	The aeronautical journal/Aeronautical journal	2,023	cc-by	21,041	Sliding mode corrector for jet UAV control Xinhua Wang University of Nottingham, Aerospace Engineering, Nottingham, United Kingdom Email: wangxinhua04@gmail.com Abstract: A sliding mode corrector is presented for disturbance rejection in position sensing using relatively accurate velocity measurement. The corrector design is based on a robust second- order sliding mode (2-sliding mode), which makes the fusion of position and velocity on a sliding surface to reject disturbance. Even when the frequency bands of disturbance and actual position signal overlap, or large-magnitude disturbance exists, the corrector can still provide the accurate and smoothed estimate of position. The proposed corrector is applied to a jet UAV navigation and control. In the unmanned aerial vehicle (UAV) system, the disturbances exist in position and attitude measurements, and the uncertainties exist in the system dynamics. For the UAV trajectory tracking control, the system model is constructed in the earth-ﬁxed frame, and the constructed model is ﬁt for observer design to estimate system uncertainties. The control laws are designed according to the correction of position and attitude by the correctors and the estimation of system uncertainties by an existing observer. Finally, the ﬂight experiment demonstrates the eﬀectiveness of the proposed method. Keywords: Sliding mode corrector; position sensing; jet UAV; system uncertainties Sliding mode corrector for jet UAV control Xinhua Wang University of Nottingham, Aerospace Engineering, Nottingham, United Kingdom Email: wangxinhua04@gmail.com Sliding mode corrector for jet UAV control Xinhua Wang University of Nottingham, Aerospace Engineering, Nottingham, United Kingdom Email: wangxinhua04@gmail.com 1 Introduction This paper considers correction of stochastic disturbance in position sensing and application to jet UAV navigation and control. This interest was motivated by the enormous civil and military applications of such ﬁxed wing UAVs. It is one of the most attractive research focuses because the dynamical system of a jet UAV has many prominent features including powerful thrust provision, payload augmentation, high-speed ﬂight and a high maneuverability [1,2,3]. UAV large-range ﬂight needs information of global position, attitude and dynamic model, also ﬂying velocity and angular velocity are necessary. However, in many cases, disturbances exist in position and attitude sensing, and uncertainties are inevitable in system modelling. These bring challenge for control. In ﬂight control systems, rational desired attitude is important for safe ﬂight, and the deter- mination of desired attitude needs the information of actual position and attitude [4]. However, disturbances in position and attitude sensing render the incorrect desired attitude, and the un- wanted control command is generated. Constant sensing disturbance can be overcome through initial calibration. Comparing to constant sensing disturbance, time-varying position disturbance is more likely to rend a serious mismatch between desired attitude and actual position, and it causes dangerous ﬂight. Furthermore, the frequency bands of disturbance and actual position signal may overlap, and disturbance cannot be separated from actual position signal using the usual low-pass ﬁlters. GPS (Global positioning system) can provide global position information with accuracy of several meters or even tens of meters [5,6]. Adverse environmental inﬂuences may contaminate GPS signals [6], and the position accuracy may become worse. Velocity is also important for UAV navigation and control. GPS can measure device velocity with two diﬀerent accuracies: 1) large-error velocity by the diﬀerence method with accuracy of a meter per second due to position accuracy and noise eﬀect; 2) accurate velocity by Doppler shift measurement with accuracy of a few centimeters, or even the accuracy approaching 5mm/s is possible [7,8]. Alternatively, accurate velocity of device can be measured by a Doppler Radar sensor with accuracy of a few centimeters [9]. Hence, measuring Doppler shift is a preferred way to get velocity. Except for sensing, velocity can be estimated from position using the observers or diﬀerentiators [10,11]. However, relatively accurate measurement of position is required. INS (Inertial navigation system) can estimate position and velocity through integrations from acceleration measurement. Nomenclature pm(t) position measurement p0(t) actual position d1(t) disturbance in position measurement L1 upper-bound of position disturbance vm(t) velocity measurement v0(t) actual velocity d2(t) disturbance in velocity measurement L2 upper-bound of velocity disturbance ε upper-bound of sensor accuracy ratio e1 sliding variable e2 sliding variable k1 corrector parameter k2 corrector parameter k3 corrector parameter ω1 position disturbance frequency ρ(ω1) disturbance rejection ratio x position in earth-ﬁxed frame x-direction y position in earth-ﬁxed frame y-direction z position in earth-ﬁxed frame z-direction ϕ roll angle θ pitch angle 1 ψ yaw angle α angle of attack β sideslip angle U linear velocity in body frame axis xb V linear velocity in body frame axis yb W linear velocity in body frame axis zb Γ the earth-ﬁxed frame Λ the body frame m UAV mass g gravity acceleration ΩΛ angular rate vector ΩΓ Euler angle derivative vector F total external force Fjet thrust by jet engine Fw aerodynamic forces on the ﬁxed wing Ff aerodynamic forces on the fuselage Fr forces created by the rudders Fe forces created by the elevators Fd uncertainties and external disturbances τ total moment τw moments created by the ﬁxed wings τr moments created by the rudders τe moments created by the elevators τd moments due to the uncertainties and external disturbances ρ air density Sw area of the half wing CL0 ﬁxed wing lift coeﬃcient when the angle of attack α equals zero CLα ﬁxed wing lift coeﬃcient due to the angle of attack α δi ﬁxed wing aileron deﬂection CLδi lift coeﬃcient due to the aileron deﬂection δi CD0 ﬁxed wing drag coeﬃcient when α = δi= 0 Aw aspect ratio of the ﬁxed wing ew value of the Oswald’s eﬃciency factor τwa ﬁxed wing aerodynamic moment τwc ﬁxed wing control torque Sf fuselage equivalent cross-sectional area Lf lift force generated by the fuselage Df drag force generated by the fuselage Clf fuselage lift coeﬃcient Cdf fuselage drag coeﬃcient 2 2 Se area of the elevator δe elevator deﬂection Cleα eleviator lift coeﬃcient due to the angle of attack α and the deﬂectio Cde0 drag coeﬃcient when α + δe= 0 Ae aspect ratio of the elevator ee the Oswald’s eﬃciency factor τea elevator aerodynamic moment τec elevator control torque Sr area of the rudders Clrβ rudder lift coeﬃcient due to the sideslip angle β δr rudder deﬂection Clrδr rudder lift coeﬃcient due to the deﬂection δr Cdr0 rudder drag coeﬃcient when β = δr= 0 Ar aspect ratio of the rudder er rudder Oswald’s eﬃciency factor τra rudder aerodynamic moment τrc rudder control torque ∆x uncertainty in x-direction ∆y uncertainty in y-direction ∆z uncertainty in z-direction ∆ϕ uncertainty in roll ∆θ uncertainty in pitch ∆ψ uncertainty in yaw y∗1 measurement for position/angle y∗2 measurement for velocity/angular velocity xd reference position in x-direction yd reference position in y-direction zd reference position in z-direction ϕd desired roll angle θd desired pitch angle ψd desired yaw angle λ∗1 parameter of extended observer λ∗2 parameter of extended observer α∗ parameter of extended observer kp1 controller parameter in position dynamics kp2 controller parameter in position dynamics ka1 controller parameter in attitude dynamics ka2 controller parameter in attitude dynamics 3 3 1 Introduction However, measurement error or non-zero mean noise in acceleration through integrations cause velocity and position to drift over time. The observer-based INS methods were used to estimate unknown variables in navigation [12,13]. However, position signals are limited to be local, but not global. For attitude information, an IMU (Inertial Measurement Unit) can determine attitude angle from accurate angular velocity through integration, but angle drift happens. Meanwhile, the outputs of the accelerometers and the magnetometer in IMU can determine the large-error pitch, roll and yaw angles [14]. Uncertainties in UAV ﬂight dynamics include: aerodynamic disturbance, unmodelled dynamics and parametric uncertainties. These uncertainties bring challenges for control system design. The uncertainty in a system can be estimated by an extended state observer [15,16]. However, accurate position measurement is required as the input of observer. Even velocity can be use for estima- tion, disturbance in position cannot still be corrected. For a jet UAV ﬂight control system, an 4 integral-uncertainty observer was designed to estimate the attitude angles and attitude dynam- ic uncertainty, and an augmented observer was used to estimate the ﬂying velocity and position dynamic uncertainty [17]. However, drift may happen for long-time ﬂight due to eﬀect of distur- bance and actuator vibrations on the IMU. Meanwhile, the augmented observer can only reduce high-frequency noise, low or mid frequency disturbances still exist. In order to reduce disturbances in position and attitude, the popular methods of GPS/INS based on KF (Kalman ﬁlter) or EKF (Extended Kalman ﬁlter) are used for signal fusion to overcome the limits of individual measurements based on optimization of a recursive least mean square error [18,19,20,21]. Thus, measurement accuracy is improved. For KF or EKF, the relatively accurate system models are needed. Furthermore, the uncertainty in noise statistics limits the performance. In addition, for EKF, system model linearization may cause ﬁltering divergence, and the derivation of the Jacobian matrices are nontrivial. A ﬁnite-time-convergent signal corrector was designed for position correction in a quadrotor UAV control system [22]. The signal corrector is complex, and the ﬁnite-time convergence cannot be implemented in engineering practice. Furthermore, the parameters’ selection is sensitive to the estimate performance. In this paper, a corrector based on robust 2-sliding mode is presented to correct position distur- bance using relatively accurate velocity measurement. The 2-sliding mode can reduce the estimate errors of corrector and make the fusion of position and velocity on a linear sliding surface. 2.2 Attitude sensing The gyroscopes in IMU provide the relatively accurate angular velocities, e.g., their accuracy is about L2 = 10◦/hr=0.003◦/s. The accuracy of attitude angles from IMU is relatively large, e.g., about L1 = 1.0◦. Therefore, L2 L1 = 0.003 1 = 0.003 ≪1, and we can select an upper-bound of sensor accuracy ratio 0.003 ≤ε = 0.003 ≪1 to satisfy max { L2 L1 } ≤ε ≪1. 2.3 Eﬀect of sensing disturbance on safe ﬂight Sensing disturbance has a serious impact on safe ﬂight. Disturbance in position sensing may generate incorrect desired attitude angles, and they are mismatched to actual position trajectory. Therefore, the determined attitude is unwanted, and it may be dangerous. In addition, in position measurement, the frequency bands of actual position p0(t) and sensing disturbance d1(t) may have intersections: the disturbance d1(t) may be in low, mid or high frequency bands. It is impossible for the usual ﬁlters to separate d1(t) from the actual position signal p0(t). Questions: How to reject disturbance d1(t) in position measurement pm(t) using the relatively accurate velocity vm(t)? Also, how to reject the disturbance in angle measurement using the relatively accurate angular velocity? 1 Introduction Position disturbance is reduced further on the sliding surface. Not only the corrector can reject high- frequency noise, but also the low and mid frequency disturbances are reduced largely. Therefore, the corrector can reject low/mid/high frequency disturbances, and it is unrelated to the types of actual position signals. Due to the existence of linear sliding surface in the 2-sliding mode, the estimate outputs from the corrector are accurate and smoothed. The contributions of the proposed corrector include: 1) the corrector can reject position distur- bance in low/mid/high frequency bands; 2) due to the continuity of 2-sliding mode, the estimate outputs from the corrector are smoothed and accurate, and they can be used directly for control without any additional ﬁlters; 3) due to the robust sliding mode, the corrector parameters are highly inclusive to change of disturbance and signal; 4) because only switch logic and linear func- tions are used in the corrector, the corrector can be implemented easily in the current hardware of computational environments. The proposed corrector is applied to navigation and control of a jet UAV. In the UAV ﬂight test, the following adverse conditions are considered: disturbances in the measurements of GPS position and IMU attitude angles, and uncertainties in the UAV ﬂight dynamics. For the UAV trajectory tracking control, the UAV system model is constructed in the earth-ﬁxed frame [23]. Furthermore, the model is ﬁt for observer design to estimate the system uncertainties. The correctors are adopted to correct the disturbances in GPS position and IMU attitude angles. In addition, an existing extended state observer [24] is used to estimate the uncertainties in the UAV ﬂight dynamics. The performance of corrector is compared to the KF-based signal fusion methods [21,25]. Moreover, based on the correction and estimation, the desired attitude is determined, and the control laws are designed to drive the UAV to achieve the ﬂight mission. 5 5 2 Problem description The problem considered in this paper is to reject disturbance in position and attitude The problem considered in this paper is to reject disturbance in position and attitude sensing. 2.1 Position and velocity sensing The problem considered in this paper is to reject disturbance in position and attitude sensing. 2.1 Position and velocity sensing GPS provides position of a device, and accurate velocity can be determined by GPS with Doppler shift measurement or by a Doppler Radar sensor. Deﬁne the position measurement: pm(t) = p0(t) + d1(t), where, p0(t) is the actual position; d1(t) is the disturbance in position measurement, and supt∈[0,∞) \|d1(t)\| ≤L1 < ∞. Deﬁne the position measurement: pm(t) = p0(t) + d1(t), where, p0(t) is the actual position; d1(t) is the disturbance in position measurement, and supt∈[0,∞) \|d1(t)\| ≤L1 < ∞. Deﬁne the velocity measurement: vm(t) = v0(t) + d2(t), where, v0(t) is the actual velocity; d2(t) is the disturbance in velocity measurement, and supt∈[0,∞) \|d2(t)\| ≤L2 < ∞. Deﬁne the velocity measurement: vm(t) = v0(t) + d2(t), where, v0(t) is the actual velocity; d2(t) is the disturbance in velocity measurement, and supt∈[0,∞) \|d2(t)\| ≤L2 < ∞. Remark 2.1: The accuracy L1 of GPS position sensing is usually a meter, a few meters or even tens of meters. Doppler shift measurement enables velocity accuracy L2 of a few centimeters per second, even the accuracy approaching 5mm/s (i.e., 0.005m/s) is possible [8]. Therefore, L2 ≪1 holds. When using the consistent unit standard, we can get L2 ≪L1, i.e., the sensor accuracy ratio L2 L1 ≪1. Furthermore, due to the reliability of Doppler measurement, velocity accuracy usually remains unchanged. However, the position accuracy may become worse because of diﬀerent environmental inﬂuences, i.e., L1 may increase. Therefore, there exists a small constant ε > 0, i.e., the upper- bound of sensor accuracy ratio, such that the sensor accuracy inequality max { L2 L1 } ≤ε ≪1 holds. 2.2 Attitude sensing 3.1 Preliminary The related background is presented here. Lemma 3.1 (Sliding mode with prescribed convergence law) [26,27]: The following system is 6 considered: considered: ˙e1 = e2 ˙e2 = φ (t) + γ (t) ˙u (1) ˙e1 = e2 ˙e2 = φ (t) + γ (t) ˙u (1) (1) ˙e2 = φ (t) + γ (t) ˙u (1) where, e1 and e2 are the states; u is the controller, and ˙u in the system; \|φ (t)\| ≤Φ, 0 < Γm ≤ γ (t) ≤ΓM, Φ > 0. A 2-sliding control algorithm is as follows: ˙u = { −u, if \|u\| > 1; −VMsign [e2 + g(e1)] , if \|u\| ≤1 (2) ˙u = { −u, if \|u\| > 1; V i [ ( )] if \| \| ≤1 (2) (2) u = { −VMsign [e2 + g(e1)] , if \|u\| ≤1 (2) where, g(e1) is smooth everywhere except on e1 = 0, for example, g(e1) = k1 \|e1\|αsign(e1), α ∈ [0.5, 1); VM > Φ+sup[g′(e1)g(e1)] Γm . Then, we get the ﬁnite-time convergence law ˙e1 = −g(e1) (i.e., sliding surface e2 + g(e1) = 0), and there exists a ﬁnite time ts > 0, for t ≥ts, such that where, g(e1) is smooth everywhere except on e1 = 0, for example, g(e1) = k1 \|e1\|αsign(e1), α ∈ [0.5, 1); VM > Φ+sup[g′(e1)g(e1)] Γm . Then, we get the ﬁnite-time convergence law ˙e1 = −g(e1) (i.e., sliding surface e2 + g(e1) = 0), and there exists a ﬁnite time ts > 0, for t ≥ts, such that e1 = 0 and e2 = 0 (3) (3) e1 = 0 and e2 = 0 Remark 3.1: For system (1), when we select φ (t) = 0 and γ (t) = 1, it becomes ˙e1 = e2 ˙e2 = ˙u (4) ˙e1 = e2 ˙e2 = ˙u (4) ˙e1 = e2 ˙e2 = ˙u (4) Then, the 2-sliding control algorithm (2) is expressed by Then, the 2-sliding control algorithm (2) is expressed by ˙u = ˙e2 = { −e2, if \|e2\| > 1; −VMsign [e2 + g(e1)] , if \|e2\| ≤1 (5) (5) 3.2 Corollary on 2-sliding mode system 3.2 Corollary on 2-sliding mode system Combining the system (4) and the 2-sliding control algorithm (5), we get the following corollary on a 2-sliding mode with prescribed convergence law. 3.1 Preliminary Corollary 3.1 (Sliding mode with prescribed ﬁnite-time convergence law): A 2-sliding mode system is as follows: ˙e1 = e2 ˙e2 = { −e2, if \|e2\| > 1; −VM · sign [e2 + g(e1)] , if \|e2\| ≤1 (6) ˙e2 = { −e2, if \|e2\| > 1; (6) (6) where, e1 and e2 are the sliding variables; g(e1) is smooth everywhere except on e1 = 0, for example, g(e1) = k1 \|e1\|αsign(e1), α ∈[0.5, 1); VM > sup [g′ (e1) g (e1)]. Then, we get the ﬁnite- time convergence law ˙e1 = −g(e1) (i.e., sliding surface e2 + g(e1) = 0), and there exists a ﬁnite time ts > 0, for t ≥ts, such that 7 (7) e1 = 0 and e2 = 0 (7) e1 = 0 and e2 = 0 e1 = 0 and e2 = 0 Remark 3.2: For system (6), the parameter selection condition VM > sup [g′ (e1) g (e1)] is too strict. In order to relax the parameter selection conditions, we can use a linear convergence law ˙e1 = −k1e1 for the nonlinear ˙e1 = −g(e1), and only VM > k1 > 0 will be required. In the following section, we will give a theorem on 2-sliding mode system with linear convergence law to be exponentially stable. 3.3 Notation: “a (ω) : b1 →b2 as ω : c1 →c2” means that function a (ω) varies monotonically increasing or decreasing from b1 to b2 as ω increases from c1 to c2. 4 Robust 2-sliding mode system Before we present the design of sliding mode corrector, we give a 2-sliding mode system, and a Theorem is presented as follows. Theorem 4.1 (Sliding mode with prescribed linear convergence law): The 2-sliding mode system is as follows: ˙e1 = e2 ˙e2 = { −e2, if \|e2\| > 1; −k2sign (e2 + k1e1) , if \|e2\| ≤1 (8a) or ˙e1 = e2 ˙e2 = { −k3sign (e2) , if \|e2\| > 1; −k2sign (e2 + k1e1) , if \|e2\| ≤1 (8b) ˙e1 = e2 ˙e2 = { −e2, if \|e2\| > 1; −k2sign (e2 + k1e1) , if \|e2\| ≤1 (8a) ˙e2 = { −e2, if \|e2\| > 1; −k2sign (e2 + k1e1) , if \|e2\| ≤1 (8a) (8a) or ˙e1 = e2 ˙e2 = { −k3sign (e2) , if \|e2\| > 1; −k2sign (e2 + k1e1) , if \|e2\| ≤1 (8b) (8b) where, e1 and e2 are the sliding variables; k2 > k1 > 0, and k3 > 0. Then, we get the linear convergence law ˙e1 = −k1e1 (i.e., sliding surface e2 + k1e1 = 0), and the system (8a) or (8b) is exponentially stable, i.e., lim t→∞e1 = 0 and lim t→∞e2 = 0 (9) (9) oof of Theorem 4.1 is presented in Appendix. ■ The proof of Theorem 4.1 is presented in Appendix. ■ The proof of Theorem 4.1 is presented in Appendix. ■ In fact, for (8b), when \|e2\| > 1, we use ˙e2 = −k3sign(e2) for ˙e2 = −e2 to speed up e2 convergence and overcome disturbance, where, k3 > 0. Simulation example (sliding mode with prescribed linear convergence law): For the sliding mode system (8b), we select k1 = 1, k2 = 10, and k3 = 5. Then, we get e1 and e2 in Figure 1. Figure 1 illustrates the fast convergence of e1 and e2. 8 8 0 20 40 60 80 100 time(s) -5 -4 -3 -2 -1 0 1 2 3 4 5 e2, e1 e2 e1 Figure 1: Sliding variables e1 and e2. Figure 1: Sliding variables e1 and e2. In the following, we give a robust 2-sliding mode system considering existence of multiple distur- bances, and a Theorem is presented as follows. 4 Robust 2-sliding mode system Theorem 4.2 (Robust 2-sliding mode): A 2-sliding mode system considering the unknown bounded disturbances is as follows: Theorem 4.2 (Robust 2-sliding mode): A 2-sliding mode system considering the unknown bounded disturbances is as follows: ˙e1 = e2 ˙e2 = { −k3sign [e2 −d2(t)] −d3(t), if \|e2 −d2(t)\| > 1; −k2sign [e2 −d2(t) + k1(e1 −d1(t))] −d4(t), if \|e2 −d2(t)\| ≤1 (10) ˙e2 = { −k3sign [e2 −d2(t)] −d3(t), if \|e2 −d2(t)\| > 1; −k2sign [e2 −d2(t) + k1(e1 −d1(t))] −d4(t), if \|e2 −d2(t)\| ≤1 (10) (10) where, e1 and e2 are the sliding variables; the unknown bounded disturbances di(t) satisfy supt∈[0,∞) \|di(t)\| ≤Li < ∞(i = 1, 2, 3, 4); L2 ≪1, and there exists a constant ε > 0 such that max { L2 L1 } ≤ε ≪1; k1 = 2ε1−r, where r ∈ ( 0, 1 2 ] ; k2 > k1 + L4, and k3 > L3. Then, the eﬀect of disturbances is rejected, and the variables e1 and e2 of system (10) are in the bounds as follows: supt∈[0,∞) \|di(t)\| ≤Li < ∞(i = 1, 2, 3, 4); L2 ≪1, and there exists a constant ε > 0 such that max { L2 L1 } ≤ε ≪1; k1 = 2ε1−r, where r ∈ ( 0, 1 2 ] ; k2 > k1 + L4, and k3 > L3. 4 Robust 2-sliding mode system Then, the eﬀect of disturbances is rejected, and the variables e1 and e2 of system (10) are in the bounds as follows: lim t→∞\|e1\| ≤ρ(ω1)L1; lim t→∞\|e2\| ≤L2 (11) (11) where, ω1 is the angular frequency of disturbance d1(t); and the rejection ratio is expressed by ρ(ω1) = 1 √ 1 + 1 4ε2r−2ω2 1 + 1 2εr (12) (12) The rejection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: The rejection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: i) In [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞, where, ω0 = 4ε1−2r √ 1 −1 4ε2r; ii) In (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0, where, ωc = ε1−1 2 r√4−εr 1−1 2 εr < 4ε1−1 2 r ≪1; iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1 due to 0 < ε ≪1) as ω1 : 0 →ωc, a i) In [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞, where, ω0 = 4ε1−2r √ 1 −1 4ε2r; ii) In (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0, where, ωc = ε1−1 2 r√4−εr 1−1 2 εr < 4ε1−1 2 r ≪1 iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1 d frequency band is suﬃciently small due to ωc ≪1. iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1 due to 0 < ε ≪1) as ω1 : 0 →ωc, and this frequency band is suﬃciently small due to ωc ≪1. iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1 due to 0 < ε ≪1) as ω1 : 0 →ωc, and this frequency band is suﬃciently small due to ωc ≪1. The proof of Theorem 4.2 is presented in Appendix. ■ Remark 4.1: From 0 < ε ≪1 and r ∈ ( 0, 1 2 ] , we can get that both the rejection ratio ρ(ω1) in frequency band [ω0, ∞) and the frequency ω0 are small enough. Therefore, the disturbance 9 9 d1 (t) is reduced at very small rejection ratio in the large frequency band [ω0, ∞). 4 Robust 2-sliding mode system In fact, the disturbance bound L2 in velocity sensing through Doppler eﬀect is a few centimeters or a few millimeters, and the disturbance bound L1 in position sensing is usually about a few meters or even tens of meters. Thus, the sensing accuracy inequality L2 L1 ≪1 holds, and there exists ε > 0 such that max { L2 L1 } ≤ε ≪1. When disturbance d1 (t) becomes worse, i.e., L1 increases, the inequality L2 L1 ≤ε ≪1 still holds. Therefore, the frequency band [ω0, ∞) covers the low/mid/high frequency bands, and the disturbance d1(t) in position sensing in [ω0, ∞) is rejected suﬃciently by the corrector. Furthermore, the disturbance d1 (t) can still be rejected largely in the other frequency bands. In the following, we consider the position disturbance d1(t) is rejected to the maximum extent in a given frequency band, and the disturbance in the other bands can still be rejected largely. We will determine the corrector parameter k1 to get the minimum value of the rejection ratio in the given frequency band [ωreq, ∞), and a Theorem is presented as follows. Theorem 4.3 (Suﬃcient disturbance rejection in given frequency band): The sliding mode system (10) is considered, where, the unknown bounded disturbances di(t) satisfy supt∈[0,∞) \|di(t)\| ≤Li < ∞(i = 1, 2, 3, 4); L2 ≪1, and there exists a constant ε > 0 such that max { L2 L1 } ≤ε ≪1. 4 Robust 2-sliding mode system For the a given ωreq > 0, if we select k3 > L3, k2 > k1 + L4 and k1 = 1/xmin, where, xmin is the unique solution to ω2 reqxmin(1 + ω2 reqx2 min)−3 2 −ε = 0 (13) (13) in the range ( 1 √ 2ωreq , ∞), then, the eﬀect of disturbances is rejected, especially it is rejected suf- ﬁciently in the frequency band [ωreq, ∞), and the variables e1 and e2 of system (10) are in the bounds as follows: lim t→∞\|e1\| ≤ρ(ω1)L1; lim t→∞\|e2\| ≤L2 (14) (14) where, the rejection ratio is expressed by where, the rejection ratio is expressed by ρ(ω1) = 1 √ 1 + x2 minω2 1 + ε · xmin (15) (15) The rejection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: i) I [ ) ( ) The rejection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: jection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: i) In [ω0, ∞), ρ(ω1) : ρmin →ε · xmin as ω1 : ωreq →∞. In [ω0, ∞), ρ(ω1) : ρmin →ε · xmin as ω1 : ωreq →∞. ii) In (ωc, ω0), ρ(ω1) : 1 →ρmin as ω1 : ωc →ωreq, where, ωc = √ 2ε xmin √ 1−1 2 εxmin 1−ε·xmin . iii) In [0, ωc], ρ(ω1) : 1 + ε · xmin →1 as ω1 : 0 →ωc. where, ρ i = 1 + ε · x i (16) ii) In (ωc, ω0), ρ(ω1) : 1 →ρmin as ω1 : ωc →ωreq, where, ωc = √ 2ε xmin √ 1−1 2 εxmin 1−ε·xmin . iii) In [0, ωc], ρ(ω1) : 1 + ε · xmin →1 as ω1 : 0 →ωc. where, (16) The proof of Theorem 4.3 is presented in Appendix. ■ The proof of Theorem 4.3 is presented in Appendix. ■ 10 5 Design of sliding mode corrector This frequency ﬃi tl ll d t ≪1 i) In [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞, where, ω0 = 4ε1−2r √ 1 −1 4ε2r; i) In [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞, where, ω0 = 4ε1−2r √ 1 −1 4ε2r; ii) In (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0, where, ωc = ε1−1 2 r√4−εr 1−1 2 εr < 4ε1−1 2 r ≪1; iii) In [0 ω ] ρ(ω ) : 1 + 1εr →1 (i e ρ(ω ) ≈1) as ω : 0 →ω This frequency band iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1) as ω1 : 0 →ωc. This frequency band is suﬃciently small due to ωc ≪1. iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1) as ω1 : 0 →ωc. This frequency band is suﬃciently small due to ωc ≪1. The proof of Theorem 5.1 is presented in Appendix. ■ 5 Design of sliding mode corrector According to Theorem 4.2, and considering disturbance in position sensing, a sliding mode corrector is designed to reject the disturbance, and a theorem is presented as follows. Theorem 5.1 (Sliding mode corrector): Suppose position measurement is pm(t) = p0(t) + d1(t), and velocity measurement is vm(t) = v0(t)+d2(t); p0(t) is the actual position, and v0(t) is the actual velocity; the unknown bounded disturbances d1(t) and d2(t) satisfy supt∈[0,∞) \|di(t)\| ≤Li < ∞, where, i = 1, 2; L2 ≪1, and there exists a constant ε > 0 such that max { L2 L1 } ≤ε ≪1; supt∈[0,∞) \| ˙v0(t)\| ≤L3 < ∞. The sliding mode corrector is designed as follows: ˙x1 = x2 ˙x2 = { −k3sign [x2 −vm(t)] , if \|x2 −vm(t)\| > 1; −k2sign [x2 −vm(t) + k1(x1 −pm(t))] , if \|x2 −vm(t)\| ≤1 (17) ˙x2 = { −k3sign [x2 −vm(t)] , if \|x2 −vm(t)\| > 1; −k2sign [x2 −vm(t) + k1(x1 −pm(t))] , if \|x2 −vm(t)\| ≤1 (17) (17) where, x1 and x2 are the corrector variables; the corrector parameters satisfy k1 = 2ε1−r (where r ∈ ( 0, 1 2 ] ), k2 > k1 + L3 and k3 > L3. Then, the disturbance d1(t) is rejected, and the corrector estimate outputs satisfy: lim t→∞\|x1 −p0(t)\| ≤ρ(ω1)L1; lim t→∞\|x2 −v0(t)\| ≤L2 (18) (18) where, ω1 is the angular frequency of disturbance d1(t), and the rejection ratio is expressed by lar frequency of disturbance d1(t), and the rejection ratio is expressed by ρ(ω1) = 1 √ 1 + 1 4ε2r−2ω2 1 + 1 2εr (19) (19) jection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: i) In [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞, where, ω0 = 4ε1−2r √ 1 −1 4ε2r; ii) In (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0, where, ωc = ε1−1 2 r√4−εr 1−1 2 εr < 4ε1−1 2 r ≪1; iii) In [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (i.e., ρ(ω1) ≈1) as ω1 : 0 →ωc. Remark 5.2 (Parameters regulation of corrector (17)): Remark 5.2 (Parameters regulation of corrector (17)): 1) The selection of k3 > L3, k2 > k1 + L3 and k1 = 2ε1−r (where, max { L2 L1 } ≤ε ≪1 and r ∈ ( 0, 1 2 ] ) makes the corrector stable: k3 > L3 and k2 > k1 + L3 make the corrector estimate errors satisfy the convergence law (90); k1 = 2ε1−r makes the sliding surface stable, and it further reduces the corrector estimate error. Remark 5.2 (Parameters regulation of corrector (17)): 1) The selection of k3 > L3, k2 > k1 + L3 and k1 = 2ε1−r (where, max { L2 L1 } ≤ε ≪1 and r ∈ ( 0, 1 2 ] ) makes the corrector stable: k3 > L3 and k2 > k1 + L3 make the corrector estimate errors satisfy the convergence law (90); k1 = 2ε1−r makes the sliding surface stable, and it further reduces the corrector estimate error. 1) The selection of k3 > L3, k2 > k1 + L3 and k1 = 2ε1−r (where, max { L2 L1 } ≤ε ≪1 and r ∈ ( 0, 1 2 ] ) makes the corrector stable: k3 > L3 and k2 > k1 + L3 make the corrector estimate errors satisfy the convergence law (90); k1 = 2ε1−r makes the sliding surface stable, and it further reduces the corrector estimate error. 2) The upper-bound of sensor accuracy ratio 0 < ε ≪1 and r ∈ ( 0, 1 2 ] make the rejection ratio ρ(ω1) in frequency band [ω0, ∞) and the frequency ω0 suﬃciently small, and the suﬃciently rejectable frequency ω1 ∈[ω0, ∞) may be in the low/mid/high frequency bands. 2) The upper-bound of sensor accuracy ratio 0 < ε ≪1 and r ∈ ( 0, 1 2 ] make the rejection ratio ρ(ω1) in frequency band [ω0, ∞) and the frequency ω0 suﬃciently small, and the suﬃciently rejectable frequency ω1 ∈[ω0, ∞) may be in the low/mid/high frequency bands. 3) Furthermore, the selection of parameter r aﬀects the rejection ratio ρ(ω1) and the suﬃciently rejectable frequency band [ω0, ∞). In fact: 3) Furthermore, the selection of parameter r aﬀects the rejection ratio ρ(ω1) and the suﬃciently rejectable frequency band [ω0, ∞). In fact: i) Due to 0 < ε ≪1, the smaller r is, the bigger εr ∈(0, 1) is, and ω0 decreases. The proof of Theorem 5.1 is presented in Appendix. ■ For the a given ωreq > 0, if we select k3 > L3, k2 > k1 + L3 and k1 = 1/xmin, where, xmin is the unique solution to ω2 reqxmin(1 + ω2 reqx2 min)−3 2 −ε = 0 (20) (20) in the range ( 1 √ 2ωreq , ∞), then, the disturbance d1(t) is rejected, especially it is rejected suﬃciently in the frequency band [ωreq, ∞), and the corrector estimate outputs satisfy: in the range ( 1 √ 2ωreq , ∞), then, the disturbance d1(t) is rejected, especially it is rejected suﬃciently in the frequency band [ωreq, ∞), and the corrector estimate outputs satisfy: (21) lim t→∞\|x1 −p0(t)\| ≤ρ(ω1)L1; lim t→∞\|x2 −v0(t)\| ≤L2 (21) lim t→∞\|x1 −p0(t)\| ≤ρ(ω1)L1; lim t→∞\|x2 −v0(t)\| ≤L2 (21) h d b where, the rejection ratio is expressed by ρ(ω1) = 1 √ 1 + x2 minω2 1 + ε · xmin (22) (22) The rejection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: The rejection ratio ρ(ω1) is a monotonically decreasing function of ω1 ∈[0, ∞), and it satisﬁes: In [ωreq, ∞), ρ(ω1) : ρmin →ε · xmin as ω1 : ωreq →∞. ii) In (ωc, ωreq), ρ(ω1) : 1 →ρmin as ω1 : ωc →ωreq, where, ωc = √ 2ε xmin √ 1−1 2 εxmin 1−ε·xmin . iii) In [0, ωc], ρ(ω1) : 1 + ε · xmin →1 as ω1 : 0 →ωc. where, ρmin = 1 √ 1 + ω2reqx2 min + ε · xmin (23) (23) According to Theorem 4.3 and the system error (123) in the proof of Theorem 5.1, we can get the bounds of the correction errors in (21). This concludes the proof of Theorem 5.2. ■ According to Theorem 4.3 and the system error (123) in the proof of Theorem 5.1, we can get the bounds of the correction errors in (21). This concludes the proof of Theorem 5.2. ■ Remark 5.2 (Parameters regulation of corrector (17)): The proof of Theorem 5.1 is presented in Appendix. ■ Remark 5.1 (Analysis of corrector (17)): The sensing accuracy inequality 0 < ε ≪1 and r ∈ ( 0, 1 2 ] guarantee that the rejection ratio ρ(ω1) in frequency band [ω0, ∞) is small enough, and [ω0, ∞) covers the low/mid/high frequency bands. Therefore, the disturbance d1(t) in [ω0, ∞) can be rejected suﬃciently by the corrector. Furthermore, the disturbance d1(t) in the low frequency band (ωc, ω0) is still reduced largely. Only the approximate constant disturbances in the extreme low frequency band [0, ωc] are not rejected. In fact, signals in the extreme low frequency band [0, ωc] is approximate constant due to suﬃciently small ωc ≪1. Therefore, the position disturbance d1(t) can be rejected suﬃciently by the corrector even when the disturbance frequency covers low/mid/high frequency bands. From Theorem 4.3, considering suﬃcient disturbance rejection in a given frequency band, we get the optimal sliding mode corrector, and a theorem is presented as follows. 11 Theorem 5.2 (Optimal sliding mode corrector): The corrector (17) and the measurement signals in Theorem 5.1 are considered, where, the unknown bounded disturbances di(t) satisfy supt∈[0,∞) \|di(t)\| ≤Li < ∞(i = 1, 2); L2 ≪1, and there exists a constant ε > 0 such that max { L2 L1 } ≤ε ≪1; supt∈[0,∞) \| ˙v0(t)\| ≤L3 < ∞. Remark 5.2 (Parameters regulation of corrector (17)): Therefore, the frequency band [ω0, ∞) becomes relatively wider. i) Due to 0 < ε ≪1, the smaller r is, the bigger εr ∈(0, 1) is, and ω0 decreases. Therefore, the frequency band [ω0, ∞) becomes relatively wider. ii) Conversely, the larger r is, the smallerεr ∈(0, 1) is, and ω0 increases. Therefore, the frequency band [ω0, ∞) is reduced. ii) Conversely, the larger r is, the smallerεr ∈(0, 1) is, and ω0 increases. Therefore, the frequency band [ω0, ∞) is reduced. 12 Figure 2: Jet UAV prototype. Figure 2: Jet UAV prototype. x E y E z E b x E b y E b z E mg 1L 1 D 2 L 2 D 2 eL 2 e D 1 eL 1 e D 2 rL 2 r D 1 rL 1 r D cF T V fL f D b a ft y t qt q t L p t L rt L Figure 3: UAV aerodynamic mesh, forces and moments. Figure 3: UAV aerodynamic mesh, forces and moments. iii) Minimum range and minimum value of ρ(ω1): From ρ(ω1) = 1 √ 1+ 1 4 ε2r−2ω2 1 + 1 2εr, we know that ρ(ω1) ∈ ( 1 2εr, 1 + 1 2εr] when ω1 ∈[0, ∞). Due to 0 < ε ≪1 and r ∈ ( 0, 1 2 ] , when we select r = 1 2, we can get the minimum value of min {εr} = ε 1 2 . Therefore, the minimum value of ρ(ω1) is min {ρ(ω1)} = 1 2ε 1 2 , and the minimum range is ρ(ω1) ∈ ( 1 2ε 1 2 , 1 + 1 2ε 1 2 ] when ω1 ∈[0, ∞). iii) Minimum range and minimum value of ρ(ω1): From ρ(ω1) = 1 √ 1+ 1 4 ε2r−2ω2 1 + 1 2εr, we know that ρ(ω1) ∈ ( 1 2εr, 1 + 1 2εr] when ω1 ∈[0, ∞). Due to 0 < ε ≪1 and r ∈ ( 0, 1 2 ] , when we select r = 1 2, we can get the minimum value of min {εr} = ε 1 2 . Therefore, the minimum value of ρ(ω1) is min {ρ(ω1)} = 1 2ε 1 2 , and the minimum range is ρ(ω1) ∈ ( 1 2ε 1 2 , 1 + 1 2ε 1 2 ] when ω1 ∈[0, ∞). 6 Application to Jet UAV navigation and control An RC-model-based F/A-18 Hornet prototype is used [17], which is shown in Figure 2, and the forces and torques of UAV are described in Figure 3. 6.1 Modelling of jet UAV ﬂight dynamics 6.1 Modelling of jet UAV ﬂight dynamics For the UAV trajectory tracking control, the modelling is considered in the earth-ﬁxed frame [23]. Furthermore, the constructed model is ﬁt for observer design to estimate system uncertainties. Let Γ = (Ex, Ey, Ez) denote the earth-ﬁxed frame, and Λ = (Eb x, Eb y, Eb z) denote the body frame of the UAV. ΘΓ = [ϕ θ ψ]T ∈ℜ3 describes the UAV roll, pitch and yaw angles (Euler angles), and ΩΓ = [ ˙ϕ ˙θ ˙ψ]T . We use sθ for sin θ and cθ for cos θ. RΓΛis the transformation matrix representing the orientation of the body frame Λ with respect to the earth-ﬁxed frame Γ: 13 RΓΛ =   cθcψ cψsθsϕ −sψcϕ cψsθcϕ + sψsϕ cθsψ sψsθsϕ + cψcϕ sψsθcϕ −cψsϕ −sθ sϕcθ cϕcθ   (24) (24) Let α and β be the angle of attack and the sideslip angle, respectively, we can get α = θ −arctan−1(W/U), β = arcsin−1(V/VT ) (25) (25) s the linear velocity in body frame Λ, and VT = √ U2 + V 2 + W 3. where, υΛ = (U, V, W) is the linear velocity in body frame Λ, and VT = √ U2 + V 2 + W 3. Deﬁne pΓ = (x, y, z) and υΓ = ( ˙x, ˙y, ˙z) as the position and velocity vectors of center of gravity, respectively, relative to the earth-ﬁxed frame Γ; ΩΛ is the angular rate vector of the airframe expressed in the body frame Λ; m ∈ℜis the UAV mass, and J =diag{Jxb, Jyb, Jzb} ∈ℜ3×3 is the UAV inertia matrix. Then, the dynamic equations for the jet UAV subjected to force F ∈ℜ3 and torque τ ∈ℜ3 are given by ˙pΓ = υΓ m · ˙υΓ = F + mgEz J · ˙ΩΛ = −ΩΛ × (JΩΛ) + τ (26) ˙pΓ = υΓ m · ˙υΓ = F + mgEz J · ˙ΩΛ = −ΩΛ × (JΩΛ) + τ (26) (26) where, Ez = [ 0 0 1 ]T . 6.1 Modelling of jet UAV ﬂight dynamics The relation between the angular rate vector ΩΛ = [ pΛ qΛ rΛ ]T and the Euler angle derivative vector ΩΓ = [ ˙ϕ ˙θ ˙ψ]T is given by ΩΛ = ZΩΓ or ΩΓ = Z−1ΩΛ (27) (27) where, Z =   1 0 −sθ 0 cϕ sϕcθ 0 −sϕ cϕcθ  , Z−1 =   1 sϕsθ/cθ cϕsθ/cθ 0 cϕ −sϕ 0 sϕ/cθ cϕ/cθ   (28) (28) The total external force F consists of the thrust Fjet generated by the jet engine, aerodynamic forces on the ﬁxed wing Fw, aerodynamic forces on the fuselage Ff, the forces created by the rudders Fr, the forces created by the elevators Fe, and uncertainties and external disturbances Fd. These forces are expressed in body frame Λ, and they are transformed by RΓΛ to be expressed in the earth-ﬁxed frame Γ as follows: F = RΓΛ(Fjet + Fw + Ff + Fr + Fe + Fd) (29) (29) The total moment τ consists of the moments created by the ﬁxed wings τw, the moments created by the rudders τr, the moments created by the elevators τe, and moments due to the uncertainties and external disturbances τd: τ = τw + τr + τe + τd (30) (30) 14 The aerodynamic parameters of the UAV are from [17]. The CFD (Computational Fluid Dynam- ics) simulation was performed, and the results from the wind tunnel tests were compared. For the boundary conditions, the method of free-stream boundary condition based on Riemann invariants was utilized [28]: no-slip viscous ﬂow condition; the linearized one-dimensional Euler equations; the free-stream values for determination of the value of the Riemann invariants; and the symmet- rical boundary condition for the symmetrical UAV. The ANSYS Fluent was used for the CFD simulation, and the design steps included meshing ﬂuid ﬁeld, ﬂuent simulation and post-processing [29]. 1) Thrust by Jet engine: The thrust of engine in body frame is expressed by Fjet = [ Fc 0 0 ]T (31) (31) 2) Aerodynamics of ﬁxed wings Deﬁne q = 0.5ρ(U2 + W 2), where, ρ is the air density. 3) Fuselage The parameters of fuselage lift and drag are described as follows: The parameters of fuselage lift and drag are described as follows: Lf = qSfClf, Clf = Clfαα, Df = qSfCdf, Cdf = Cdf0 + Cdfαα (36) (36) where, Sf is the fuselage equivalent cross-sectional area; Lf and Df are the lift and drag forces generated by the fuselage, respectively; Clf is the lift coeﬃcient; Cdf is the drag coeﬃcient; Cdf0 is the constant in the coeﬃcient of drag force. Then the force vector Ff on the fuselage in body frame is expressed by Ff =   Lfsα −Dfcα 0 −Lfcα −Dfsα   (37) 4) Elevator Ff =   Lfsα −Dfcα 0 −Lfcα −Dfsα   (37) (37) 6.1 Modelling of jet UAV ﬂight dynamics The lift and drag forces generated by the ﬁxed wings are, respectively Li = qSwCLi, CLi = CL0 + CLαα + CLδiδi Di = qSwCDi, CDi = CD0 + C2 Li/(πAwew) ew = 1.78(1 −0.045A0.68 w ) −0.46 (32) (32) where i = 1, 2; Sw is the area of the half wing; CL0 is the lift coeﬃcient when the angle of attack α equals zero; CLα is the lift coeﬃcient due to the angle of attack α; δi is the aileron deﬂection, and CLδi is the lift coeﬃcient due to the aileron deﬂection δi; CD0 is the drag coeﬃcient when α = δi = 0; Aw is the aspect ratio of the ﬁxed wing; ew is the value of the Oswald’s eﬃciency factor. The expression of lift and drag coeﬃcients is considered as valid for low angles of attack. Then the aerodynamic force vector Fw on the ﬁxed wings in body frame can be written as Then the aerodynamic force vector Fw on the ﬁxed wings in body frame can be writ Fw =   (L1 + L2)sα −(D1 + D2)cα 0 −(L1 + L2)cα −(D1 + D2)sα   (33) (33) The ﬁxed-wing moment τw includes the aerodynamic moment τwa and control torque τwc around the body axis Eb x, i.e., τw = τwa + τwc, where, The ﬁxed-wing moment τw includes the aerodynamic moment τwa and control torque τwc around the body axis Eb x, i.e., τw = τwa + τwc, where, τwa =   lw(D1 −D2)sα lc[(L2 + L1)cα + (D2 + D1)sα] lw[(L1 −L2)sα + (D2 −D1)cα]   (34) (34) and τwc =   lwqSwCLδ1.2(δ1 −δ2)cα 0 0   (35) (35) 4) Elevator The parameters of elevator lift and drag are described as follows: The parameters of elevator lift and drag are described as follows: Le = qSeCle, Cle = Cleα(α + δe) De = qSeCde, Cde = Cde0 + C2 le/(πAeee) ee = 1.78(1 −0.045A0.68 e ) −0.46 (38) (38) where Se is the area of the elevator, δe is the elevator deﬂection; Cleα is the lift coeﬃcient due to the angle of attack α and the deﬂection δe; Cde0 is the drag coeﬃcient when α + δe = 0; Ae is the aspect ratio of the elevator; ee is the value of the Oswald’s eﬃciency factor. Then the force vector Fe on the elevator in body frame is expressed by Fe =   Lesα −Decα 0 −Lecα −Desα   (39) (39) The elevator moment τe includes the aerodynamic moment τea and control torque τec in the body axis Eb y, i.e., τe = τea + τec, where, The elevator moment τe includes the aerodynamic moment τea and control torque τec in the body axis Eb y, i.e., τe = τea + τec, where, axis Eb y, i.e., τe = τea + τec, where, τea =   0 −leDesα 0   (40) τea =   0 −leDesα 0   (40) and τec =   0 −leqSeCleα(α + δe)cα 0   (41) (40) and τec =   0 −leqSeCleα(α + δe)cα 0   (41) (41) 16 5) Rudders 5) Rudders Deﬁne ¯q = 0.5ρ(U 2 + V 2). The lift and drag forces generated by the rudders, respectively Lr = ¯qSrClr, Clr = Clrββ + Clrδrδr Dr = ¯qSrCdv, Cdr = Cdr0 + C2 lr/(πArer) er = 1.78(1 −0.045A0.68 r ) −0.46 (42) (42) where, Sr is the area of the rudders; Clrβ is the lift coeﬃcient due to the sideslip angle β; δr is the rudder deﬂection, and Clrδr is the lift coeﬃcient due to the deﬂection δr; Cdr0 is the drag coeﬃcient when β = δr = 0; Ar is the aspect ratio of the rudder; er is the value of the Oswald’s eﬃciency factor. 5) Rudders 6.2 Measurements 5) Rudders Then the aerodynamic force vector Fr on the rudders in body frame can be expressed by Fr =   Lrsβ −Drcβ Lrcβ + Drsβ 0   (43) (43) The rudder moment τr includes the aerodynamic moment τra and control torque τrc in body axis Eb z, i.e., τr = τra + τrc, where, τra =   0 0 lrDrsβ   (44) and τrc =   0 0 lr¯qSr(Clrββ + Clrδrδr)cβ   (45) τra =   0 0 lrDrsβ   (44) (44) and and τrc =   0 0 lr¯qSr(Clrββ + Clrδrδr)cβ   (45) (45) 6) UAV motion equations in the earth-ﬁxed frame considering system uncertainties According to [23], from (27) and (26), we get ˙ΩΓ = ( Z−1)′ ΩΛ + Z−1 ˙ΩΛ = ( Z−1)′ ZΩΓ −Z−1J−1 [ZΩΓ × (JZΩΓ)] + Z−1J−1τ (46) (46) The total moment τ can be categorized into the control torque τc, uncertain moment τd and other moments τother, i.e., The total moment τ can be categorized into the control torque τc, uncertain moment τd and other moments τother, i.e., τ = τc + τother + τd (47) (47) τ = τc + τother + τd From (35), (41) and (45), we get τc = τwc + τec + τrc (48) (48) τc = τwc + τec + τrc 17 and from (34), (40) and (44), we get τother = τwa + τea + τra (49) τother = τwa + τea + τra (49) τother = τwa + τea + τra τother = τwa + τea + τra In system (46), considering (47), we deﬁne In system (46), considering (47), we deﬁne ( Z−1)′ ZΩΓ −Z−1J−1 [ZΩΓ × (JZΩΓ)] + Z−1J−1τother deﬁne =   aϕ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) aθ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) aψ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β )   (50) (50)  ψ ( ) Z−1J−1τc deﬁne = [ τϕ τθ τψ ]T (51) In (29), we deﬁne Z−1J−1τc deﬁne = [ τϕ τθ τψ ]T (51) In (29), we deﬁne RΓΛ(Fw + Ff + Fr + Fe)/m deﬁne =   Fxa(ϕ, θ, ψ, α, β) Fya(ϕ, θ, ψ, α, β) F (ϕ θ ψ β)   (52) Z−1J−1τc deﬁne = [ τϕ τθ τψ ]T (51) (51) In (29), we deﬁne RΓΛ(Fw + Ff + Fr + Fe)/m deﬁne =   Fxa(ϕ, θ, ψ, α, β) Fya(ϕ, θ, ψ, α, β) Fza(ϕ, θ, ψ, α, β)   (52) RΓΛ(Fw + Ff + Fr + Fe)/m deﬁne =   Fxa(ϕ, θ, ψ, α, β) Fya(ϕ, θ, ψ, α, β) Fza(ϕ, θ, ψ, α, β)   (52) (52) Then, the Jet UAV motion equations written in terms of the centre of mass C in the earth-ﬁxed frame are Then, the Jet UAV motion equations written in terms of the centre of mass C in the earth-ﬁxed frame are ¨x = cθcψFc/m + Fxa(ϕ, θ, ψ, α, β) + ∆x ¨y = cθsψFc/m + Fya(ϕ, θ, ψ, α, β) + ∆y ¨z = −sθFc/m + Fza(ϕ, θ, ψ, α, β) + g + ∆z (53) ¨x = cθcψFc/m + Fxa(ϕ, θ, ψ, α, β) + ∆x ¨y = cθsψFc/m + Fya(ϕ, θ, ψ, α, β) + ∆y ¨z = −sθFc/m + Fza(ϕ, θ, ψ, α, β) + g + ∆z (53) (53) ¨ϕ = aϕ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) + τϕ + ∆ϕ ¨θ = aθ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) + τθ + ∆θ ¨ψ = aψ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) + τψ + ∆ψ (54) (54) where, m is the mass of the UAV; g is the gravity acceleration; (∆x, ∆y, ∆z) and (∆ϕ, ∆θ, ∆ψ) are the bounded uncertainties in the position and attitude dynamics, respectively; τϕ, τθ and τψ are the control torques for roll, pitch and yaw dynamics, respectively, deﬁned in (51). 6.2 Measurements For the jet UAV, a GPS receiver provides the global position and the velocity. An IMU gives the attitude angle and angular velocity. The measurement outputs are expressed by For the jet UAV, a GPS receiver provides the global position and the velocity. An IMU gives the attitude angle and angular velocity. The measurement outputs are expressed by y∗1 = ∗+ d∗1(t), y∗2 = ˙∗+ d∗2(t) (55) (55) where, ∗i = {x, y, z, ϕ, θ, ψ}, and ˙∗= { ˙x, ˙y, ˙z, ˙ϕ, ˙θ, ˙ψ}; d∗1(t) denotes the disturbances in position and angle measurements, and supt∈[0,∞) \|d∗1(t)\| ≤L∗1 < ∞; d∗2(t) denotes the disturbances in the 18 Figure 4: Scheme of control system Figure 4: Scheme of control system measurements of ﬂying velocity and angular velocity, and supt∈[0,∞) \|d∗2(t)\| ≤L∗2 < ∞; L∗2 ≪1, and max { L∗1 L∗2 } ≤ε∗≪1. The corrector (17) is used to reject the measurement disturbances and to estimate the actual (x, y, z, ϕ, θ, ψ). 6.3 Controller design In this section, the control laws are derived for UAV trajectory tracking and attitude stabilization. The position, attitude and system uncertainties are reconstructed by the presented corrector and an existing extended state observer. The scheme of control system with correction and estimation is shown in Figure 4: 1) The correctors estimate position and attitude angles, and the disturbances are rejected from the mea- surements; 2) The extended state observers estimate the uncertainties in position and attitude dynamics, respectively; 4) According to the reference trajectory and estimation from the correctors and observers, the position controller drives the position dynamics; 5) From the estimation of po- sition, attitude, uncertainties, and the reference trajectory, the desired attitude is determined; 6) According to the desired attitude and estimation from the correctors and observers, the attitude controller drives the attitude dynamics. 1) Error systems 1) Error systems 1) Error systems Suppose the reference trajectory and its ﬁnite order derivatives are bounded, and can be generated directly. For the reference trajectory Xd = (xd, yd, zd), let ex = x−xd, ey = y −yd, and ez = z −zd, then the system error for position dynamics (53) is ¨ep = up + Ξp + ∆p (56) (56) ¨ep = up + Ξp + ∆p where, 19 ep =   ex ey ez  , up =   upx upy upz  =   cθcψ cθsψ −sθ  Fc/m, Ξp =   Fxa(ϕ, θ, ψ, α, β) −¨xd Fya(ϕ, θ, ψ, α, β) −¨yd Fza(ϕ, θ, ψ, α, β) + g −¨zd  , ∆p =   ∆x ∆y ∆z   (57) (57) For the desired attitude angle Θd = (ϕd, θd, ψd), let eϕ = ϕ −ϕd, eθ = θ −θd, and eψ = ψ −ψd, then the system error for attitude dynamics (54) is For the desired attitude angle Θd = (ϕd, θd, ψd), let eϕ = ϕ −ϕd, eθ = θ −θd, and eψ = ψ −ψd, then the system error for attitude dynamics (54) is ¨ea = ua + Ξa + ∆a (58) where, ¨ea = ua + Ξa + ∆a (58) ¨ea = ua + Ξa + ∆a (58) where, ea =   eϕ eθ eψ  , ua =   τϕ τθ τψ  , ∆a =   ∆ϕ ∆θ ∆ψ  , Ξa =   aϕ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) −¨ϕd aθ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) −¨θd aψ ( ϕ, θ, ψ, ˙ϕ, ˙θ, ˙ψ, α, β ) −¨ψd   (59) (59) 2) Extended state observers for the uncertainties ∆p and ∆a 2) Extended state observers for the uncertainties ∆p and ∆a 2) Extended state observers for the uncertainties ∆p and ∆a The following extended state observers are used [24]: The following extended state observers are used [24]: ˙x∗1 = x∗2 −λ∗1 \|x∗1 −y∗2\| 1+α∗ 2 sign(x∗1 −y∗2) + H∗ ˙x∗2 = −λ∗2 \|x∗1 −y∗2\|α∗sign(x∗1 −y∗2) (60) (60) From Theorem 1 in [24], there exist a ﬁnite time ts > 0 such that, for t ≥ts, x∗1 = ˙∗, x∗2 = ∆∗ (61) (61) where, ∗= {x, y, z, ψ, θ, ϕ} and ˙∗= { ˙x, ˙y, ˙z, ˙ϕ, ˙θ, ˙ψ}; λ∗1, λ∗2 > 0, and α∗∈(0, 1); The measurement y∗2 deﬁned in (55) is the observer input signal; where, ∗= {x, y, z, ψ, θ, ϕ} and ˙∗= { ˙x, ˙y, ˙z, ˙ϕ, ˙θ, ˙ψ}; λ∗1, λ∗2 > 0, and α∗∈(0, 1); The measurement y∗2 deﬁned in (55) is the observer input signal; 20 Hx = upx + Fxa(bϕ, bθ, bψ, α, β) Hy = upy + Fya(bϕ, bθ, bψ, α, β) Hz = upz + Fza(bϕ, bθ, bψ, α, β) −g Hϕ = τϕ + aϕ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) Hθ = τθ + aθ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) Hψ = τψ + aψ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) (62) Hx = upx + Fxa(bϕ, bθ, bψ, α, β) Hy = upy + Fya(bϕ, bθ, bψ, α, β) Hz = upz + Fza(bϕ, bθ, bψ, α, β) −g Hϕ = τϕ + aϕ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) Hθ = τθ + aθ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) Hψ = τψ + aψ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) (62) Hx = upx + Fxa(bϕ, bθ, bψ, α, β) Hy = upy + Fya(bϕ, bθ, bψ, α, β) Hz = upz + Fza(bϕ, bθ, bψ, α, β) −g Hϕ = τϕ + aϕ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) Hθ = τθ + aθ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) Hψ = τψ + aψ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) (62) (62) and (bϕ, bθ, bψ) are from the outputs of the sliding mode correctors. From (60) and (61), we get b∆p = [ xx2 xy2 xz2 ]T (63) (63) and and and b∆a = [ xψ2 xθ2 xϕ2 ]T (64) (64) 2) Extended state observers for the uncertainties ∆p and ∆a 2) Extended state observers for the uncertainties ∆p and ∆a The extended state observers [24] are used to estimate the uncertainties in position dynamics (53) and attitude dynamics (54). For the observers, the measurements of ﬂying velocity and angular velocity y∗2 = ˙∗+ d∗2(t) (where, ˙∗= { ˙x, ˙y, ˙z, ˙ϕ, ˙θ, ˙ψ}) are the input signals. Then, the uncertainty ∆p = [ ∆x ∆y ∆z ]T in the position dynamics and the uncertainty ∆a = [ ∆ϕ ∆θ ∆ψ ]T in the attitude dynamics are estimated. The continuous extended state observers can provide smooth and accurate estimations of the uncertainties in the system dynamics, reducing high frequency vibrations. 3) Controller design for position dynamics The radius of curvature is The combination of lift force from the wings, elevator and fuselage can provide the centripetal force. The radius of curvature is r = ˙x2 + ˙y2 \| ˙x¨y −¨x ˙y\| (72) r = x2 + y2 \| ˙x¨y −¨x ˙y\| (72) and the centripetal force is (72) \|xy −xy\| ( ) and the centripetal force is fcentri = m( ˙x2 + ˙y2) r = m \| ˙x¨y −¨x ˙y\| (73) fcentri = m( ˙x2 + ˙y2) r = m \| ˙x¨y −¨x ˙y\| (73) Also the centripetal force can be expressed by (73) fcentri = m(x + y ) r = m \| ˙x¨y −¨x ˙y\| (73) Also, the centripetal force can be expressed by r Also, the centripetal force can be expressed by Also, the centripetal force can be expressed by Also, the centripetal force can be expressed by fcentri = Lwef cos ϕd (74) where, Lwef = −(L1 +L2)cα −(D1 +D2)sα −Lecα −Desα −Lfcα −Dfsα. 3) Controller design for position dynamics 3) Controller design for position dynamics 3) Controller design for position dynamics 3) Controller design for position dynamics For position dynamics (53), to track the reference trajectory Xd = (xd, yd, zd), when we select the following controller, the position system error (56) will converge asymptotically to zero: up = −bΞp −b∆p −kp1bep −kp2b˙ep (65) (65) where bex = bx −xd, b˙ex = b˙x −˙xd, bey = by −yd, b˙ey = b˙y −˙yd, bez = bz −zd, b˙ez = b˙z −˙zd; kp1, kp2 > 0; (bx, by, bz, b˙x , b˙y,b˙z, bϕ, bθ, bψ) are from the outputs of the correctors; b∆p is from the outputs of the extended state observer; and where bex = bx −xd, b˙ex = b˙x −˙xd, bey = by −yd, b˙ey = b˙y −˙yd, bez = bz −zd, b˙ez = b˙z −˙zd; kp1, kp2 > 0; (bx, by, bz, b˙x , b˙y,b˙z, bϕ, bθ, bψ) are from the outputs of the correctors; b∆p is from the outputs of the extended state observer; and bep =   bex bey bez  ,b˙ep =   b˙ex b˙ey b˙ez  , bΞp =   Fxa(bϕ, bθ, bψ, α, β) −¨xd Fya(bϕ, bθ, bψ, α, β) −¨yd Fza(bϕ, bθ, bψ, α, β) −g −¨zd   (66) (66) From (57), we get the engine thrust From (57), we get the engine thrust From (57), we get the engine thrust Fc = m ∥up∥2 = m √ u2px + u2py + u2pz (67) (67) 4) Desired attitude angles From (56) and (65), we get 4) Desired attitude angles 4) Desired attitude angles From (56) and (65), we get ¨ez = −sθFc/m + Fza(bϕ, bθ, bψ, α, β) + g −¨zd + b∆z = −kp1bez −kp2b˙ez (68) ¨ez = −sθFc/m + Fza(bϕ, bθ, bψ, α, β) + g −¨zd + b∆z = −kp1bez −kp2b˙ez (68) ¨ez = −sθFc/m + Fza(bϕ, bθ, bψ, α, β) + g −¨zd + b∆z = −kp1bez −kp2b˙ez (68) (68) 21 Then, the desired pitch angle is Then, the desired pitch angle is θd = arcsin m(Fza(bϕ, bθ, bψ, α, β) + g −¨zd + b∆z + kp1bez + kp2b˙ez) Fc (69) (69) Fc Also, from (56) and (65), we get Also, from (56) and (65), we get Also, from (56) and (65), we get Also, from (56) and (65), we get ¨ex = cθcψFc/m + Fxa(bϕ, bθ, bψ, α, β) + b∆x = −kp1bex −kp2b˙ex ¨ey = cθsψFc/m + Fya(bϕ, bθ, bψ, α, β) + b∆y = −kp1bey −kp2b˙ey (70) (70) Then, the desired yaw angle is Then, the desired yaw angle is Then, the desired yaw angle is ψd = arctan Fya(bϕ, bθ, bψ, α, β) + b∆y + kp1bey + kp2b˙ey Fxa(bϕ, bθ, bψ, α, β) + b∆x + kp1bex + kp2b˙ex (71) (71) The combination of lift force from the wings, elevator and fuselage can provide the centripetal force. 4) Determination of upper-bound of sensor accuracy ratio: From the sensor accuracy, we can get supt∈[0,∞) \|d1(t)\| ≤L1 = 3(m), supt∈[0,∞) \|d2(t)\| ≤L2 = 0.1(m/s). The upper-bound of sensor accuracy ratio ε = 0.034 can be selected such that the sensor accuracy inequality L2 L1 = 0.1 3 ≤ε = 0.034 ≪1 holds. 7 Simulation examples We use two examples to illustrate the sliding mode corrector presented in Theorems 5.1 and 5.2. Example 1 (Sliding mode corrector design from Theorem 5.1): Example 1 (Sliding mode corrector design from Theorem 5.1): 1) Sensor outputs and actual values: ) Sensor outputs and actual values: 1) Sensor outputs and actual values: 1) Sensor outputs and actual values: Measurement signals for position and velocity: pm(t) = p0(t) + d1(t), vm(t) = v0(t) + d2(t) where, d1(t) and d2(t) are the disturbances in position and velocity measurements, respectively; Suppose the actual position: p0 (t) = 10 + 20 sin (t); and the actual velocity: v0 (t) = 20 cos (t) Measurement signals for position and velocity: pm(t) = p0(t) + d1(t), vm(t) = v0(t) + d2(t) where, d1(t) and d2(t) are the disturbances in position and velocity measurements, respectively; Measurement signals for position and velocity: pm(t) = p0(t) + d1(t), vm(t) = v0(t) + d2(t) where, d1(t) and d2(t) are the disturbances in position and velocity measurements, respectively Suppose the actual position: p0 (t) = 10 + 20 sin (t); where, d1(t) and d2(t) are the disturbances in position and velocity measurements, respectively; Suppose the actual position: p0 (t) = 10 + 20 sin (t); and the actual velocity: v0 (t) = 20 cos (t). 2) Disturbance in position measurement: 2) Disturbance in position measurement: Position sensing disturbance d1(t) = d11(t) + d12(t) includes disturbance d11(t) and stochastic noise d12(t). We consider the following three types of disturbance d11(t): (a) d11(t) = 2 sin(4t) + cos(9t); (a) d11(t) = 2 sin(4t) + cos(9t); (b) d11(t) = 6 sin(4t) + 3 cos(9t), and stochastic noise 3d12(t); (disturbance magnitude increases, i.e., L1 increases) (c) d11(t) = 1.5 cos(0.2t)+0.5 sin(0.6t)+2 sin(4t)+cos(9t). (disturbance in low and mid frequency bands) (c) d11(t) = 1.5 cos(0.2t)+0.5 sin(0.6t)+2 sin(4t)+cos(9t). (disturbance in low and m bands) 3) Controller design for position dynamics Then, we get the desired roll angle (74) ϕd = arccos fcentri Lwef (75) (75) 5) Controller design for attitude dynamics er design for attitude dynamics 5) Controller design for attitude dynamics For attitude dynamics (54), to track the desired attitude Θd = (ψd, θd, ϕd), when we select the following controller, the attitude system error (58) will converge asymptotically to zero: ua = −bΞa −b∆a −ka1bea −ka2b˙ea (76) where, ka1, ka2; beϕ = bϕ −ϕd, b˙eϕ = b˙ϕ −˙ϕd, beθ = bθ −θd, b˙eθ = b˙θ −˙θd, beψ = bψ −ψd, b˙eψ = b˙ψ −˙ψd; bea = [ beϕ beθ beψ ]T ; ˙ea = [ b˙eϕ b˙eθ b˙eψ ]T ; (bϕ, bθ, bψ) are from the outputs of the correctors; b∆a is from the outputs of the extended state observers; and ua = −bΞa −b∆a −ka1bea −ka2b˙ea (76) where, ka1, ka2; beϕ = bϕ −ϕd, b˙eϕ = b˙ϕ −˙ϕd, beθ = bθ −θd, b˙eθ = b˙θ −˙θd, beψ = bψ −ψd, b˙eψ = b˙ψ −˙ψd; bea = [ beϕ beθ beψ ]T ; ˙ea = [ b˙eϕ b˙eθ b˙eψ ]T ; (bϕ, bθ, bψ) are from the outputs of the correctors; b∆a is from the outputs of the extended state observers; and (76) bΞa =   aϕ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) −¨ϕd aθ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) −¨θd aψ ( bϕ, bθ, bψ, ˙ϕ, ˙θ, ˙ψ, α, β ) −¨ψd   (77) (77) 22 3) Disturbance in velocity measurement: 3) Disturbance in velocity measurement: Velocity sensing disturbance d2(t) = d21(t) + d22(t) + d23(t) includes time-varying disturbance d21(t), constant disturbance d22(t), and stochastic noise d23(t). We suppose d21(t) = 0.05 cos(0.3t)+ 0.03 sin(0.6t) and d22(t) = 0.02. 4) Determination of upper-bound of sensor accuracy ratio: 5) Corrector parameters’ selection: From the corrector parameter selection in the Remark 5.2 of Theorem 5.1, we select r = 1 2 to get the minimum value and minimum range of rejection ratio ρ(ω1): min {ρ(ω1)} = 1 2ε 1 2 , and ρ(ω1) ∈ ( 1 2ε 1 2 , 1 + 1 2ε 1 2 ] when ω1 ∈[0, ∞). Therefore, from ε = 0.034 and r = 1 2, we can determine the corrector parameter k1 = 2ε1−r = 2 × 0.0341−0.5 = 0.36. Therefore, from ε = 0.034 and r = 1 2, we can determine the corrector parameter k1 = 2ε1−r = 2 × 0.0341−0.5 = 0.36. According to supt∈[0,∞) \| ˙v0(t)\| ≤L3 = 10, k2 > k1+L3 and k3 > L3, we select k2 = 100, k3 = 100. According to supt∈[0,∞) \| ˙v0(t)\| ≤L3 = 10, k2 > k1+L3 and k3 > L3, we select k2 = 100, 6) Rejection ratio and disturbance frequency bands: 6) Rejection ratio and disturbance frequency bands: From ε = 0.034 and r = 1 2, the rejection ratio is expressed by From ε = 0.034 and r = 1 2, the rejection ratio is expressed by 23 ρ(ω1) = 1 √ 1 + 1 4ε2r−2ω2 1 + 1 2εr = 1 √ 1 + 7.35ω2 1 + 0.09 and we get: g ω0 = 4ε1−2r √ 1 −1 4ε2r = 4 √ 1 −1 4 × 0.034 = 3.98(rad/s) ωc = ε1−1 2 r√4−εr 1−1 2 εr = 0.0341−1 4 √ 4−0.0340.5 1−1 2 ×0.0340.5 = 0.17(rad/s) Therefore, the rejection ratio in the diﬀerent frequency bands of [0, ∞) can be described by: Therefore, the rejection ratio in the diﬀerent frequency bands of [0, ∞) can be described by: i) In [3.98rad/s, ∞), ρ(ω1) : 0.18 →0.09 as ω1 : 3.98 →∞(rad/s); ii) In (0.17, 3.98rad/s), ρ(ω1) : 1 →0.18 as ω1 : 0.17 →3.98(rad/s); iii) In [0, 0.17rad/s], ρ(ω1) : 1.09 →1 (i.e., ρ(ω1) ≈1) as ω1 : 0 →0.17(rad/s). i) In [3.98rad/s, ∞), ρ(ω1) : 0.18 →0.09 as ω1 : 3.98 →∞(rad/s); ii) In (0.17, 3.98rad/s), ρ(ω1) : 1 →0.18 as ω1 : 0.17 →3.98(rad/s); Comparison with signal fusion based on Kalman ﬁlter: Comparison with signal fusion based on Kalman ﬁlter: We compare the sliding mode corrector with KF-based method. For this example, there are only two measurement signals, and no system model is given, we use the direct KF-based signal fusion method [25]. The position measurement is pm(t) = p0(t) + d1(t), and the velocity measurement is vm(t) = v0(t) + d2(t). According to the Taylor’s expansion, position and velocity in discrete system can be expressed approximately by p0(k) ≈p0(k −1) + v0(k −1) · ∆T v0(k) ≈v0(k −1) (78) (78) where, ∆T is the sampling time, and k is the sample step. where, ∆T is the sampling time, and k is the sample step. where, ∆T is the sampling time, and k is the sample step. Comparison with signal fusion based on Kalman ﬁlter: Deﬁne X (k) = [ p0(k) v0(k) ]T , A = [ 1 ∆T 0 1 ] , the above relation can be described by a matrix system: Deﬁne X (k) = [ p0(k) v0(k) ]T , A = [ 1 ∆T 0 1 ] , the above relation can be described by a matrix system: system: system: X (k) = A · X (k −1) (79) X (k) = A · X (k −1) (79) For pm(t) = p0(t) + d1(t) and vm(t) = v0(t) + d2(t), we get (79) X (k) A X (k 1) (79) For pm(t) = p0(t) + d1(t) and vm(t) = v0(t) + d2(t), we get For pm(t) = p0(t) + d1(t) and vm(t) = v0(t) + d2(t), we get pm(k) = p0(k) + d1(k); vm(k) = v0(k) + d2(k) (80) (80) Deﬁne H = [ 1 0 0 1 ] , V (k) = [ d1(k) d2(k) ]T , then the measurement outputs can be expressed by Deﬁne H = [ 1 0 0 1 ] , V (k) = [ d1(k) d2(k) ]T , then the measurement outputs can be expressed by Y (k) = H · X (k) + V (k) (81) Y (k) = H · X (k) + V (k) (81) (81) Therefore, the Kalman ﬁlter for signal integration is designed as follows: Therefore, the Kalman ﬁlter for signal integration is designed as follows: Therefore, the Kalman ﬁlter for signal integration is designed as follows: X(k\|k −1) = A · X(k −1\|k −1) X(k\|k −1) = A · X(k −1\|k −1) 24 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -6 -4 -2 0 2 4 Errors(m) Measurement error Error by EKF Error by corrector 2.2 2.4 24 26 28 (a) 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -15 -10 -5 0 5 10 15 Errors(m) Measurement error Error by EKF Error by corrector 3.6 3.8 4 -6 -4 -2 0 2 (b) 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -5 0 5 10 Errors(m) Measurement error Error by EKF Error by corrector 2 2.5 22 24 26 28 30 32 34 7.6 7.8 8 28 30 32 (c) - Simulation on position disturbance rejection. Comparison with signal fusion based on Kalman ﬁlter: (a) Distur ance rejection when L1 = 9. (c) Disturbance rejection cluded. 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -6 -4 -2 0 2 4 Errors(m) Measurement error Error by EKF Error by corrector 2.2 2.4 24 26 28 (a) 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -15 -10 -5 0 5 10 15 Errors(m) Measurement error Error by EKF Error by corrector 3.6 3.8 4 -6 -4 -2 0 2 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -6 -4 -2 0 2 4 Errors(m) Measurement error Error by EKF Error by corrector 2.2 2.4 24 26 28 (a) ( ) 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -15 -10 -5 0 5 10 15 Errors(m) Measurement error Error by EKF Error by corrector 3.6 3.8 4 -6 -4 -2 0 2 (b) 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -5 0 5 10 Errors(m) Measurement error Error by EKF Error by corrector 2 2.5 22 24 26 28 30 32 34 7.6 7.8 8 28 30 32 (c) Figure 5: Example 1 - Simulation on position disturbance rejection. (a) Disturbance rejection when L1 = 3. (b) Disturbance rejection when L1 = 9. (c) Disturbance rejection when low-frequency disturbance is also included. (b) 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -5 0 5 10 Errors(m) Measurement error Error by EKF Error by corrector 2 2.5 22 24 26 28 30 32 34 7.6 7.8 8 28 30 32 (c) (c) Figure 5: Example 1 - Simulation on position disturbance rejection. Analysis of simulation results: Analysis of simulation results: The disturbance rejections in position sensing are presented in Figure 5. Figure 5(a) describes the disturbance rejection when the disturbance d1(t) is in the frequency band that is rejected suﬃciently. In fact, the frequency band of disturbance d1(t) is ω1 ∈[4rad/s, ∞), and [4rad/s, ∞) ⊂ [3.98rad/s, +∞). We know that in frequency band [3.98rad/s, ∞), the minimum rejection ratio is obtained, i.e., ρ(ω1) ∈ ( 1 2ε 1 2 , ε 1 2 ] = (0.09, 0.18]. Therefore, the position disturbance is rejected suﬃciently. Figure 5(b) presents the disturbance rejection when the magnitude of disturbance d1(t) increases. Even the position sensing accuracy becomes worse (L1 : 3m →9m), the corrector with the original parameters can still reject the disturbance suﬃciently. Figure 5(c) shows the disturbance rejection when the position disturbance d1(t) covers the low/min/high frequency bands. In the disturbance d1(t), the mid/high frequency part is reject- ed suﬃciently. Even the low frequency disturbance exists, the eﬀect of disturbance can still be rejected largely. In fact, in the position disturbance d1(t) = d11(t) + d12(t) (where, d11(t) = 1.5 cos(0.2t) + 0.5 sin(0.6t) + 2 sin(4t) + cos(9t) and the high-frequency stochastic noise d12(t)): Figure 5(c) shows the disturbance rejection when the position disturbance d1(t) covers the low/min/high frequency bands. In the disturbance d1(t), the mid/high frequency part is reject- ed suﬃciently. Even the low frequency disturbance exists, the eﬀect of disturbance can still be rejected largely. In fact, in the position disturbance d1(t) = d11(t) + d12(t) (where, d11(t) = 1.5 cos(0.2t) + 0.5 sin(0.6t) + 2 sin(4t) + cos(9t) and the high-frequency stochastic noise d12(t)): 1) the part 2 sin(4t) + cos(9t) is within the frequency band [3.98rad/s, ∞), and the rejection ratio is minimum, i.e., ρ(ω1) ∈(0.09, 0.18]; therefore, this part of disturbance is rejected suﬃciently; d12(t) is also rejected suﬃciently due to its high frequency. 1) the part 2 sin(4t) + cos(9t) is within the frequency band [3.98rad/s, ∞), and the rejection ratio is minimum, i.e., ρ(ω1) ∈(0.09, 0.18]; therefore, this part of disturbance is rejected suﬃciently; d12(t) is also rejected suﬃciently due to its high frequency. 2) the part 1.5 cos(0.2t) + 0.5 sin(0.6t) is within the frequency band (0.17, 3.98rad/s], and the rejection ratio is ρ(ω1) ∈[0.18, 1rad/s); therefore, this part of disturbance is still reduced. Comparison with signal fusion based on Kalman ﬁlter: (a) Disturbance rejection when L1 = 3. (b) Disturbance rejection when L1 = 9. (c) Disturbance rejection when low-frequency disturbance is also included. 25 P(k\|k −1) = A · P(k −1\|k −1)AT + Q K(k) = P(k\|k −1)HT H · P(k\|k −1)HT + R X(k\|k) = X(k\|k −1) + K(k)(Y (k) −H · X(k\|k −1)) P(k\|k) = (I −K(k)H)P(k\|k −1) (82) (82) here, I = [ 1 0 0 1 ] ; Q = [ 1 3qc∆T 3 1 2qc∆T 2 1 2qc∆T 2 qc∆T ] where, I = [ 1 0 0 1 ] ; Q = [ 1 3qc∆T 3 1 2qc∆T 2 1 2qc∆T 2 qc∆T ] is the process noise covariance matrix, and qc is the here, I = [ 1 0 0 1 ] ; Q = [ 1 3qc∆T 3 1 2qc∆T 2 1 2qc∆T 2 qc∆T ] is the process noise covariance matrix, and qc is the power spectral density of the input white noise; and R is the measurement noise covariance matrix. In the simulation, the power spectral density of the input white noise is selected as qc = 1 [25], R = [ 0.8 0 0 0.8 ] , P (0\|0) = [ 0.1 0 0 0.1 ] and the sampling time is ∆T = 0.008(sec). Example 2 (Sliding mode corrector design from Theorem 5.2): 3) Disturbance in velocity sensing: 3) Disturbance in velocity sensing: Velocity sensing disturbance d2(t) = d21(t) + d22(t) + d23(t) includes time-varying disturbance d21(t), constant disturbance d22(t), and stochastic noise d23(t). We suppose d21(t) = 0.05 cos(0.3t)+ 0.03 sin(0.6t) and d22(t) = 0.02. 1) Sensor outputs and actual values: Sensing signals for position and velocity are pm(t) = p0(t) + d1(t) and vm(t) = v0(t) + d2(t), re- spectively, where, d1(t) and d2(t) are the disturbances in position and velocity sensing, respectively; the actual position: p0 (t) = 10 + 20 sin (t); and the actual velocity: v0 (t) = 20 cos (t). the actual position: p0 (t) = 10 + 20 sin (t); and the actual velocity: v0 (t) = 20 cos (t). the actual position: p0 (t) = 10 + 20 sin (t); and the actual velocity: v0 (t) = 20 cos (t). 2) Disturbance in position sensing: Position sensing disturbance d1(t) = d11(t) + d12(t) includes time-varying disturbance d11(t) and stochastic noise d12(t), and we suppose d11(t) = 2 sin(1.5) + cos(3t). Analysis of simulation results: 2) the part 1.5 cos(0.2t) + 0.5 sin(0.6t) is within the frequency band (0.17, 3.98rad/s], and the rejection ratio is ρ(ω1) ∈[0.18, 1rad/s); therefore, this part of disturbance is still reduced. From Figure 5, we can also ﬁnd that the estimate outputs of corrector are accurate and smoothed even stochastic noise exists in position and velocity measurements. In addition, if initial calibration is done for position sensing, the corrector error keeps small from the beginning; and if there is no initial calibration for position sensing, the corrector error can still converge to the small bound. The corrector performance is compared with the estimate results of the KF-based method. Comparing to the corrector, due to the existence of widely frequency-band disturbance in position sensing, the obviously large estimate errors exist in the outputs of the KF, although it can reduce the eﬀect of disturbance to some extent. Example 2 (Sliding mode corrector design from Theorem 5.2): 26 This example illustrates the position disturbance d1(t) is rejected to the maximum extent within a given frequency band, and the disturbance in the other bands can still be reduced largely. The corrector parameters are determined to get an optimal value of rejection ratio in the given frequency band. 1) Sensor outputs and actual values: 4) Corrector parameters’ selection: In this example, we suppose the disturbance d1(t) is required to be rejected suﬃciently in the given frequency band [ωreq, ∞) = [1rad/s, ∞), and a small rejection ratio is obtained in this frequency band. From ωreq = 1rad/s, we can get the unique solution xmin to 12xmin(1 + 0.52x2 min)−3 2 −0.034 = 0 in the range ( 1 √ 2×1, ∞), i.e., xmin = 5.26, and k1 = 1/xmin = 0.19. According to supt∈[0,∞) \| ˙v0(t)\| ≤L3 = 20, k2 > k1+L3 and k3 > L3, we select k2 = 100, k3 = 100. 5) Rejection ratio and disturbance frequency bands: 5) Rejection ratio and disturbance frequency bands: From the sensor accuracy L1 = 3(m) and L2 = 0.1(m/s), the upper-bound of sensor accuracy ratio ε = 0.034 is selected such that the sensor accuracy inequality L2 L1 = 0.1 3 ≤ε = 0.034 ≪1 holds. From ε = 0.034 and xmin = 5.26, the rejection ratio can be expressed by ρ(ω1) = 1 √ 1 + x2 minω2 1 + ε · xmin = 1 √ 1 + 27.67ω2 1 + 0.18 Therefore, the rejection ratio in the diﬀerent frequency bands of [0, ∞) can be described by: Therefore, the rejection ratio in the diﬀerent frequency bands of [0, ∞) can be described by: ) [ / ) ( ) ( / ) i) In [1rad/s, ∞), ρ(ω1) : 0.37 →0.18 as ω1 : 1 →∞(rad/s); ii) In (0.13, 1rad/s), ρ(ω1) : 1 →0.37 as ω1 : 0.13 →1 (rad/s), in which, ωc = √ 2ε xmin √ 1−1 2 εxmin 1−ε·xmin = 0.13(rad/s); 27 27 0 1 2 3 4 5 6 7 8 9 10 -20 0 20 40 Correction(m) Measured EKF Corrected Real 0 1 2 3 4 5 6 7 8 9 10 time(s) -5 0 5 Errors(m) Measurement error Error by EKF Error by corrector 3.4 3.6 3.8 -4 -2 0 2 4 Figure 6: Example 2 - Simulation on position disturbance rejection for the given frequency band. Figure 6: Example 2 - Simulation on position disturbance rejection for the given frequency band. iii) In [0, 0.13rad/s], ρ(ω1) : 1.18 →1 as ω1 : 0 →0.13 (rad/s). iii) In [0, 0.13rad/s], ρ(ω1) : 1.18 →1 as ω1 : 0 →0.13 (rad/s). The disturbance rejection for example 2 is presented in Figure 6. Due to the relatively small rejection ratio ρ(ω1) ∈(0.18, 0.37] when ω1 ∈[1rad/s, ∞), the disturbance d1 (t) in position sensing is rejected suﬃciently in the given frequency band [1rad/s, ∞). Also, we can ﬁnd that the corrector has strong robustness against stochastic noise from the measurements of position and velocity, and the estimate output is smoothed. Thus, the plot performance conﬁrms the results of numerical calculation. For example 1, when ω1 = 1rad/s, we can get ρ(ω1) = 1 √ 1+7.35ω2 1 + 0.09 = 0.44rad/s. Therefore, in the given frequency band [1rad/s, ∞), the rejection ratio ρ(ω1) it satisﬁes ρ(ω1) : 0.44 →0.09 as ω1 : 1 →∞(rad/s). 5) Rejection ratio and disturbance frequency bands: The rejection performance of example 1 is a little worse than that of example 2 near the frequency ω1 due to 0.44 > 0.37. However, in low frequency band, the method in example 1 can get better performance because ρ(ω1) : 1.09 →1 (i.e., ρ(ω1) ≈1) as ω1 : 0 →0.17(rad/s); while, for example 2, we can get ρ(ω1) : 1.18 →1 as ω1 : 0 →0.13 (rad/s). For the whole frequency range [0, ∞), the corrector in example 1 can get ρ(ω1) : 1.09 →0.09 as ω1 : 0 →∞, while the corrector in example 2 can get ρ(ω1) : 1.18 →0.18 as ω1 : 0 →∞. 8 Experiment on jet UAV navigation and control In this section, an experiment on a jet UAV ﬂight is presented. The jet UAV prototype (An RC- model-based F/A-18 Hornet) shown in Figure 2 is used for the ﬂight test [17]. A JetCat P200-SX jet engine is adopted to provide the thrust, and the engine starter includes: Jet-tronic ECU for fuel control; electronic valve; electronic starting gas valve; electronic fuel valve; fuel tubing, tubing connector set, ﬁlters, and cable set; 2 cell, 3300mA LiPoly battery pack; and starting gas tank. The engine can provide 220N (52 lbs) thrust for 112000 RPM, and RPM range: 33000∼112000 RPM. A Gumstix microcomputer is used for data collection and signal processing from sensors. The ﬂight control system implementation on the hardware is shown in Figure 7. An Arduino Mega 2560 is taken as the driver board, which has multiple PWM output channels. The input voltage is 28 GPS receiver Laser Altimeter Servos Jet-tronic ECU IMU AOA sensor Arduino Mega Airspeed sensor Jet engine Provide position and velocity signals Provide higher altitude signals Provide the lower altitude signals Provide bias angles of elevators, rudders and ailerons Provide the thrust force Drive jet engine Measure angle of attack Measure the relative wind speed Measure angular velocities and angles Gumstix Figure 7: Control system hardware. Gumstix Jet-tronic ECU Drive jet engine Figure 7: Control system hardware. 7∼12V. The control update time is 5ms. The FUTABA S3001 servos are adopted to control the deﬂections of ailerons, elevators and rudders. A 10Hz GPS MediaTek MT3329 is selected as the GPS receiver. A 9Hz VTI SCP1000 altimeter with 10cm resolution is utilized for above the sea level altitude measurements at higher altitudes. A 12Hz SF02-F laser altimeter is used for altitude measurements at lower altitudes with 40m range. A 10kHz Xsens MTI AHRS provides the 3-axial accelerations, the angular rates and the earth’s magnetic ﬁeld. A 192kHz kpilot 32 digital air speed sensor is utilized to obtain the relative wind speed. A 100kHz 4239-01 AOA sensor is used to measure the angle of attack. 7∼12V. The control update time is 5ms. The FUTABA S3001 servos are adopted to control the deﬂections of ailerons, elevators and rudders. A 10Hz GPS MediaTek MT3329 is selected as the GPS receiver. A 9Hz VTI SCP1000 altimeter with 10cm resolution is utilized for above the sea level altitude measurements at higher altitudes. 8 Experiment on jet UAV navigation and control A 12Hz SF02-F laser altimeter is used for altitude measurements at lower altitudes with 40m range. A 10kHz Xsens MTI AHRS provides the 3-axial accelerations, the angular rates and the earth’s magnetic ﬁeld. A 192kHz kpilot 32 digital air speed sensor is utilized to obtain the relative wind speed. A 100kHz 4239-01 AOA sensor is used to measure the angle of attack. The jet UAV parameters are described in Table 1. The jet UAV parameters are described in Table 1. Measurements of position and velocity: A 10Hz GPS MediaTek MT3329 (without aid) provides position at accuracy of 3m and velocity at accuracy of 0.1m/s. Measurements of attitude angle and angular velocity: A 10kHz Xsens MTI AHRS provides atti- tude angles and angular velocity, in which: roll/pitch accuracy: 0.5◦, yaw accuracy: 1.0◦; angular accuracy: 9◦/hr=0.0025◦/s Desired ﬂight trajectory: The desired ﬂight trajectory consists of takeoﬀ, climb, cruise in a circle with the radius 500m and the height 300m, and landing back, which is shown in Figure 8(a). In the experiment, considering measurement disturbances and the uncertainties in the UAV ﬂight dynamics, the jet UAV is controlled to track the reference trajectory. The position and velocity are obtained from the GPS receiver, and the attitude angle and the angular velocity are measured by the IMU. The corrected positions from the correctors and the system uncertainty estimations from the extended state observers are used for determination of the desired attitude and design of the controllers. The controllers (65) and (76) drive the UAV to track the reference trajectory. The performance of position correction by the correctors is compared with the EKF-based method [21]. 8.1 Design of correctors 8.1.1 Determination of upper-bound of sensor accuracy ratio: 8.1.1 Determination of upper-bound of sensor accuracy ratio: From the position sensor accuracy, we get supt∈[0,∞) \|d∗1(t)\| ≤L∗1 = 3(m), supt∈[0,∞) \|d∗2(t)\| ≤ L∗2 = 0.1(m/s), where, ∗= {x, y, z}. The upper-bound of sensor accuracy ratio ε = 0.034 can be selected such that the sensor accuracy inequality L2 L1 = 0.1 3 ≤ε∗= 0.034 ≪1 holds. 29 From the attitude sensor accuracy, we get supt∈[0,∞) \|d∗1(t)\| ≤L∗1 = 0.5◦, supt∈[0,∞) \|d∗2(t)\| ≤ L∗2 = 0.0025◦/s, where, ∗= {ϕ, θ}; and supt∈[0,∞) \|dψ1(t)\| ≤Lψ1 = 1◦, supt∈[0,∞) \|dψ2(t)\| ≤Lψ2 = 0.0025◦/s. For the roll/pitch, the upper-bound of sensor accuracy ratio ε∗= 0.005 can be selected such that the sensor accuracy inequality L2 L1 = 0.0025 0.5 ≤ε∗= 0.005 ≪1 holds, where, ∗= {ϕ, θ}. For the yaw, the upper-bound of sensor accuracy ratio εψ = 0.0025 can be selected such that the sensor accuracy inequality L2 L1 = 0.0025 1 ≤εψ = 0.0025 ≪1 holds. From the attitude sensor accuracy, we get supt∈[0,∞) \|d∗1(t)\| ≤L∗1 = 0.5◦, supt∈[0,∞) \|d∗2(t)\| ≤ L∗2 = 0.0025◦/s, where, ∗= {ϕ, θ}; and supt∈[0,∞) \|dψ1(t)\| ≤Lψ1 = 1◦, supt∈[0,∞) \|dψ2(t)\| ≤Lψ2 = 0.0025◦/s. The jet UAV parameters are described in Table 1. For the roll/pitch, the upper-bound of sensor accuracy ratio ε∗= 0.005 can be selected such that the sensor accuracy inequality L2 L1 = 0.0025 0.5 ≤ε∗= 0.005 ≪1 holds, where, ∗= {ϕ, θ}. For the yaw, the upper-bound of sensor accuracy ratio εψ = 0.0025 can be selected such that the sensor accuracy inequality L2 L1 = 0.0025 1 ≤εψ = 0.0025 ≪1 holds. Table 1 UAV Parameters Table 1 UAV Parameters Symbol Quantity Value md dry weight of UAV 22.5kg g gravity of acceleration 9.8m/s2 ρ air density 1.225kg/m3 lw wingspan 1.92m Sw ﬁxed wing area 1.58m2 CL1,20 ﬁxed wing lift coeﬃcient (α = 0) 0.3145 CD1,20 ﬁxed wing drag coeﬃcient (α = 0) 0.1634 CL1,2α ﬁxed wing lift coeﬃcient due to α 0.5122 CLδ1,2 ﬁxed wing lift coeﬃcient due to δi 0.1634 CDδ1,2 ﬁxed wing drag coeﬃcient due to δi 0.0025 Aw ﬁxed wing aspect ratio 2.67 lf fuselage length 2.35m Sf fuselage equivalent area 0.69m2 Clfα fuselage lift coeﬃcient due to α 0.1573 Cdf0 fuselage drag coeﬃcient (α = 0) 0.0096 Cdfα fuselage drag coeﬃcient due to α 0.0152 Se elevator area 0.36m2 Cleα elevator lift coeﬃcient due to α 0.6103 Cde0 elevator drag coeﬃcient (α = 0) 0.0046 Ae elevator aspect ratio 1.15 Sr rudder area 0.38m2 Clrβ rudder force coeﬃcient due to β 0.3261 Clrδr rudder lift coeﬃcient due to δr 0.0075 Cdr0 rudder drag coeﬃcient (β = δr= 0) 0.0046 Ar rudder aspect ratio 1.56 Jx moment inertia about axis Eb x 18Nm Jy moment inertia about axis Eb y 18Nm Jz moment inertia about axis Eb z 34Nm 8.1.2 Corrector parameters selection: 8.1.2 Corrector parameters selection: For the position, we select r = 1 2 to get the minimum value and minimum range of rejection ratio ρ(ω1): min {ρ(ω∗1)} = 1 2ε 1 2∗= 1 20.0340.5 = 0.09, and ρ(ω∗1) ∈ ( 1 2ε 1 2∗, 1 + 1 2ε 1 2∗ ] = (0.09, 1.09] when ω∗1 ∈[0, +∞), where, ∗= {x, y, z}. 30 From ε∗= 0.034 and r = 1 2, we can determine the corrector parameter k∗1 = 2ε1−r ∗ = 2 × 0.0341−0.5 = 0.36. From ε∗= 0.034 and r = 1 2, we can determine the corrector parameter k∗1 = 2ε1−r ∗ = 2 × 0.0341−0.5 = 0.36. According to supt∈[0,∞) \| ˙v∗0(t)\| ≤L∗3 = 50, k∗2 > k∗1 + L∗3 and k∗3 > L∗3, we select k∗2 = 100, k∗3 = 100, where, ∗= {x, y, z}. For the attitude, we know that εϕ = 0.005, εθ = 0.005 and εψ = 0.0025, we select r = 1 2. Then, we get the minimum rejection ratio min {ρ(ω∗1)} = 1 2ε 1 2∗= 1 20.0050.5 = 0.035, and ρ(ω∗1) ∈ (0.035, 1.035] when ω∗1 ∈[0, ∞), where, ∗= {ϕ, θ}; and min {ρ(ωψ1)} = 1 2ε 1 2 ψ = 1 20.00250.5 = 0.025, and ρ(ωψ1) ∈(0.025, 1.025] when ωψ1 ∈[0, ∞). Therefore: From ε∗= 0.005 and r = 1 2, we can determine k∗1 = 2ε1−r = 2 × 0.0051−0.5 = 0.14, where, ∗= {ϕ, θ}. From ε∗= 0.005 and r = 1 2, we can determine k∗1 = 2ε1−r = 2 × 0.0051−0.5 = 0.14, where, ∗= {ϕ, θ}. m εψ = 0.0025 and r = 1 2, we can determine kψ1 = 2ε1−r = 2 × 0.00251−0.5 = 0.1. From εψ = 0.0025 and r = 1 2, we can determine kψ1 = 2ε1−r = 2 × 0.00251−0.5 = 0.1. For the other parameters, to overcome the eﬀect of angular accelerations on the correctors, we select the relatively large k∗2 = 10, k∗3 = 10, where, ∗= {ϕ, θ, ψ}. 8.1.2 Corrector parameters selection: Therefore, we get the corrector parameters: Therefore, we get the corrector parameters: Therefore, we get the corrector parameters: Correctors for position: k∗1 = 0.36, k∗2 = 100, k∗3 = 100, where, ∗= {x, y, z}; Correctors for position: k∗1 = 0.36, k∗2 = 100, k∗3 = 100, where, ∗= {x, y, z}; Correctors for attitude: kϕ1 = 0.14, kθ1 = 0.14, kψ1 = 0.1; k∗2 = 10, k∗3 = 10, where, ∗= {ϕ, θ, ψ}. Correctors for position: k∗1 0.36, k∗2 100, k∗3 100, where, {x, y, z}; Correctors for attitude: kϕ1 = 0.14, kθ1 = 0.14, kψ1 = 0.1; k∗2 = 10, k∗3 = 10, where, ∗= {ϕ, θ, ψ}. 8.1.3 Rejection ratio and disturbance frequency bands 8.1.3 Rejection ratio and disturbance frequency bands 1) Rejection ratios in the disturbance frequency bands for position: 1) Rejection ratios in the disturbance frequency bands for position: From ε∗= 0.034 and r = 1 2, where, ∗= {x, y, z}, the rejection ratio is expressed by 1) Rejection ratios in the disturbance frequency bands for position: From ε∗= 0.034 and r = 1 2, where, ∗= {x, y, z}, the rejection ratio is expressed by ρ(ω∗1) = 1 √ 1 + 1 4ε2r−2 ∗ ω2 ∗1 + 1 2εr ∗= 1 √ 1 + 7.35ω2 ∗1 + 0.09 Therefore, the rejection ratios in the diﬀerent frequency bands of [0, +∞) can be described by: i) In [3.98rad/s, ∞), ρ(ω∗1) : 0.18 →0.09 as ω∗1 : 3.98 →∞(rad/s); ii) In (0.17, 3.98rad/s), ρ(ω∗1) : 1 →0.18 as ω∗1 : 0.17 →3.98(rad/s); iii) In [0, 0.17rad/s], ρ(ω∗1) : 1.09 →1 (i.e., ρ(ω∗1) ≈1) as ω∗1 : 0 →0.17(rad/s). i) In [3.98rad/s, ∞), ρ(ω∗1) : 0.18 →0.09 as ω∗1 : 3.98 →∞(rad/s); ii) In (0 17 3 98rad/s) ρ(ω 1) : 1 →0 18 as ω 1 : 0 17 →3 98(rad/s); i) In [3.98rad/s, ∞), ρ(ω∗1) : 0.18 →0.09 as ω∗1 : 3.98 →∞(rad/s); ii) In (0.17, 3.98rad/s), ρ(ω∗1) : 1 →0.18 as ω∗1 : 0.17 →3.98(rad/s); 2) Rejection ratios in the disturbance frequency bands for attitude ( ϕ, θ): From ε∗= 0.005 and r = 1 2, where, ∗= {ϕ, θ}, the rejection ratio is expressed by 2) Rejection ratios in the disturbance frequency bands for attitude ( ϕ, θ): From ε∗= 0.005 and r = 1 2, where, ∗= {ϕ, θ}, the rejection ratio is expressed by ρ(ω∗1) = 1 √ 1 + 1 4ε2r−2 ∗ ω2 1 + 1 2εr ∗= 1 √ 1 + 50ω2 ∗1 + 0.035 Therefore, the rejection ratios in the diﬀerent frequency bands can be described by: i) In [4rad/s, ∞), ρ(ω∗1) : 0.07 →0.035 as ω∗1 : 4 →∞(rad/s); ii) In (0.04, 4rad/s), ρ(ω∗1) : 1 →0.07 as ω∗1 : 0.04 →4(rad/s); i) In [4rad/s, ∞), ρ(ω∗1) : 0.07 →0.035 as ω∗1 : 4 →∞(rad/s); ii) In (0.04, 4rad/s), ρ(ω∗1) : 1 →0.07 as ω∗1 : 0.04 →4(rad/s); 31 (a) (b) AV ﬂight based on correction. (a) 3D navigation trajectories. (b) Position comparison directions. 8.2 Parameters of observers and controllers: 8.2 Parameters of observers and controllers: 8.2 Parameters of observers and controllers: According to the selection rules of observer parameters [24], we select the extended state observer parameters: λ1∗= 4, λ∗2 = 20, α∗= 0.6, where, ∗= {x, y, z, θ, ϕ, ψ}. According to the properties and tests of engine and digital servos, we select the control law parameters: kp1 = 16, kp2 = 8, ka1 = 25, ka2 = 8. 8.3 Analysis of UAV navigation and control performance: 8.1.3 Rejection ratio and disturbance frequency bands (a) (a) (a) (b) Fi 8 UAV ﬂi ht b d ti ( ) 3D i ti t j t i (b) P iti (b) (b) Figure 8: UAV ﬂight based on correction. (a) 3D navigation trajectories. (b) Position comparison in the three directions. Figure 9: Attitude correction. Figure 9: Attitude correction. 32 iii) In [0, 0.04rad/s] ρ(ω∗1) : 1.035 →1 (i.e., ρ(ω∗1) ≈1) as ω∗1 : 0 →0.04(rad/s). iii) In [0, 0.04rad/s] ρ(ω∗1) : 1.035 →1 (i.e., ρ(ω∗1) ≈1) as ω∗1 : 0 →0.04(rad/s). 3) Rejection ratios in the disturbance frequency bands for attitude ( ψ): From εψ = 0.0025 and r = 1 2, the rejection ratio is expressed by 3) Rejection ratios in the disturbance frequency bands for attitude ( ψ From εψ = 0.0025 and r = 1 2, the rejection ratio is expressed by ρ(ωψ1) = 1 √ 1 + 1 4ε2r−2 ψ ω2 ψ1 + 1 2εr ψ = 1 √ 1 + 100ω2 ψ1 + 0.025 Therefore, the rejection ratios in the diﬀerent frequency bands can be described by: i) In [4rad/s, ∞), ρ(ωψ1) : 0.05 →0.025 as ωψ1 : 4 →∞(rad/s); ii) In (0.023, 4rad/s), ρ(ωψ1) : 1 →0.05 as ωψ1 : 0.023 →4(rad/s); iii) In [0, 0.023rad/s], ρ(ωψ1) : 1.025 →1 (i.e., ρ(ωψ1) ≈1) as ωψ1 : 0 →0.023(rad/s). i) In [4rad/s, ∞), ρ(ωψ1) : 0.05 →0.025 as ωψ1 : 4 →∞(rad/s); ii) In (0.023, 4rad/s), ρ(ωψ1) : 1 →0.05 as ωψ1 : 0.023 →4(rad/s); i) In [4rad/s, ∞), ρ(ωψ1) : 0.05 →0.025 as ωψ1 : 4 →∞(rad/s); ii) I (0 023 4 d/ ) ( ) 1 0 05 0 023 4( d/ ) 8.3 Analysis of UAV navigation and control performance: Figure 8(a) shows the comparison of the ﬂight trajectories, including the measured from GPS, the reference trajectory, and the estimations by the corrector and the EKF-based method. Meanwhile, the trajectory comparisons in the three directions are shown in Figure 8(b): Due to the eﬀect of adverse conditions, e.g., engine vibration and communication, the actual measurement disturbances in position from GPS were about 10m. The estimate errors by the corrector were less than 1m, while the estimate errors by the KF were about 3m. From the estimate errors and the above numerical calculation, we can ﬁnd that the position disturbances were mainly within the frequency band [3.98rad/s, +∞). Therefore, the disturbances in position measurements were rejected suﬃciently by the correctors, and the correctors provided the relatively accurate and smoothed correction outputs. The attitude angle comparisons in the three directions are shown in Figure 9. During the UAV ﬂight, the actual measurement disturbances in attitude from the IMU were about 3◦∼4◦. The corrector estimate errors for attitude angle were less than 0.2◦. Then, comparing the above calculation, we can ﬁnd that the attitude disturbances were mainly within the frequency band [4rad/s, +∞). Therefore, the disturbances in the attitude measurements were rejected suﬃciently by the correctors due to the very small rejection ratio in this frequency band. From the ﬂight test, we can ﬁnd that the correctors also reduced the sensing disturbances from the eﬀect of UAV vibrations, and the jet UAV remained in the safe ﬂight condition throughout the ﬂight. 9 Conclusions In this paper, a sliding mode corrector has been presented, which can correct disturbance in position measurement using relatively accurate velocity. The performance of the corrector was demonstrated by two simulation examples and a jet UAV ﬂight test: i) It succeeded in rejecting the disturbances 33 largely in position and attitude sensing, even though the disturbances are in the low/mid/high frequency bands. ii) The experimental test veriﬁed the validity of the corrector’s providing accurate and smoothed estimate of position and attitude. iii) The estimate outputs from the correctors can be used directly by the control system without any additional ﬁlters. The merits of the corrector include its model free, bounded corrector gains, the accurate and smoothed estimate outputs and strong parameter inclusion to change of disturbance and signal. References [1] Panagiotou, P. & Yakinthos, K. Aerodynamic eﬃciency and performance enhancement of ﬁxed-wing UAVs. Aerosp. Sci. Technol., 2020, 99, pp 105575. [2] Footohi, P., Bouskela, A. & Shkarayev, S.V. Aerodynamic design of long-range VTOL UAV. 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Select the Lyapunov function candidate as V = 1 2 (e2 + k1e1)2 (83) V = 1 2 (e2 + k1e1)2 (83) 2 Then, if \|e2\| ≤1, taking the derivative of V , we get Then, if \|e2\| ≤1, taking the derivative of V , we get ˙V = (e2 + k1e1) {−k2sign (e2 + k1e1) + k1e2} = −k2 \|e2 + k1e1\| + k1e2 (e2 + k1e1) ≤−k2 \|e2 + k1e1\| + k1 \|e2\| \|e2 + k1e1\| ≤−(k2 −k1) \|e2 + k1e1\| = − √ 2 (k2 −k1) V 1 2 (84) = − √ 2 (k2 −k1) V 1 2 (84) (84) We know that k2 > k1 > 0. Therefore, there exists a time ts, for t ≥ts, such that V = 0, i.e., the sliding variables are on the sliding surface e2 + k1e1 = 0. Then, from the relation ˙e1 = e2, we get the following convergence law: ˙e1 = −k1e1 (85) ˙e1 = −k1e1 (85) Therefore, lim t→∞e1 = 0. Furthermore, from ˙e2 = −k2sign(e2 + k1e1), we get lim t→∞e2 = 0. This concludes the proof. ■ Proof of Theorem 4.2: Determination of e2 range For (10), when \|e2 −d2(t)\| > 1, we get Proof of Theorem 4.2: nation of e2 range Determination of e2 range f 2 g , when \|e2 −d2(t)\| > 1, we get For (10), when \|e2 −d2(t)\| > 1, we get For (10), when \|e2 −d2(t)\| > 1, we get ), when \|e2 −d2(t)\| > 1, we get ˙e2 = −k3sign(e2 −d2(t)) −d3(t) (86) ˙e2 = −k3sign(e2 −d2(t)) −d3(t) (86) (86) Then, it achieves a diﬀerential inclusion Then, it achieves a diﬀerential inclusion ˙e2 ∈−k3sign(e2 −d2(t)) + [−L3, L3] (87) (87) From Lemma 8 in [10] and k3 > L3, there exists a ﬁnite time ts, for t ≥ts, such that \|e2\| ≤L2 (88) where, supt∈[0,∞) \|d2(t)\| ≤L2. References & Lin, Y.P. Trajectory estimation based on extended state observer with Fal-ﬁlter. Aeronaut. J., 2015, 119, (1218), pp.1017-1031. [16] Panchal, B., Subramanian, K., & Talole, S.E. Robust missile autopilot design using two time- scale separation. IEEE Trans. Aerosp. Electron. 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CFD analysis of the ﬂow around the X-31 aircraft at high angle of attack, Aerosp. Sci. Technol., 2012, 20, (1), pp 38-51. 35 [29] Wang, X., Chen, Z., & Yuan, Z. Modeling and control of an agile tail-sitter aircraft, J. Frank. Inst., 2015, 352, 5437-5472. [29] Wang, X., Chen, Z., & Yuan, Z. Modeling and control of an agile tail-sitter aircraft, J. Frank. Appendix Then, there exists a time ts > 0, for t ≥ts, we get 36 (89) \|e2 −d2(t)\| ≤\|e2\| + \|d2(t)\| ≤2L2 \|e2 −d2(t)\| ≤\|e2\| + \|d2(t)\| ≤2L2 (89) \|e2 −d2(t)\| ≤\|e2\| + \|d2(t)\| ≤2L2 (89) Therefore, due to L2 ≪1, the inequality \|e2 −d2(t)\| ≤1 holds for t ≥ts. Then, for system (10), according to the 2-sliding mode system (8b) in Theorem 4.1 and k2 > k1 + L4, the sliding variables e1 and e2 are on the sliding surface e2 −d2(t) + k1(e1 −d1(t)) = 0, i.e., we get the following convergence law: ˙e1 = −k1e1 + k1d1(t) + d2(t) (90) ˙e1 = −k1e1 + k1d1(t) + d2(t) (90) E1(s) = L[e1], D1(s) = L[d1(t)] and D2(s) = L[d2(t)], we get ˙e1 = −k1e1 + k1d1(t) + d2(t) (90) Deﬁning the Laplace transforms E1(s) = L[e1], D1(s) = L[d1(t)] and D2(s) = L[d2(t)], we get (90) sE1(s) = −k1E1(s) + k1D1(s) + D2(s) (91) (91) Therefore, the error variable e1 is expressed by Therefore, the error variable e1 is expressed by E1(s) = k1 s + k1 D1(s) + 1 s + k1 D2(s) (92) (92) For the disturbance d1(t), the transfer function k1 s+k1 can be taken as a ﬁlter, the disturbance d1(t) is the input, and e1(t) is the output. The selection of k1 should try to reduce the eﬀect of d1(t) by considering the eﬀect of disturbance d2(t) from the velocity measurement. For the disturbance d1(t), the transfer function k1 s+k1 can be taken as a ﬁlter, the disturbance d1(t) is the input, and e1(t) is the output. The selection of k1 should try to reduce the eﬀect of d1(t) by considering the eﬀect of disturbance d2(t) from the velocity measurement. Suppose the disturbance d2(t) includes time varying part d21(t) and constant part d22, i.e., d2(t) = d21(t) + d22; the angular frequency variable of d21(t) is supposed to be ω2. We deﬁne supt∈[0,∞) \|d21(t)\| ≤L21 < ∞, supt∈[0,∞) \|d22(t)\| ≤L22 < ∞, and L2 = L21 + L22 ≪1. Taking Laplace transform for d2(t), we get D2(s) = D21(s) + d22 s , where, D2(s) = L[d2(t)] and D21(s) = L[d21(t)] . Then, (92) can be expressed by d2(t) d21(t) + d22; the angular frequency variable of d21(t) is supposed to be ω2. We deﬁne supt∈[0,∞) \|d21(t)\| ≤L21 < ∞, supt∈[0,∞) \|d22(t)\| ≤L22 < ∞, and L2 = L21 + L22 ≪1. Appendix Then, (95) can be rewritten by lim t→∞\|e1\| ≤ ( 1 √ 1 + ω2 1x2 + L21 L1 x √ 1 + ω2 2x2 + L22 L1 x ) L1 (96) (96) In (96), for all the ω2 ∈[0, ∞), we have In (96), for all the ω2 ∈[0, ∞), we have L21 L1 x √ 1 + ω2 2x2 ≤L21 L1 x (97) Therefore, for (96) and L2 L1 ≤ε, we get Therefore, for (96) and L2 L1 ≤ε, we get Therefore, for (96) and L2 L1 ≤ε, we get lim t→∞\|e1\| ≤ ( 1 √ 1 + ω2 1x2 + L21 + L22 L1 x ) L1 = ( 1 √ 1 + ω2 1x2 + L2 L1 x ) L1 = ( 1 √ 1 + ω2 1x2 + ε · x ) L1 (98) Deﬁne the rejection ratio as ρ(ω1, x) = 1 √ 1 + ω2 1x2 + ε · x (99) Therefore, (98) can be expressed by li \| \| ≤( )L (100) ρ(ω1, x) = 1 √ 1 + ω2 1x2 + ε · x (99) by (99) Therefore, (98) can be expressed by Therefore, (98) can be expressed by lim t→∞\|e1\| ≤ρ(ω1, x)L1 (100) In the rejection ratio (99), deﬁne ( ) 1 ( ) (101) lim t→∞\|e1\| ≤ρ(ω1, x)L1 (100) lim t→∞\|e1\| ≤ρ(ω1, x)L1 (100) In the rejection ratio (99), deﬁne ρ1(ω1, x) = 1 √ 1 + ω2 1x2 ; ρ2(ω1, x) = ε · x (101) (101) Taking the derivative for ρ1(ω1, x) and ρ2(ω1, x) about x, respectively, we get Taking the derivative for ρ1(ω1, x) and ρ2(ω1, x) about x, respectively, we get dρ1(ω1, x) dx = −ω2 1x(1 + ω2 1x2)−3 2 < 0 (102) (102) dρ2(ω1, x) dx = ε > 0 (103) (103) We know that, ρ1(ω1, x) is the monotonically decreasing function of x from ρ1(ω1, 0) = 1, and ρ2(ω1, x) is monotonically increasing function of x from ρ2(ω1, 0) = 0 (See ρ1(ω1, x) and ρ2(ω1, x) in Figure 10). Therefore, ρ1(ω1, x) and ρ2(ω1, x) will intersect at a point, and we deﬁne the point x deﬁne = x0. We know that, ρ1(ω1, x) is the monotonically decreasing function of x from ρ1(ω1, 0) = 1, and ρ2(ω1, x) is monotonically increasing function of x from ρ2(ω1, 0) = 0 (See ρ1(ω1, x) and ρ2(ω1, x) in Figure 10). Appendix Taking Laplace transform for d2(t), we get D2(s) = D21(s) + d22 s , where, D2(s) = L[d2(t)] and D21(s) = L[d21(t)] . Then, (92) can be expressed by E1(s) = k1 s + k1 D1(s) + 1 s + k1 (D21(s) + d22 s ) = k1 s + k1 D1(s) + k1 s + k1 D21(s) k1 + d22 s(s + k1) (93) (93) ess of corrector estimate error Boundness of corrector estimate error Boundness of corrector estimate error Deﬁne d1(t) = U1 sin(ω1t) and d21(t) = U21 sin(ω2t). For (93), from the frequency analysis of ﬁrst-order ﬁlter, we can get Deﬁne d1(t) = U1 sin(ω1t) and d21(t) = U21 sin(ω2t). For (93), from the frequency analysis of ﬁrst-order ﬁlter, we can get lim t→∞e1 = U1 √ 1 + ( ω1 k1 )2 sin(ω1t + ϕ1) + U21/k1 √ 1 + ( ω2 k1 )2 sin(ω2t + ϕ2) + d22 k1 (94) (94) where, ϕ1 = −tan−1 ω1 k1 and ϕ2 = −tan−1 ω2 k1 . We know that U1 ≤L1, U21 ≤L21 and \|d22\| ≤L22. Therefore, for (94), we get where, ϕ1 = −tan−1 ω1 k1 and ϕ2 = −tan−1 ω2 k1 . We know that U1 ≤L1, U21 ≤L21 and \|d22\| ≤L22. Therefore, for (94), we get lim t→∞\|e1\| ≤ L1 √ 1 + ( ω1 k1 )2 + L21/k1 √ 1 + ( ω2 k1 )2 + L22 k1 (95) (95) 37 Deﬁne x = 1 k1 . Then, (95) can be rewritten by Deﬁne x = 1 k1 . Appendix Therefore, ρ1(ω1, x) and ρ2(ω1, x) will intersect at a point, and we deﬁne the point x deﬁne = x0. In the following, we consider to determine ω1 and x to make ρ(ω1, x) equal a given rejection ratio ρ0 at the intersection point x0, i.e., 38 Figure 10: Rejection ratio. Figure 10: Rejection ratio. Figure 10: Rejection ratio. Figure 10: Rejection ratio. ρ(ω1, x0) = 1 √ 1 + ω2 1x2 0 + ε · x0 = ρ0 (104) ρ(ω1, x0) = 1 √ 1 + ω2 1x2 0 + ε · x0 = ρ0 (104) and ρ1(ω1, x0) = ρ2(ω1, x0) = ρ0 2 ρ1(ω1, x0) = ρ2(ω1, x0) = ρ0 2 (105) holds at the the intersection point x0. From (104) and (105), we have holds at the the intersection point x0. From (104) and (105), we have 1 √ 1 + ω2 1x2 0 = ρ0 2 ε · x0 = ρ0 2 (106) Solving the equations in (106), we get 1 √ 1 + ω2 1x2 0 = ρ0 2 ε · x0 = ρ0 2 1 √ 1 + ω2 1x2 0 = ρ0 2 ε · x0 = ρ0 2 (106) 1 √ 1 + ω2 1x2 0 = ρ0 2 ε · x0 = ρ0 2 (106) Solving the equations in (106), we get Solving the equations in (106), we get Solving the equations in (106), we get x0 = ρ0 2ε ω1 = 2ε ρ2 0 √( 4 −ρ2 0 ) deﬁne = ω0 (107) ω1 = 2ε ρ2 0 √( 4 −ρ2 0 ) deﬁne = ω0 (107) (107) The rejection ratio ρ0 at intersection point should satisfy ρ0 ∈(0, 1). Moreover, due to 0 < ε ≪1, the selection of ρ0 should make ω0 bounded. Due to 2 √( 4 −ρ2 0 ) > 1 is bounded, we can select ρ0 to make ε ρ2 0 ≤1. Therefore, ρ0 ≥ε 1 2 and ρ0 ∈(0, 1) need to hold. We select ρ0 = εr ∈(0, 1) (where, r ∈ ( 0, 1 2 ] ) to satisfy the above conditions. Appendix Then, at the intersection point, x0 and ω1 in (107) can be expressed respectively by x0 = 1 2εr−1 ω1 = 4ε1−2r √ 1 −1 4ε2r deﬁne = ω0 (108) ω1 = 4ε1−2r √ 1 −1 4ε2r deﬁne = ω0 (108) (108) where, r ∈ ( 0, 1 2 ] . For ρ(ω1, x), when x = x0 and ω1 ≥ω0, we get where, r ∈ ( 0, 1 2 ] . For ρ(ω1, x), when x = x0 and ω1 ≥ω0, we get ρ(ω1, x) = 1 √ 1 + ω2 1x2 0 + ε · x0 39 ≤ 1 √ 1 + ω2 0x2 0 + ε · x0 = ρ0 (109) (109) Therefore, the error variable e1 is in the bound: Therefore, the error variable e1 is in the bound: Therefore, the error variable e1 is in the bound: lim t→∞\|e1\| ≤ρ0L1 (110) (110) lim t→∞\|e1\| ≤ρ0L1 Due to 0 < ε ≪1 and r ∈ ( 0, 1 2 ] , we can get ρ0 = εr ≪1. Also, the frequency ω0 = 4ε1−2r √ 1 −1 4ε2r can be small enough. Thus, the disturbance d1(t) is rejected suﬃciently in the frequency band [ω0, ∞). Due to 0 < ε ≪1 and r ∈ ( 0, 1 2 ] , we can get ρ0 = εr ≪1. Also, the frequency ω0 = 4ε1−2r √ 1 −1 4ε2r can be small enough. Thus, the disturbance d1(t) is rejected suﬃciently in the frequency band [ω0, ∞). Determination of corrector parameter k1 Determination of corrector parameter k1 Determination of corrector parameter k1 Because k1 = 1 x0 , the parameter k1 is selected as Because k1 = 1 x0 , the parameter k1 is selected as k1 = 1 x0 = 2ε1−r (111) (111) Rejection ratio for frequency band ω1 ∈[0, ∞) Rejection ratio for frequency band ω1 ∈[0, ∞) Rejection ratio for frequency band ω1 ∈[0, ∞) When the corrector parameter k1 = 1 x0 = 2ε1−r is selected, i.e., x = x0 = 1 2εr−1, the rejection ratio (99) is described by: When the corrector parameter k1 = 1 x0 = 2ε1−r is selected, i.e., x = x0 = 1 2εr−1, the rejection ratio (99) is described by: ρ(ω1) deﬁne = ρ(ω1, x0)\|x=x0 = 1 √ 1 + 1 4ε2r−2ω2 1 + 1 2εr (112) (112) We ﬁnd that the rejection ratio ρ(ω1) in (112) is a monotonically decreasing function of distur- bance frequency ω1 ∈[0, ∞), and it satisﬁes: i) When ω1 = ω0 = 4ε1−2r √ 1 −1 4ε2r, we have ρ(ω0) = ρ0 = εr; and ρ(ω1) →1 2εr as ω1 →∞. Therefore, in the frequency band [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞. i) When ω1 = ω0 = 4ε1−2r √ 1 −1 4ε2r, we have ρ(ω0) = ρ0 = εr; and ρ(ω1) →1 2εr as ω1 →∞. Therefore, in the frequency band [ω0, ∞), ρ(ω1) : εr →1 2εr as ω1 : ω0 →∞. ii) When ω1 = ωc = c1−1 2 r√4−εr 1−1 2εr , we have ρ(ωc) = 1. Therefore, in the frequency band (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0. ii) When ω1 = ωc = c1−1 2 r√4−εr 1−1 2εr , we have ρ(ωc) = 1. Therefore, in the frequency band (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0. ii) When ω1 = ωc = c1−1 2 r√4−εr 1−1 2εr , we have ρ(ωc) = 1. Therefore, in the frequency band (ωc, ω0), ρ(ω1) : 1 →εr as ω1 : ωc →ω0. iii) When ω1 = 0, we have ρ(0) = 1 + 1 2εr; and ρ(0) ≈1 due to 0 < ε ≪1 and r ∈ ( 0, 1 2 ] . Therefore, in the frequency band [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (or ρ(ω1) ≈1) as ω1 : 0 →ωc. Determination of corrector parameter k1 iii) When ω1 = 0, we have ρ(0) = 1 + 1 2εr; and ρ(0) ≈1 due to 0 < ε ≪1 and r ∈ ( 0, 1 2 ] . Therefore, in the frequency band [0, ωc], ρ(ω1) : 1 + 1 2εr →1 (or ρ(ω1) ≈1) as ω1 : 0 →ωc. We know that ωc = ε1−1 2 r√4−εr 1−1 2 εr < ε1−1 2 r√ 4 1−1 2 = 4ε1−1 2 r ≪1 because of 0 < ε ≪1 and r ∈ ( 0, 1 2 ] . Therefore, the frequency band [0, ωc] is suﬃciently small. In general, the disturbance d1(t) in position measurement can be rejected suﬃciently by the corrector even the the disturbance frequency covers the low/mid/high frequency bands. This concludes the proof. ■ Proof of Theorem 4.3: We know that the rejection ratio (99) is a monotonically decreasing function of frequency ω1. Therefore, in the given frequency band [ωreq, ∞), the rejection ratio satisﬁes 40 Figure 11: ρ(ω1, x) curve and its minimum value. Figure 11: ρ(ω1, x) curve and its minimum value. ρ(ω1, x) = 1 √ 1 + ω2 1x2 + ε · x ≤ 1 √ 1 + ω2reqx2 + ε · x = ρ(ωreq, x) (113) (113) and lim t→∞\|e1\| ≤ρ(ωreq, x)L1 (114) In the following, we will determine x to get min {ρ(ωreq, x)}. Taking the derivatives for ρ(ωreq, x) about x, we get lim t→∞\|e1\| ≤ρ(ωreq, x)L1 (114) (114) In the following, we will determine x to get min {ρ(ωreq, x)}. Taking the derivatives for ρ(ωreq, x) about x, we get In the following, we will determine x to get min {ρ(ωreq, x)}. Taking the derivatives for ρ(ωreq, x) about x, we get dρ(ωreq, x) dx = −ω2 reqx(1 + ω2 reqx2)−3 2 + ε (115) d2ρ(ωreq, x) dx2 = 2ω4 req(1 + ω2 reqx2)−5 2 [ x2 − 1 2ω2req ] (116) dρ(ωreq, x) dx = −ω2 reqx(1 + ω2 reqx2)−3 2 + ε (115) (115) d2ρ(ωreq, x) dx2 = 2ω4 req(1 + ω2 reqx2)−5 2 [ x2 − 1 2ω2req ] (116) (116) According to (115) and (116), ρ(ωreq, x) is the convex function about x when x2 < 1 2ω2req , and ρ(ωreq, x) is the concave function about x when x2 > 1 2ω2req . In order to get the minimum value of ρ(ωreq, x), the selection of x should make ρ(ωreq, x) about x be concave function, i.e., d2ρ(ωreq,x) dx2 > 0 holds. Therefore, from (116), the following inequality should be satisﬁed: x2 > 1 2ω2req (117) (117) Then, it follows that Then, it follows that x > 1 √ 2ωreq deﬁne = xinf (118) (118) Therefore, ρ(ωreq, x) is the concave function in the range x ∈(xinf, ∞), and xinf is the curve inﬂection point (See Figure 11). From the concave property of ρ(ωreq, x) in the range x ∈(xinf, ∞) and 0 < ε ≪1, the minimum value ρmin of ρ(ωreq, x) exists when dρ(ωreq,x) dx = 0, i.e., dρ(ωreq, x) dx = −ω2 reqx(1 + ω2 reqx2)−3 2 + ε = 0 (119) (119) Deﬁne the solution to (119) in x ∈(xinf, ∞) is xmin. Proof of Theorem 4.3: Then, the minimum value ρmin of ρ(ωreq, x) can be expressed by Deﬁne the solution to (119) in x ∈(xinf, ∞) is xmin. Then, the minimum value ρmin of ρ(ωreq, x) can be expressed by 41 ρmin deﬁne = min {ρ(ωreq, x)} = 1 √ 1 + ω2reqx2 min + ε · xmin (120) (120) It means that, when we select the corrector parameter k1 = 1/xmin, and xmin is the solution to (119) in the range x ∈(xinf, ∞), the rejection ratio can be expressed by It means that, when we select the corrector parameter k1 = 1/xmin, and xmin is the solution to (119) in the range x ∈(xinf, ∞), the rejection ratio can be expressed by ρ(ω1) deﬁne = ρ(ω1, x)\|x=xmin = 1 √ 1 + x2 minω2 1 + ε · xmin (121) (121) The rejection ratio ρ(ω1) is a monotonically decreasing function of disturbance frequency ω1, and it satisﬁes: The rejection ratio ρ(ω1) is a monotonically decreasing function of disturbance frequency ω1, and it satisﬁes: i) In [ωreq, ∞), ρ(ω1) : ρmin →ε · xmin as ω1 : ωreq →∞. ii) In (ωc, ωreq), ρ(ω1) : 1 →ρmin as ω1 : ωc →ωreq, where, ωc = √ 2ε xmin √ 1−1 2 εxmin 1−ε·xmin . iii) In [0, ωc], ρ(ω1) : 1 + ε · xmin →1 as ω1 : 0 →ωc. Therefore, the disturbance d1 (t) in the frequency band [ωreq, ∞) is rejected suﬃciently. This concludes the proof. ■ Proof of Theorem 5.1: Deﬁne e1 = x1 −p0(t), and e2 = x2 −v0(t). Then, the system error can be expressed by Proof of Theorem 5.1: Deﬁne e1 = x1 −p0(t), and e2 = x2 −v0(t). Then, the system error can be expressed by Deﬁne e1 = x1 −p0(t), and e2 = x2 −v0(t). Proof of Theorem 4.3: Then, the system error can be expressed by ˙e1 = e2 ˙e2 =      −k3sign [e2 −d2(t)] −˙v0(t), if \|e2 −d2(t)\| > 1; −k2sign [e2 −d2(t) + k1(e1 −d1(t))] −˙v0(t), if \|e2 −d2(t)\| ≤1 (122) (122) Deﬁne d3(t) = d4(t) = ˙v0(t), the system error (122) is rewritten as ˙e1 = e2 ˙e2 =      −k3sign(e2 −d2(t)) −d3(t), if \|e2 −d2(t)\| > 1; −k2sign [e2 −d2(t) + k1(e1 −d1(t))] −d4(t), if \|e2 −d2(t)\| ≤1 (123) (123) According to Theorem 4.2, we can get the bounds of the estimate errors (18) and the other conclusions. This concludes the proof. ■ 42
https://openalex.org/W4311477349	https://www.actabiomedica.ru/jour/article/download/3804/2410	Russian	null	Mechanisms of autoimmune pathology in post-COVID syndrome	Acta biomedica scientifica	2,022	cc-by	13,721	Автор, ответственный за переписку: Орлова Елизавета Андреевна, e-mail: elizaveta.a.orlova@gmail.com Ключевые слова: COVID-19, SARS-CoV-2, постковидный синдром, аутоим мунные заболевания, молекулярная мимикрия, SOX13, белок E Статья получена: 20.07.2022 Статья принята: 03.10.2022 Статья опубликована: 08.12.2022 Для цитирования: Орлова Е.А., Кондратов И.Г., Огарков О.Б., Колесникова Л.И. Меха низмы аутоиммунной патологии постковидного синдрома. Acta biomedica scientifica. 2022; 7(5-1): 62-76. doi: 10.29413/ABS.2022-7.5-1.8 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 РЕЗЮМЕ Орлова Е.А., Кондратов И.Г., Огарков О.Б., Колесникова Л.И. Орлова Е.А., Кондратов И.Г., Огарков О.Б., Колесникова Л.И. Одним из отсроченных последствий инфекции SARS-CoV-2 является постко- видный синдром - развивающиеся у реконвалесцентов COVID-19 полиморф ные нарушения различных систем организма, которые сохраняются более четырёх недель после перенесённой острой инфекции. В силу инфекционной природы заболевания отдельного внимания требуют осложнения со сторо ны иммунной системы, особенно сопутствующая и впервые возникающая аутоиммунная патология. В этом обзоре проводится анализ современного состояния вопроса постковидных осложнений, обсуждаются молекулярные особенности вируса SARS-CoV-2 и механизмы, лежащие в основе нарушений иммунного ответа в период острой инфекции COVID-19 и возникновения аутоиммунных и аутовоспалительных состояний при реконвалесценции. Особое внимание уделено молекулярной мимикрии антигенных детерми нант вируса SARS-CoV-2, структурно сходных с эпитопами аутоантигенов человека. Приведены современные данные о постковидных аутоиммунных осложнениях со стороны гуморального иммунитета и эндокринной систе мы, а также репродуктивных нарушений, с которыми сталкиваются паци енты мужского пола. Впервые высказывается гипотеза о роли структурной гомологии аутоантигена SOX13 человека (HMG box factor SOX13), ассоции рованного с сахарным диабетом, и белка Е оболочки SARS-CoV-2 в развитии постковидных аутоиммунных патологий. В силу структурного сходства двух белков и перекрывания их иммуногенных регионов мы предполагаем, что повышенный риск развития сахарного диабета и нарушений со стороны репродуктивной системы у мужчин после перенесённого COVID-19 могут быть связаны с перекрёстной иммунологической реактивностью. ФГБНУ «Научный центр проблем здоровья семьи и репродукции человека» (664003, г. Иркутск, ул. Тимирязева, 16, Россия) ФГБНУ «Научный центр проблем здоровья семьи и репродукции человека» (664003, г. Иркутск, ул. Тимирязева, 16, Россия) Ключевые слова: COVID-19, SARS-CoV-2, постковидный синдром, аутоим мунные заболевания, молекулярная мимикрия, SOX13, белок E Key words: COVID-19, SARS-CoV-2, post-acute COVID-19 syndrome, autoimmune diseases, molecular mimicry, SOX13, Eprotein Received: 20.07.2022 Accepted: 03.10.2022 PubNshed: 08.12.2022 мунные заболевания, молекулярная мимикрия, SOX13, белок E Статья получена: 20.07.2022 Статья принята: 03.10.2022 Статья опубликована: 08.12.2022 Для цитирования: Орлова Е.А., Кондратов И.Г., Огарков О.Б., Колесникова Л.И. Меха низмы аутоиммунной патологии постковидного синдрома. Acta biomedica scientifica. 2022; 7(5-1): 62-76. doi: 10.29413/ABS.2022-7.5-1.8 Infectious diseases 62 Инфекционные болезни 62 Инфекционные болезни Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 For citation: Orlova E.A., Kondratov I.G., Ogarkov O.B., Kolesnikova L.I. Mechanisms of autoimmune pathology in post-covid syndrome. Acta biomedica scientifica. 2022; 7(5-1): 62-76. doi: 10.29413/ABS.2022-7.5-1.8 ВВЕДЕНИЕ Постковидный синдром (англ. long COVID-19 или post-acute COVID-19) - это состояние, характеризу ющееся сохранением клинических симптомов, связан ных с инфекцией SARS-CoV-2, по крайней мере, спустя четыре недели после перенесённой острой инфекции. Затрагиваются дыхательная, нервная, сердечно-сосуди стая, эндокринная системы, возникают нарушения обо няния и вкуса и другие клинические симптомы. Перво начально постковидный синдром чаще фиксировался у пациентов, госпитализированных с тяжёлой формой заболевания, однако долгосрочные осложнения затро нули также амбулаторных и бессимптомных пациентов [1]. Имеющиеся заболевания лёгких, пожилой возраст, ожирение [2] и женский пол [3] считаются факторами по вышенного риска развития постковидных осложнений. Распространёнными признаками, наблюдаемыми вплоть до года после COVID-19, являются утомляемость, одыш ка, стойкая потеря обоняния или вкуса, миалгия, голов ная боль, кашель, диарея, боль в груди, депрессия, из менение состава респираторного микробиома и общее ухудшение качества жизни [4, 5]. ABSTRACT Orlova E.A., Kondratov I.G., Ogarkov O.B., Kolesnikova L.I. One of the delayed consequences of SARS-CoV-2 infection is post-acute COVID-19 - polymorphic disorders of various organ systems that affect COVID-19 convalescents and persist for more than four weeks after an acute infection. Due to the infectious nature of the COVID-19, we would like to pay special attention to complications from the immune system, especially concomitant and new-onset autoimmune pathology. This review analyzes the current state of the issue of post-acute COVID-19 complications, discusses the molecular features of the SARS-CoV-2 vi rus and the mechanisms underlying the impaired immune response during acute COVID-19 infection and the occurrence of autoimmune and autoinflammatory con ditions during convalescence. Particular attention is paid to the molecular mimicry of antigenic determinants of the SARS-CoV-2 virus, which are structurally similar to the epitopes of human autoantigens. The current data on post-acute COVID-19 autoimmune complications from humoral immunity and the endocrine system, as well as reproductive disorders faced by male patients are presented. For the first time, we hypothesize a role of the structural homology of the human SOX13 autoan tigen (HMG box factor SOX13) associated with diabetes mellitus and SARS-CoV-2 envelope (E) protein in the development of the post-acute COVID-19 autoimmune pathologies. Due to the structural similarity of the two proteins and the overlap of their immunogenic regions, we suggest that the increased risk of developing diabetes mellitus and reproductive disorders in men after suffering from COVID-19 may be associated with immunological cross-reactivity. Scientific Centre for Family Health and Human Reproduction Problems (Timiryazeva str. 16, Irkutsk 664003, Russian Federation) Corresponding author: Elizaveta A. Orlova, e-mail: elizaveta.a.orlova@gmail.com Corresponding author: Elizaveta A. Orlova, e-mail: elizaveta.a.orlova@gmail.com Key words: COVID-19, SARS-CoV-2, post-acute COVID-19 syndrome, autoimmune diseases, molecular mimicry, SOX13, Eprotein For citation: Orlova E.A., Kondratov I.G., Ogarkov O.B., Kolesnikova L.I. Mechanisms of autoimmune pathology in post-covid syndrome. Acta biomedica scientifica. 2022; 7(5-1): 62-76. doi: 10.29413/ABS.2022-7.5-1.8 63 Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 ности [12]. Они способствуют уклонению вируса от им мунной системы и манипулированию иммунными реак циями, включая задержку интерферон-опосредованной защиты и производства вируснейтрализующих антител и, напротив, ответственны за стимулирование клеточно го механизма репликации вируса. Таким образом повы шается вирусная нагрузка [13] и способность вируса вы зывать неадекватные иммунные реакции, такие как гипе рактивация врождённого иммунитета. Всё это приводит к отягощению течения COVID-19 и может стать причиной развития острого респираторного дистресс-синдрома (ОРДС), гиперкоагуляционного синдрома, гипоперфузии органов, септического шока, полиорганной недостаточ ности и в итоге привести к смерти, а у выживших пациен тов - к долгосрочным постковидным осложнениям [14]. ПОЧЕМ У У РЕКОНВАЛЕСЦЕНТОВ COVID-19 м о ж е т в о з н и к а т ь а у т о и м м у н н а я п а т о л о г и я ? Часто сообщалось о быстром исчезновении специ фических нейтрализующих антител против SARS-CoV-2 у выживших пациентов с COVID-19 [15] или их отсутствии у бессимптомно переболевших пациентов [16], а иссле дование посмертных образцов лимфоидной ткани паци ентов, умерших от тяжёлого течения инфекции, показа ло значительное её поражение [17], атрофию или пол ное отсутствие зародышевых центров, истощение спец ифических подмножеств фолликулярных Т-хелперов (Tfh-клетки) и В-клеток, экспрессирующих белок Bcl6 [18]. Tfh-клетки и Bcl6+ B-клетки участвуют в образова нии антител, а наличие зародышевых центров во вторич ных лимфоидных тканях, в свою очередь, необходимо для переключения их изотипа и отбора высокоаффин ных В-клеток с длительной продолжительностью жиз ни. Любопытно, что описанные у пациентов с тяжёлым течением COVID-19 изменения происходят не локально в месте инфильтрации, а системно затрагивают всю лим фоидную ткань, что указывает на влияние на организм некоторых циркулирующих факторов, препятствующих нормальной дифференцировке клеток. Прогрессирую щая лимфоцитопения коррелирует с неконтролируе мым высвобождением провоспалительных цитокинов и хемокинов, в частности с увеличением IL-6, IL-8 [19], TNF-a [18]. Вероятно, избыточный синтез провоспали тельных цитокинов в месте дифференцировки клеток может способствовать нарушению образования заро дышевых центров и снижению аффинности и устойчи вости антител против SARS-CoV-2 при COVID-19. Фундаментальной характеристикой иммунной си стемы является её аутотолерантность - способность от личать собственные молекулярные структуры от чуже родных и оставаться ареактивной к ним. Определённые триггеры могут нарушать функционирование сдержива ющих механизмов и обращать деструктивный потенциал иммунной системы против собственных антигенов. По следствием этого является иммуноопосредованное по вреждение тканей и органов, приводящее к системному воспалению и развитию аутовоспалительных (затраги вающих в основном механизмы врождённого иммуни тета) или аутоиммунных (основанных в первую очередь на нарушении адаптивного иммунитета) заболеваний. Потеря толерантности к определённым аутоантигенам может происходить за счёт реализации разных молеку лярных и клеточных механизмов: повреждение физиче ских гистогематических барьеров; нарушение работы регуляторных иммунных клеток, продуцирующих про тивовоспалительные цитокины (IL-4, IL-10 и трансформи рующий фактор роста бета (TGF-P)); активация ауторе активных T- и B-лимфоцитов при попадании в организм перекрёстно-реагирующих микробных антигенов (моле кулярная мимикрия); переключение на новые эпитопы в ходе реализации противоинфекционного иммунного ответа; появление неоантигенов. Распространённым эк зогенным фактором, способным запускать аутоиммуни тет, являются вирусные патогены. Определённые типы вирусов могут вызывать активацию аутореактивных Т и В-лимфоцитов, способствуя продукции аутоантител и избыточного количества провоспалительных цитоки нов. Например, наличие вируса Эпштейна - Барр и пар- вовируса B19 коррелирует с тиреоидитом Хашимото [22], Т-лимфотропного вируса человека и пенистого вируса человека - с болезнью Грейвса [23], а вируса простого герпеса - с аутоиммунным энцефалитом [24]. ПРЕДПОСЫЛКИ ИММУННОЙ ПАТОЛОГИИ ПРИ COVID-19 щим истощением Т-клеток, а также парадоксальное раз витие иммунных реакций - замедленный интерфероно- вый ответ (IFN типа I и III), сопровождаемый усиленной продукцией цитокинов (рис. 1). За подавление раннего интерферонового ответа отвечают вирусные белки ORF3b и SARS-CoV-2-PLpro. ORF3b - неструктурный белок, отличается от ортологов других коронавирусов длиной из-за возникшего у SARS- CoV-2 стоп-кодона, что делает его более мощным ан тагонистом IFN I типа [10]. Папаиноподобная протеаза SARS-CoV-2-PLpro отщепляет убиквитин-подобный бе лок ISG15 от IRF3 (регуляторный фактор интерферона 3), таким образом ослабляя транскрипцию IFN I типа [9]. ПРЕДПОСЫЛКИ ИММУННОЙ ПАТОЛОГИИ ПРИ COVID-19 Ответом организма на тяжёлое инфекционное или неинфекционное поражение является синдром си стемного воспалительного ответа, за которым следует длительный компенсаторный противовоспалительный каскад - глобальная дезактивация иммунной системы, задачей которой является восстановление гомеоста за. Инфекция SARS-CoV-2 может привести к нарушению тонкого баланса между активацией иммунитета и имму носупрессией, поскольку вирус использует своеобраз ную стратегию уклонения от врождённого иммунитета. У пациентов с тяжёлым течением COVID-19 наблюдает ся неадекватный иммунный профиль - увеличение ко личества клеток врождённого иммунитета с сопутствую- Это состояние имеет сходство с постострыми вирус ными синдромами, описанными у пациентов, перенёс ших инфекции SARS и MERS [6], однако имеет уникаль ные черты патогенеза. Особенности SARS-CoV-2, такие как сайт фуринового расщепления S-белка [7] и дина мические конформационные изменения в его структу ре при слиянии с клеткой хозяина [8], папаиноподобная протеаза (SCoV2-PLpro) [9], ORF3b [10] и некоторые не структурные белки [11], уникальны для нового коронави- руса и являются факторами его повышенной вирулент immune cell supression SARS-CoV -2 В cells-Bcl6 follicular helper (V h) ce lls Cytokine storm ,:ч! C'rtr* S ' : • l/li Vkvi rrpitCMUon TLR3. TLR7 OU SA R S- Co v-2 proteins Pro-inflammatory Cytokine (e g- II -6 0RF3o, SCoV2-PLpro. Nspt/10/16 and iL 8 n r м Cleaves ISC 15 from IRF3 and inhibits production of Type 1 and Type III IFN response X Antibody response IRf-3 IF N s production (Тур* I end ІІО Lung epithelial cell РИС. 1. FIG. 1. Нарушение иммунного ответа, вызванное SARS-CoV-2 [12] SARS-CoV-2 manipulates host immune response [12] FIG. 1. SARS-CoV-2 manipulates host immune response [12] FIG. 1. SARS-CoV-2 manipulates host immune response [12] Нарушение иммунного ответа, вызванное SARS-CoV-2 [12] 64 Инфекционные болезни Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 лительных цитокинов и поддержанию вызванного раз ными компонентами SARS-CoV-2 «порочного круга». Вли яние вируса на молекулярные пути, регулируемые MAPK, CDK, GFR, может иметь потенциальные последствия у па циентов в постковидном периоде. Поскольку SARS-CoV-2 может инфицировать широкий спектр тканей человека, долговременным осложнением COVID-19 может оказать ся ингибирование пролиферации клеток в тканях с высо кой скоростью митоза, таких как репродуктивные и эн докринные органы, эпителий слизистой оболочки и со судов, а также нейрогенные области мозга [12]. щим истощением Т-клеток, а также парадоксальное раз витие иммунных реакций - замедленный интерфероно- вый ответ (IFN типа I и III), сопровождаемый усиленной продукцией цитокинов (рис. 1). ПОЧЕМ У У РЕКОНВАЛЕСЦЕНТОВ COVID-19 м о ж е т в о з н и к а т ь а у т о и м м у н н а я п а т о л о г и я ? Известно, что неспец ифическая активация Т-клеток при вирусных инфекциях (вирус Эпштейна - Барр, вирусы Коксаки, ротавирусы, эн теровирусы, вирус гриппа А) связана с развитием таких аутоиммунных заболеваний как ревматоидный артрит, сахарный диабет (СД) 1-го типа, рассеянный склероз [27]. F. Angileri и соавт. (2020) обратили внимание на три белка человека, имеющих гомологию с вирусными бел ками, и, возможно, связанными с аносмией, лейкопе нией и поражением сосудов при COVID-19. Это OR7D4 (одорантный рецептор 7D4) - один из наиболее важ ных рецепторов на плазматической мембране обоня тельных сенсорных нейронов, PARP9 (представитель группы поли(АДФ-рибоза)-полимераз), участвующий в дифференцировке и функционировании B-клеток и макрофагов, и SLC12A6 - котранспортёр хлорида калия в эндотелиальных клетках сосудов. G. Lucchese и A. Floel (2020) сообщили ещё о трёх белках комплек са пре-Бётцингера (DAB1, SURF1 и AIFM), экспрессиру емых в нейронах дыхательного центра ствола головно го мозга, которые, как показывает анализ in silico, име ют общие потенциально антигенные эпитопы с белками SARS-CoV-2 (N и S). Эти три последовательности отсут ствуют у других коронавирусов человека (HKU1 и OC43), которые могут вызывать повреждение лёгких, но обыч но не связаны с дыхательной недостаточностью. Авторы отмечают, что все три изученных белка могут участво вать в дисфункции комплекса пре-Бётцингера, и пред полагают, что иммунная агрессия в отношении DAB1, AIFM1 и SURF1 может способствовать нейрогенной ды хательной недостаточности у пациентов с COVID-19. V. Vasilevska и соавт. (2021) в систематическом обзоре сообщают о связи между инфекцией SARS-CoV-2 и вто ричным возникновением анти-NMDAR-энцефалита. В ка честве основной причины аутоиммунного ответа про тив рецепторов NMDA головного мозга авторы также предполагают мимикрию неструктурных белков NSP8 и NSP9 вируса с эпитопами субъединиц GluN1 и GluN2a рецептора NMDA (N-метил^-аспарагиновой кислоты). Анти-NMDAR-энцефалит, опосредованный антитела ми к субъединице GluN1, является распространённой формой аутоиммунного энцефалита, характеризующей ся проявлением неврологических и психозоподобных симптомов [29]. Патогенез заболевания заключается в интернализации перекрёстно связанных на мембра не NMDAR-IgG, вследствие чего рецепторы становятся р ( ) р р [ ] Современные данные подтверждают также идею о том, что аутоиммунные осложнения могут быть вызва ны инфекцией SARS-CoV-2. В 2020 г. M. Gregorova и соавт. описали клинический случай пациента с COVID-19, у кото рого развилась рецидивирующая вентиляторно-ассоци ированная пневмония, вызванная штаммом Pseudomonas aeruginosa, а постковидные симптомы в виде одышки и усталости не проходили спустя 8 месяцев после выздо ровления. В данном случае SARS-CoV-2-индуцированная гиперактивация иммунной системы дополнительно сти мулировалась рецидивирующей инфекцией P. aeruginosa, что привело к «сторонней» активации Т-клеток, специ фичных к антигенам, не связанным ни с одной из пере несённых инфекций. ПОЧЕМ У У РЕКОНВАЛЕСЦЕНТОВ COVID-19 м о ж е т в о з н и к а т ь а у т о и м м у н н а я п а т о л о г и я ? Авторы предполагают, что высокий уровень циркулирующих неспецифичных Т-клеток мог сыграть решающую роль в тяжести заболевания и раз витии постковидного синдрома у этого пациента. С другой стороны, некоторые клинические симпто мы COVID-19 и постковидного синдрома, включая артрал- гии, миалгии, утомляемость, сыпь, часто обнаруживают ся при системных аутоиммунных заболеваниях. Рентгено логические особенности поражения лёгких у пациентов с COVID-19 - затемнения по типу матового стекла, - имеют сходство с пневмонией при аутоиммунных заболевани ях (например, ревматоидном артрите, системном склеро зе, эозинофильном гранулёматозе с полиангиитом) и ау товоспалительных заболеваниях (например, системном ювенильном идиопатическом артрите). Патогенез «цито- кинового шторма», развивающийся при COVID-19, анало гичен таковому при вторичном гемофагоцитарном лим- фогистиоцитозе (вГЛГ) - гипервоспалительном состоянии, ассоциированном как с аутоиммунными и аутовоспали тельными заболеваниями, так и с COVID-19. ПОЧЕМ У У РЕКОНВАЛЕСЦЕНТОВ COVID-19 м о ж е т в о з н и к а т ь а у т о и м м у н н а я п а т о л о г и я ? Помимо этого, вирус SARS-CoV-2 напрямую манипу лирует некоторыми молекулярными путями клетки-хо зяина для увеличения уровня репликации своего гено ма, что влечёт за собой тяжёлые последствия со стороны иммунной системы и других инфицируемых тканей. Про- теомное исследование выявило резкое изменение про филя фосфорилирования белков клетки при заражении: инфекция SARS-CoV-2 способствует активации рецепто ра фактора роста (GFR) [20], казеинкиназы II (CK2) и ми- тоген-активируемой протеинкиназы p38/MAPK и, напро тив, дезактивации циклинзависимых киназ CDK1, CDK2, CDK5 [21]. Такое изменение клеточной сигнализации приводит к аресту клеточного цикла в фазе S/G2 - бога тому на белки репарации и репликации и нуклеотиды и поэтому столь благоприятному для усиленной репли кации вируса. Активация p38/MAPK дополнительно ин дуцирует выработку потенциально вредных провоспа Вирусное инфицирование нарушает транскрипцион ные и посттранскрипционные механизмы и может при водить к неконтролируемому синтезу и высвобождению хемокинов и медиаторов воспаления, которое форми рует состояние, названное синдромом «цитокинового шторма». Высокие уровни различных цитокинов, таких как IL-6, INF-y и TNF, у генетически предрасположенных 65 Инфекционные болезни Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 цированного запуска аутоиммунных заболеваний. Моле кулярная мимикрия позволяет эпитопам вирусных бел ков «копировать» антигенные детерминанты организма- хозяина, что приводит к перекрёстной реакции и может вызвать потерю толерантности и развитие аутоиммуни тета. Хорошо известно, что предикторами тяжёлого те чения COVID-19 являются гипертония и сахарный диа бет, при которых наблюдается хронический стресс эн дотелиальных клеток и аномальная экспрессия на их по верхности внутриклеточных белков, например, пост трансляционно модифицированных шаперонов [28]. F. Cappello (2020), по-видимому, первым предположил, что противовирусные антитела могут перекрёстно ре агировать с эпитопами собственных белков на поверх ности плазматической мембраны эндотелиальных кле ток, подвергшихся стрессу. В свою очередь, аутоиммун ные реакции против эндотелия сосудов могут вызывать тромбоз, синдром диссеминированного внутрисосуди стого свёртывания и полиорганную недостаточность. лиц приводят к гиперактивации врождённых и адаптив ных иммунных механизмов. Попадая в воспалительный очаг с высокой концентрацией провоспалительных мо лекул, лимфоциты могут выходить из состояния анергии и активироваться даже в отсутствие специфического ан тигена - происходит «сторонняя» (bystander) активация Т-лимфоцитов [25]. Очевидно, её физиологический смысл состоит в том, что антиген-независимые Т-клетки могут выступать звеном врождённого иммунитета, секретируя IFN-y [26]; однако, аутореактивные лимфоциты, активи рованные таким образом, провоцируют аутоиммунные заболевания. Мощными стимуляторами «сторонней» активации Т-лимфоцитов являются интерлейкины IL-1, IL-2, IL-12, IL-18, IL-23, IL-27, IL-33 и передача сигналов че рез Toll-подобные рецепторы [26]. М ОЛЕКУЛЯРНАЯ МИМИКРИЯ С другой стороны, повреждение микрососу дов, вызванное системным воспалительным ответом на инфекцию SARS-CoV-2, и их повышенная проницае мость, могут вызывать нарушение гематоэнцефаличе ского барьера и облегчать перенос NMDAR-реактивных и других аутоантител в ЦНС. ру у С помощью алгоритма HMI-PRED (структурное про гнозирование взаимодействия между хозяином и пато геном на основе пространственной мимикрии) H. Yapici- Eser и соавт. (2021) было предсказано множество челове ческих белков-канидатов, под которые могут мимикри ровать белки SARS-CoV-2. Это белки, связанные с нейро трансмиссией, возбудимостью нейронов, эндоцитозом, праймированием синаптических пузырьков, основные факторы роста (FGF1, FGF2 и FGF4, VEGF2, GDNF, EGF, GLP-1, инсулиноподобный фактор роста, рецептор ин сулина и эстрогена, цилиарный нейротрофический фак тор), белки, связанные с митохондриальным транспор том, инсулин и многие другие. Безусловно, необходи мы дальнейшие исследования для обсуждения реаль ности угрозы аутоагрессии против указанных белков- кандидатов, поскольку структурная гомология является необходимым, но недостаточным условием, и необхо димо учитывать присутствие в белках B- и T-клеточных эпитопов. Для этих целей недавно был создан веб сервер CRESSP (англ. Cross Reactive Epitope Search using Structural properties of Proteins), с помощью которого становится возможным выявлять перекрёстно-реак тивные эпитопы между протеомами SARS-CoV-2 и че ловека с использованием структурных свойств белков [30]. Данным подходом были обнаружены уже 133 чело веческих белка, несущих потенциальные перекрёстно реактивные В-клеточные эпитопы, и 648 белков - с по тенциальными CD8+ Т-клеточными эпитопами. Подоб ное широкое распространение аутоантигенов предпо лагает их участие в мультиорганных проявлениях пост- ковидного синдрома. ф [ ] M.C. Sacchi и соавт. (2021) указывают, что после пе ренесённого COVID-19 значительно распростране ны также ANCA (антитела к цитоплазме нейтрофилов) и ASCA класса IgA (антитела к Saccharomyces cerevisiae), а также ANA. С ANCA, которые ошибочно нацеливают ся на белки нейтрофилов и моноцитов, обычно ассоци ированы несколько форм васкулитов [37]. ASCA часто обнаруживаются в крови пациентов с болезнью Кро на и их роль в патогенезе поражения кишечника оста ётся не до конца выясненной [38]. Эти антитела взаимо действуют с фосфопептидоманнаном клеточной стенки S. cerevisiae или другими, вероятно, аутоантигенами, об ладающими сходной структурой. M.C. Sacchi с коллега ми также показали, что пациенты с COVID-19 с высоким титром ANA и ANCA имеют наихудший клинический ис ход. P.G. Vlachoyiannopoulos и соавт. (2020) проанализи ровали 29 тяжелобольных пациентов с COVID-19 и обна ружили, что почти у 70 % развилась аутоиммунная акти вация. М ОЛЕКУЛЯРНАЯ МИМИКРИЯ Сообщается также о случае возникновения СКВ непосредственно после инфекции COVID-19 у пациент ки на фоне первично-выявленных высоких уровней ANA, ANCA и аутоантител к Ku-антигену (ДНК-связывающий белок, участвующий в репарации двухцепочечных раз рывов) [39]. М ОЛЕКУЛЯРНАЯ МИМИКРИЯ Помимо сторонней активации лимфоцитов, суще ствует другой, антиген-специфичный, путь вирус-инду- 66 Инфекционные болезни Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 ческих заболеваниях, таких как СКВ, ревматоидный ар трит, синдром Шегрена, антифосфолипидный синдром, системный склероз, воспалительные миопатии, гломе- рулонефрит, васкулит, энцефалит, аутоиммунный гепа тит, первичный билиарный холангит, воспалительное за болевание кишечника и интерстициальное заболевание лёгких [34]. На сегодняшний день множество исследова ний показали наличие аутоантител у пациентов с SARS- CoV-2, ранее не имевших аутоиммунных заболеваний, подтверждая гипотезу COVID-19-ассциированного ауто иммунитета. Y. Zhang и соавт. описали трёх пациентов с COVID-19, отягощённым коагулопатией и церебральны ми тромбами, у которых наблюдались APA (антифосфо липидные антитела; IgA к кардиолипину, IgA и IgG к Р2- гликопротеину I) [35]. Авторы заявляют, что APA могут быть причиной тромбозов у пациентов в критическом состоянии. В целом, проведённый к настоящему момен ту метаанализ встречаемости APA среди реконвалесцен- тов COVID-19 свидетельствует об их наличии почти у по ловины пациентов, при этом более высокая распростра нённость этого вида аутоантител обнаружена при тяжё лом течении инфекции [36]. ческих заболеваниях, таких как СКВ, ревматоидный ар трит, синдром Шегрена, антифосфолипидный синдром, системный склероз, воспалительные миопатии, гломе- рулонефрит, васкулит, энцефалит, аутоиммунный гепа тит, первичный билиарный холангит, воспалительное за болевание кишечника и интерстициальное заболевание лёгких [34]. На сегодняшний день множество исследова ний показали наличие аутоантител у пациентов с SARS- CoV-2, ранее не имевших аутоиммунных заболеваний, подтверждая гипотезу COVID-19-ассциированного ауто иммунитета. Y. Zhang и соавт. описали трёх пациентов с COVID-19, отягощённым коагулопатией и церебральны ми тромбами, у которых наблюдались APA (антифосфо липидные антитела; IgA к кардиолипину, IgA и IgG к Р2- гликопротеину I) [35]. Авторы заявляют, что APA могут быть причиной тромбозов у пациентов в критическом состоянии. В целом, проведённый к настоящему момен ту метаанализ встречаемости APA среди реконвалесцен- тов COVID-19 свидетельствует об их наличии почти у по ловины пациентов, при этом более высокая распростра нённость этого вида аутоантител обнаружена при тяжё лом течении инфекции [36]. недоступными для возбуждающей глутаматергической передачи. С другой стороны, повреждение микрососу дов, вызванное системным воспалительным ответом на инфекцию SARS-CoV-2, и их повышенная проницае мость, могут вызывать нарушение гематоэнцефаличе ского барьера и облегчать перенос NMDAR-реактивных и других аутоантител в ЦНС. недоступными для возбуждающей глутаматергической передачи. АУТОАНТИТЕЛА Последствием описанных вирус-индуцированных нарушений иммунной системы, а также, как предпола гают S.E. Chang и соавт. [31], взаимодействия между ви русными компонентами с собственными белками орга низма с возникновением новых межмолекулярных эпи топов, является мобилизация аутоантител. Уже в первый год пандемии появились первые сообщения о наличии аутоиммунных явлений у пациентов с тяжёлой и кри тической формой COVID-19. В первой опубликованной работе [32] авторы указали на распространённость ANA (антинуклеарных антител) и антител к цитоплазматиче ским антигенам SS-A(Ro) (SS-A (52 кДа) / SS-A (60 кДа)) - полипептидам, образующим комплекс с RoPHK, - кото рые обычно диагностируют при синдроме Шегрена и не которых разновидностях системной красной волчанки (СКВ) [33]. ANA реактивны к ядерным белкам и присут ствуют при многих системных аутоиммунных ревмати В одном из наиболее обширных продольных серо логических исследований проводился поиск новых и из учение специфичности уже известных аутоантител у го спитализированных пациентов с COVID-19 [31]. Выявлен ные у примерно половины пациентов антитела как ми нимум к одному из аутоантигенов появились у пациен тов de novo вместе с IgG к белкам SARS-CoV-2. Авторы полагают, что повышение уровня аутоантител не яв ляется просто отражением гипергаммаглобулинемии, т. к. их уровень не пропорционален общей концентра ции сывороточных IgG, а их мишенью является лишь не большое количество аутоантигенов, т. е. происходит из бирательная потеря аутотолерантности в ответ на ви 67 Инфекционные болезни Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 русную инфекцию. В данной работе помимо описанных выше традиционных аутоантигенов в качестве дополни тельных мишеней выявили рибосомальные P-белки (P0, P1 и P2), антигены щитовидной железы, белки хромати на, редкие антигены, характерные для пациентов с ау тоиммунным миозитом (MDA5, Mi-2 и тРНК-синтетазы), аутоантигены-кандидаты аутоиммунного миокардита (тропонин и MYH6), аутоантигены системного склероза (Th/To (RPP25), фибрилларин и мяРНП U11/U12) и даже ангиотензинпревращающий фермент-2 (ACE2). Аутоан тигены, которые естественным образом образуют ком плексы с молекулой структурной РНК, например, MDA5, могут служить лигандами для Toll-подобных рецепто ров (TLR7, TLR3) в клетках-хозяевах. В свою очередь, че рез экстрафолликулярный, TLR7-зависимый путь, могут активироваться аутореактивные В-клетки, как это про исходит, например, при СКВ [40]. Нуклеиновые кисло ты, высвобождаемые из разрушающихся клеток, сами по себе также могут образовывать иммунные комплек сы с вирусными или собственными антигенами, которые могут способствовать выработке аутоантител. фицировании связано с драматическим нарушением координации разных ветвей иммунитета и широким противовирусным ответом не только на структурные, но и на внутренние вирусные белки. М УЖСКОЕ БЕСПЛОДИЕ И ДИАБЕТ Согласно мировой статистике, мужчины более под вержены заболеванию COVID-19, чем женщины. В пост- ковидном периоде у пациентов мужского пола всё чаще выявляются снижение фертильности и вновь приобре тённый диабет 1-го типа. Высказываются гипотезы об ау тоиммунной природе этих осложнений. у р у В вопросе об аутоантителах интересно упомянуть также нейтрофильные внеклеточные ловушки (NETs). NETs образуются в результате нетоза - одного из ви дов запрограммированной клеточной гибели нейтро филов, и предназначены для улавливания и «запутыва ния» патогена и создания локальной высокой концен трация медиаторов воспаления и бактерицидных ком понентов. В случае COVID-19, тем не менее, NETs отяго щают патогенез заболевания [41]. Содержащиеся в них компоненты - нуклеиновые кислоты, гистоны и бакте рицидные пептиды - признаны молекулярными фраг ментами, ассоциированными с повреждениями (DAMP), и запускают воспалительные сигнал ы, усиливая «цитоки- новый шторм» (замыкается патологическая петля NETs- IL-1P). У пациентов с тяжёлой инфекцией детектируют аутоантитела против NETs [42], а через месяц после пе ренесённого заболевания отмечают снижение метабо лических резервов нейтрофилов [43]. По-видимому, ак тивация нейтрофилов с образованием NETs и развёрты вание на этом фоне антинейтрофильного и анти-NETs ау тоиммунитета могут иметь прямые последствия в виде высокого уровня циркулирующего D-димера, тромбо воспаления (иммунотромбоз) и васкулитов [44]. у р р В мужских репродуктивных органах, особен но в семенниках, высок уровень экспрессии рецеп торов ACE2 [46] и трансмембранной сериновой про теазы 2 (TMPRSS2) [47], которые используются SARS- CoV-2 для проникновения в клетку. Коэкспрессия ACE2 и TMPRSS2 в сперматогониях, клетках Сертоли и Лей- дига делает их уязвимыми для инфекции, увеличива ет риск дегенерации тестикулярной ткани и мужско го бесплодия [48]. Вирус может нарушать сперматоге нез напрямую, проникая и разрушая ACE2- и TMPRSS2- коэкспрессирующие клетки, или косвенно, приводя к ме таболическому перепрограммированию или нарушая гемато-тестикулярный барьер и вызывая аутоиммун ный орхит. Клетки Сертоли отвечают за формирование иммунологической толерантности к сперматогенным клетками, скрывая их от аутоиммунного реагирования, а также обеспечивают стабильное микроокружение, не обходимое для сперматогенеза [49]. Инфицируя клетки Сертоли, SARS-CoV-2 повреждает гемато-тестикулярный барьер и приводит к инфильтрации ткани яичка лейко цитами, которая негативно влияет на функцию тестосте- рон-продуцирующих клеток Лейдига и непосредствен но повреждает семявыносящий эпителий [50]. Продуци руемые повреждёнными клетками провоспалительные цитокины, такие как IL-6, усиливают разрушение гемато тестикулярного барьера [51] и могут активировать выра ботку аутоантител в семенных канальцах [46]. В мужских репродуктивных органах, особен но в семенниках, высок уровень экспрессии рецеп торов ACE2 [46] и трансмембранной сериновой про теазы 2 (TMPRSS2) [47], которые используются SARS- CoV-2 для проникновения в клетку. АУТОАНТИТЕЛА Наиболее часто вы являются антитела к структурным белкам SARS-CoV-2 (от метим, однако, что среди них к белку оболочки E анти тела в работе [31] выявлены только у одного пациента из 94), однако среди неструктурных NSP9 является наи более часто распознаваемым внутренним белком SARS- CoV-2. Примечательно, что уровни IgG на белки других вирусов, включая сезонные коронавирусы и SARS-CoV, не коррелируют с уровнем ACA. М УЖСКОЕ БЕСПЛОДИЕ И ДИАБЕТ Первоначально бе лок охарактеризовали как маркер СД 1-го типа [75], од нако позже разница в частоте аутоантител, идентифи цируемых у пациентов с СД обоих типов, не подтверди лась [76]. При этом в состав эпитопа, распознаваемого анти-SOX13-антителами, входит большая часть белка, а его экспрессия детектируется в большинстве тканей, с наиболее высоким уровнем в клетках поджелудочной и щитовидной железы, плаценты и почек [75]. Помимо этого, анти-SOX13-антитела обнаружены также у паци ентов с первичным билиарным циррозом, аутоиммун ным гепатитом и при мультисистемных аутоиммунных заболеваниях, СКВ и ревматоидном артрите, что указы вает на способность SOX13 вызывать разнонаправлен ный аутоиммунный ответ [77]. Недавно было показано, что экспрессия SOX13 снижена в периферических моно- нуклеарных клетках крови (PMBC) пациентов с синдро мом Клайнфельтера - крайне распространённого гене тического заболевания у мужчин, вызванного наличием дополнительной X-хромосомы. Синдром Клайнфельте ра характеризуется потерей зародышевых клеток, на рушением функции клеток Сертоли, гиперплазией кле ток Лейдига, азооспермией и другими вариабельны ми клиническими признаками, включая аутоиммунитет и СД 2-го типа. R. Cannarella и соавт. (2021) предполага ют, что в зародышевых клетках у мужчин с синдромом Клайнфельтера может происходить аналогичное пода вление экпрессии SOX13, которое приводит к апоптозу клеток и бесплодию, а также к эндокринно-органоспе цифическому гуморальному аутоиммунитету, типично му для пациентов с этим синдромом. рые, как предполагается, снижают фертильность, дей ствуя против развивающихся сперматоцитов, а посколь ку вирус вызывает также гиперкоагуляцию, предпола гается, что орхит также может быть результатом васку лита [56], или нарушений со стороны антиоксидантной системы организма [57]. Важно, что тестикулярная экс прессия ACE2 зависит от возраста: увеличивается по сле 20 лет, достигает максимума у 30-летних пациентов и практически не наблюдается у пациентов 60 лет [58]. Таким образом, молодые пациенты мужского пола под вержены более высокому риску постковидных ослож нений со стороны репродуктивной системы, чем паци енты старшего возраста. рые, как предполагается, снижают фертильность, дей ствуя против развивающихся сперматоцитов, а посколь ку вирус вызывает также гиперкоагуляцию, предпола гается, что орхит также может быть результатом васку лита [56], или нарушений со стороны антиоксидантной системы организма [57]. Важно, что тестикулярная экс прессия ACE2 зависит от возраста: увеличивается по сле 20 лет, достигает максимума у 30-летних пациентов и практически не наблюдается у пациентов 60 лет [58]. Таким образом, молодые пациенты мужского пола под вержены более высокому риску постковидных ослож нений со стороны репродуктивной системы, чем паци енты старшего возраста. C.C. Maresch и соавт. указывают на патологическую взаимосвязь между мужской фертильностью, сахарным диабетом (СД) и вирус-индуцированным воспалением [59]. М УЖСКОЕ БЕСПЛОДИЕ И ДИАБЕТ Косвенно гипергликемия усугубляет негативное влияние инфекции на мужской репродуктивный потен циал [60]. Особенно это важно в контексте формирова ния СД в виде постковидного осложнения. Недавние ра боты показывают, что у пациентов с COVID-19 всё чаще наблюдается впервые диагностированный диабет обо их типов [61]: риск его развития повышен уже после пер вых 30 дней болезни у детей, подростков [62] и взрослых [63]. Заражение различными вирусами (вирус краснухи и цитомегаловирус [64], вирус гепатита C [65], SARS-CoV [66]) часто ассоциировано с возникновением СД. Сооб щается, что инфекция SARS-CoV-2 приводит к возникно вению резистентности к инсулину и острой гипергли кемии у людей без диабета, к развитию СД у пациентов с предиабетом и тяжёлыми метаболическими осложне ниям ранее существовавшего диабета, в том числе ди абетическому кетоацидозу и гиперосмолярности [60, 67]. В этом процессе могут быть задействованы прямые и непрямые патологические механизмы. При вирусной инфильтрации панкреатических островков происхо дит разрушение ACE2-экспрессирующих р-клеток и мо жет сформироваться СД 1-го типа [61]. Резистентность к инсулину (СД 2-го типа) может также вызываться «ци- токиновым штормом» и высокими уровнями АФК [68], наблюдаемыми при COVID-19 [69, 70]. Классическая функция белка оболочки E вируса SARS-CoV-2 заключается образовании белково-липид ных каналов в мембранах промежуточного компартмен- та ЭПР-Гольджи (ERGIC) и комплекса Гольджи, влияющих на внутриклеточные ионные градиенты, которые облег чают сборку вирусных частиц [78] и высвобождение ви- риона [79]. Определение В-клеточных эпитопов и обла стей связывания MHC-I в структуре белка E позволило выявить множество перекрывающихся антигенных де терминант, покрывающих почти весь небольшой белок [80]. Ещё в начале пандемии COVID-19 учёные обратили внимание на высокое сходство трансмембранной обла сти белка E с последовательностью LytR-домена туберку- лин-подобных белков микобактерий [81] и предположи ли, что этот феномен может лежать в основе перекрёст ного иммунитета против вируса SARS-CoV-2, вызванного туберкулёзной инфекцией [82], или объяснять наблюда емое защитное действие вакцины БЦЖ [83]. М УЖСКОЕ БЕСПЛОДИЕ И ДИАБЕТ Коэкспрессия ACE2 и TMPRSS2 в сперматогониях, клетках Сертоли и Лей- дига делает их уязвимыми для инфекции, увеличива ет риск дегенерации тестикулярной ткани и мужско го бесплодия [48]. Вирус может нарушать сперматоге нез напрямую, проникая и разрушая ACE2- и TMPRSS2- коэкспрессирующие клетки, или косвенно, приводя к ме таболическому перепрограммированию или нарушая гемато-тестикулярный барьер и вызывая аутоиммун ный орхит. Клетки Сертоли отвечают за формирование иммунологической толерантности к сперматогенным клетками, скрывая их от аутоиммунного реагирования, а также обеспечивают стабильное микроокружение, не обходимое для сперматогенеза [49]. Инфицируя клетки Сертоли, SARS-CoV-2 повреждает гемато-тестикулярный барьер и приводит к инфильтрации ткани яичка лейко цитами, которая негативно влияет на функцию тестосте- рон-продуцирующих клеток Лейдига и непосредствен но повреждает семявыносящий эпителий [50]. Продуци руемые повреждёнными клетками провоспалительные цитокины, такие как IL-6, усиливают разрушение гемато тестикулярного барьера [51] и могут активировать выра ботку аутоантител в семенных канальцах [46]. Значительная часть аутоантител у 60-80 % госпита лизированных пациентов является ACA (антицитокино- выми) [31]. Подмножество ACA препятствует развитию защитного иммунного ответа при COVID-19, конкурируя за связывание цитокинов (например, IFN I типа, многих интерлейкинов) с их рецепторами на клеточной поверх ности. Учитывая нарушение интерферонового ответа, которое непосредственно вызвано неструктурными бел ками SARS-CoV-2 и описано выше, становится более яс ной связь между тяжестью заболевания и нарушенной активностью IFN [45] у лиц с дополнительным его бло кированием за счёт анти-^-антител. Орхит диагностируют у 19 % пациентов с COVID-19 [52], и даже средняя тяжесть инфекции у мужчин ассо циирована со снижением общего количества сперма тозоидов в эякуляте и их подвижности [53, 54]. Однако большинство опубликованных данных указывают на от сутствие РНК вируса в образцах спермы [52], что может указывать на воспалительную и аутоиммунную причину повреждения тестикулярной ткани [55], а не на прямое разрушение вирусом. Например, у пациентов с COVID-19 обнаруживаются антифосфолипидные антитела, кото Исследуя возможные причины стойкого аутоанти- тельного ответа у пациентов с COVID-19, S.E. Chang и со- авт. постулируют, что развитие аутоиммунитета при ин 68 Инфекционные болезни Infectious diseases A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 брионального развития и клеточной дифференциров- ки, в том числе Y^-Т-клеток [71], которые, как извест но, способствуют B-клеткам в выработке аутоантител [72]. SOX13 участвует также в онкогенезе [73], вероятно, в сперматогенезе [74] и, как установили H. Kasimiotis и со- авт. (2000), также является аутоантигеном островковых клеток (ICA12 - islet cell antigen 12). Конфликт интересов А й Авторы данной статьи сообщают об отсутствии кон фликта интересов. Авторы данной статьи сообщают об отсутствии кон фликта интересов. ЛИТЕРАТУРА 1. Montani D, Savale L, Noel N, Meyrignac O, Colle R, Gas- nier M, et al. Post-acute COVID-19 syndrom. Eur Respir Rev. 2022; 31(163): 210185. doi: 10.1183/16000617.0185-2021 1. Montani D, Savale L, Noel N, Meyrignac O, Colle R, Gas- nier M, et al. Post-acute COVID-19 syndrom. Eur Respir Rev. 2022; 31(163): 210185. doi: 10.1183/16000617.0185-2021 2. Halpin S, O'Connor R, Sivan M. Long COVID and chronic COVID syndromes. JM ed Virol. 2021; 93(3): 1242-1243. doi: 10.1002/ JMV.26587 2. Halpin S, O'Connor R, Sivan M. Long COVID and chronic COVID syndromes. JM ed Virol. 2021; 93(3): 1242-1243. doi: 10.1002/ JMV.26587 Дополнительно к этому, предсуществующие у па циентов с СД или репродуктивными нарушениями B- или T-клетки, реактивные к SOX13, могут получать акти вирующие стимулы при связывании их поверхностных рецепторов со сходными эпитопами белка Е. Вероят ность их активации многократно возрастает в агрессив ной среде «цитокинового шторма». Клональная экспан сия анти-E/SOX13 аутореактивых клеток в конечном ито ге будет приводить к развёртыванию иммунного ответа против SOX13-экспрессирующих клеток, например, под желудочной железы и, вероятно, семенников, приводя к нарушению работы данных органов. Таким образом, мы предполагаем, что кросс-реактивный иммунный от вет против белков Е и SOX13 может послужить причи ной аутоиммунных постковидных эндокринных/репро- дуктивных осложнений. 3. Huang C, Huang L, Wang Y, Li X, Ren L, Gu X, et al. 6-month consequences of COVID-19 in patients discharged from hospital: A cohort study. Lancet. 2021; 397(10270): 220-232. doi: 10.1016/ S0140-6736(20)32656-8 4. Healey Q, Sheikh A, Daines L, Vasileiou Е. Symptoms and signs of long COVID: A rapid review and meta-analysis. J Glob Health. 2022; 12: 05014. doi: 10.7189/jogh.12.05014 5. Galeeva J, Babenko V, Bakhtyev R, Baklaushev V, Balykova L, Bashkirov P, et al. 16S rRNA gene sequencing data of the upper respiratory tract microbiome in the SARS-CoV-2 infected patients. Data Brief. 2022; 40: 107770. doi: 10.1016/j.dib.2021.107770 6. Ahmed H, Patel K, Greenwood DC, Halpin S, Lewthwaite P, Salawu A, et al. Long-term clinical outcomes in survivors of severe acute respiratory syndrome and Middle East respiratory syndrome coronavirus outbreaks after hospitalisation or ICU admission: A systematic review and meta-analysis. JRehabilM ed. 2020; 52(5): jrm00063. doi: 10.2340/16501977-2694 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 и соавт. (2020) обнаружено взаимодействие антител к белку E с восьмью аутоантигенами, наиболее сильное - с митохондриальным M2 и антигенами эпителиальных клеток кишечника. Эти данные позволяют предполагать, что перекрёстная реакция между белком E SARS-CoV-2 и антигенами тканей желудочно-кишечного тракта че ловека может быть причиной желудочно-кишечных про явлений COVID-19. В работе Z. Karagoz и соавт. (2021), с использованием биоинформационного анализа сход ства белковых последовательностей, показана значи тельная гомология белка E с шестью поверхностными транспортными белками пигментного эпителия сетчат ки (MRP-4, MRP-5, RFC1, SNAT7, TAUT и MATE). Предпола гается, что иммунореактивные эпитопные участки этих белков, сходные с эпитопом белка Е, могут вызывать им мунную стимуляцию Т-цитотоксических и Т-хелперных клеток, поэтому также весьма вероятна аутоиммунная перекрёстная реакция. ния со стороны иммунной системы человека и вызывает стойкий гуморальный аутоиммунитет у более чем поло вины выживших и выздоравливающих пациентов. Часто выявляются антинуклеарные, антифосфолипидные и ан- тицитокиновые антитела, антитела к цитоплазме ней трофилов, Saccharomyces cerevisiae, цитоплазматическим антигенам SS-A(Ro), аутоантитела против №Ts и многих других аутоантигенов человека. При этом активировать гуморальный иммунитет способны как структурные бел ки вируса, так и неструктурные, такие как NSP9. Молекулярная мимикрия антигенов является одной из главных причин развития аутоиммунных патологий. Известно, что некоторые белки вируса SARS-CoV-2 мо гут имитировать эпитопы белков организма-хозяина. Мы постулируем о роли структурной гомологии HMG- мотива SOX13 человека, аутоантигена СД, и белка Е обо лочки SARS-CoV-2 в механизмах развития постковидных патологий. Мы предполагаем, что структурное сходство их эпитопов может быть связано с перекрёстной имму нологической реактивностью и развитием сахарного ди абета и нарушений со стороны репродуктивной систе мы у мужчин после перенесённого COVID-19. Учитывая приведённые выше данные, мы позволим себе выдвинуть гипотезу, что участники гуморального или, что более вероятно, клеточного иммунитета в от ношении белка E, активируемые во время инфекции COVID-19, могут распознавать также антигенные детер- минантны собственного белка SOX13, приобретая ауто иммунный потенциал. Основанием предполагать имен но клеточную природу иммунного ответа является низ кая частота детекции анти-Е-антител среди пациентов с COVID-19. Скорее всего, белок Е, несмотря на наличие B-клеточных эпитопов, является слабым модулятором гу морального иммунного ответа по сравнению с белком S. Тем не менее, выявляемые даже у небольшого количе ства пациентов антитела против белка Е могли бы при нимать участие в развитии перекрёстной реактивности. СВЯЗЬ СТРУКТУРНОЙ ГОМОЛОГИИ БЕЛКА SO X13 ЧЕЛОВЕКА И БЕЛКА Е ОБОЛОЧКИ SARS-CoV-2 С АУТО И м м УН Н ы м И п о с т к о в и д н ы м и о с л о ж н е н и я м и Обсуждая роль молекулярной мимикрии в фор мировании аутоиммнной патологии, мы предполага ем, что повышенный риск развития СД и нарушений со стороны репродуктивной системы у мужчин после перенесённого COVID-19 могут быть связаны с пере крёстной иммунологической реактивностью. Нами об наружена структурная гомология между трансмембран ным и C-доменами белка оболочки E SARS-CoV-2 c HMG- мотивом аутоантигена SOX-13 (HMG box factor SOX13). К сожалению, вирусный белок E, в отличие от S, оста ётся за рамками глобальных исследований, и всего не сколько работ посвящены анализу связанного с ним кросс-реактивного аутоиммунитета. В работе A. 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J Med Virol. 2020; 92(4): 448-454. doi: 10.1002/jmv.25693 75 Инфекционные болезни Infectious diseases Сведения об авторах Орлова Елизавета Андреевна - аспирант, младший научный сотрудник лаборатории эпидемиологически и социально-значимых инфекций, ФГБНУ «Научный центр проблем здо ровья семьи и репродукции человека», e-mail: elizaveta.a.orlova@gmail.com, https//orcid.org/0000-0003-2169-0242 Орлова Елизавета Андреевна - аспирант, младший научный сотрудник лаборатории эпидемиологически и социально-значимых инфекций, ФГБНУ «Научный центр проблем здо ровья семьи и репродукции человека», e-mail: elizaveta.a.orlova@gmail.com, https//orcid.org/0000-0003-2169-0242 дьевич - кандидат биологических наук, научный сотрудник лаборатории эпидемиологически и социально-значимых инфекций, ФГБНУ «Научный центр репродукции человека», e-mail: kondratovig@mail.ru, https//orcid.org/0000-0002-2631-4724 Кондратов Илья Геннадьевич - кандидат биологических наук, научный сотрудник лаборатории эпидемиологически и социально-значимых инф проблем здоровья семьи и репродукции человека», e-mail: kondratovig@mail.ru, https//orcid.org/0000-0002-2631-4724 Огарков Олег Борисович - доктор медицинских наук, заведующий отделом эпидемиологии и микробиологии, ФГБНУ «Научный центр проблем здоровья семьи и репродукции че ловека», e-mail: obogarkov@mail.ru, https//orcid.org/0000-0002-3168-1983 Колесникова Любовь Ильинична - доктор медицинских наук, профессор, академик РАН, научный руководитель, ФГБНУ «Научный центр проблем здоровья семьи и репродукции человека», e-mail: iphr@sbamsr.irk.ru, https//orcid.org/0000-0003-3354-2992 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 cobacterial infections and may provide specific anti-SARS-CoV-2 immunity via the BCG vaccine. Ann Diagn Pathol. 2020; 48: 151600. doi: 10.1016/j.anndiagpath.2020.151600 79. Lim KP, Liu DX. The missing link in coronavirus assembly. Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins. J Biol C hem . 2001; 276(20): 17515-17523. doi: 10.1074/jbc. M009731200 82. Madan M, Pahuja S, Mohan A, Pandey RM, Madan K, Hadda V, et al. TB infection and BCG vaccination: Are we protected from COVID-19? Public Health. 2020; 185: 91-92. doi: 10.1016/ j.puhe.2020.05.042.80 80. Thomas S. Towards determining the epitopes of the struc tural proteins of SARS-CoV-2. Methods Mol Biol. 2022; 2410: 265-272. doi: 10.1007/978-1-0716-1884-4_12 83. Netea MG, Giamarellos-Bourboulis EJ, Dominguez- Andres J, Curtis N, van Crevel R, van de Veerdonk FL, et al. Trained immunity: A tool for reducing susceptibility to and the severity of SARS-CoV-2 infection. Cell. 2020; 181(5): 969-977. doi: 10.1016/ j.cell.2020.04.042 81. Nuovo G, Tili E, Suster D, Matys E, Hupp L, Magro C. Strong hom ology between SARS-CoV-2 envelope protein and a Mycobacterium sp. antigen allows rapid diagnosis of My Information about the authors Elizaveta A. Orlova - Postgraduate, Junior Research Officer at the Laboratory of Epidemically and Socially Significant Infections, Scientific Centre for Family Health and Human Reproduction Problems, e-mail: elizaveta.a.orlova@gmail.com, https//orcid.org/0000-0003-2169-0242 Ilya G. Kondratov - Cand. Sc. (Biol.), Research Officer at the Laboratory of Epidemically and Socially Significant Infections, Scientific Centre for Family Health and Human Reproduction Problems, e-mail: kondratovig@mail.ru, https//orcid.org/0000-0002-2631-4724 ed.), Head of the Department of Epidemiology and Microbiology, Scientific Centre for Family Health and Human Reproduction Problems, e-mail: obogarkov@mail.ru, 3168-1983 Статья опубликована в рамках V Всероссийской научно-практической конференции молодых учёных с международным участием «Фундаментальные и прикладные аспекты в ме дицине и биологии». 76 Инфекционные болезни Infectious diseases
https://openalex.org/W4312275089	https://akwi.hswlu.ch/article/download/3213/2940	German	null	Editorial	Anwendungen und Konzepte der Wirtschaftsinformatik	2,018	cc-by	514	Editorial Christian Müller Liebe Leserinnen und Leser, vor Ihnen liegt nunmehr die bereits achte Ausgabe des E-Journals Anwendungen und Konzepte in der Wirtschaftsinformatik (AKWI). Diesmal werden viele charakteristische Themen der Wirtschaftsinformatik behandelt. So ist es wenig überraschend, dass SAP-Produkte vertreten sind. Die derzeit sehr hohe Bedeutung von SAP S/4HANA für betriebliche belegen drei Artikel. Im ersten wird untersucht, inwieweit sich die neue Produktgeneration von SAP SE (SAP S/4HANA) als Enabler zur Smart Factory der AUDI AG eignet. Der zweite befasst sich mit der Ablösung vom ERP-System SAP ERP Central Component 6.0 durch die (SAP Business Suite 4) SAP HANA (kurz: SAP S/4HANA). Die grundlegende Schwierigkeit war ein hoch komplexes und nur noch schwer überschaubares System an Schnittstellen. Es werden Entwicklungs- schritte einer Migrationsstrategie zur Konsolidierung einer bestehenden Schnittstellen- Landschaft auf ein SAP S/4HANA-System in Kombination mit SAP Process Orchestration aufgezeigt. Im dritten wird untersucht, inwieweit sich ein graphisches, interaktives Prozess Mining Tool mit Technologien, die über die Entwicklungsplattform im Standard einer aktuellen SAP S/4 HANA Version mit ausgeliefert werden, erstellbar ist. Eher klassisch ist die Teilautomatisierung von Akzeptanztests im Rahmen der Webentwicklung in einem agilen Umfeld, wodurch Überprüfungen von Anforderungen in hohen Tempo erreicht werden und zugleich der manuelle Prüfungsaufwand auf ein Minimum reduziert wird. Ebenfalls eine hohe ökonomische Bedeutung hat die bereichsübergreifende Personal- steuerung in Form von Leihungen und Verleihungen, einem Teilbereich der monatlichen Personalplanung. Sehr aktuelle Themen werden in drei weiteren Artikeln behandelt. Ein Beitrag im Umfeld von KI behandelt den Einsatz von Text Analytics zur Unterstützung literaturintensiver Forschungsprozesse. Leserinnen und Leser dürften Blockchain erwar- ten, obwohl ihre Bedeutung nach neueren Pressemeldungen abzunehmen scheint. In dieser Ausgabe geht es um die Potenziale von Blockchain 2.0 in der Energiewirtschaft. Als Beispiel zu Predictive Analytics wird die Nutzerbindung durch verhaltensbasierte Big- Data-Analyse erläutert. Was dies für die Menschen bedeutet wird im Artikel „Wie sieht denn nun die Zukunft der Arbeit aus? Aktivitäten des interdisziplinären Zukunftslabors CreaLab im Rahmen des Schwerpunkthemas «Zukunft der Arbeit» diskutiert. Christian Müller Konrad Marfurt Konrad Marfurt Norbert Ketterer Über Ihr Interesse an der Zeitschrift freuen wir uns und wünschen Ihnen Freude bei der Lektüre. Regensburg, Fulda, Wildau und Luzern, im Dezember 2018 Frank Herrmann, Norbert Ketterer, Konrad Marfurt und Christian Müller Frank Herrmann, Norbert Ketterer, Konrad Marfurt und Christian Müller Frank Herrmann In eigener Sache: g Alle Beiträge wurden von zwei unabhängigen Gutachtern begutachtet und von den Autoren anschließend überarbeitet. Dieser Prozess nimmt naturgemäß viel Zeit in Anspruch, da sämtliche Redakteure, Gutachterinnen und Gutachter ihre Arbeit in der immer spärlicher werdenden Freizeit leisten. Dafür gebührt ihnen unser besonderer Dank! Unsere Zeitschrift steht kostenfrei unter https://akwi.hswlu.ch als E-Journal an der Hochschule Luzern unter Federführung von Konrad Marfurt zur Verfügung. Für unsere Autoren entstehen durch die Publikation keine Kosten, allerdings erhalten sie auch keine Honorare. Damit wir unter diesen Bedingungen erfolgreich arbeiten können, reichen die Autoren druckfertige Manuskripte, die unserer Formatvorlage genügen, in deutscher oder englischer Sprache ein, ordnen diesen einer der Rubriken Grundlagen, Trends, Praxis, Kurz erklärt, Buchbesprechung oder Abschlussarbeit zu. Ferner bitten wir unsere Autoren um eine Einverständniserklärung zur Publikation und eine Selbsteinschätzung. Nach der Einreichung beginnt sofort unser Begutachtungsprozess.
https://openalex.org/W4292466853	https://www.frontiersin.org/articles/10.3389/fonc.2022.971190/pdf	English	null	Development and validation of a deep learning model to predict survival of patients with esophageal cancer	Frontiers in oncology	2,022	cc-by	7,706	OPEN ACCESS OPEN ACCESS EDITED BY Ying-Tai Chen, Chinese Academy of Medical Sciences and Peking Union Medical College, China REVIEWED BY Ran Wei, Chinese Academy of Medical Sciences and Peking Union Medical College, China Zhenhua Lu, Beijing Hospital, China CORRESPONDENCE Xunyu Xu xunyuxu@sina.com Xiao Chen nalanyu2000@163.com †These authors have contributed equally to this work and share ﬁrst authorship SPECIALTY SECTION This article was submitted to Thoracic Oncology, a section of the journal Frontiers in Oncology RECEIVED 16 June 2022 ACCEPTED 20 July 2022 PUBLISHED 10 August 2022 EDITED BY Ying-Tai Chen, Chinese Academy of Medical Sciences and Peking Union Medical College, China Chen Huang 1†, Yongmei Dai 2†, Qianshun Chen 1, Hongchao Chen 1, Yuanfeng Lin 1, Jingyu Wu 1, Xunyu Xu 1 and Xiao Chen 3* 1Shengli Clinical College of Fujian Medical University, Department of Thoracic Surgery, Fujian Provincial Hospital, Fuzhou, China, 2Shengli Clinical College of Fujian Medical University, Department of Oncology, Fujian Provincial Hospital, Fuzhou, China, 3College of Mathematics and Data Science (Software College), Minjiang University, Fuzhou, China Objective: To compare the performance of a deep learning survival network with the tumor, node, and metastasis (TNM) staging system in survival prediction and test the reliability of individual treatment recommendations provided by the network. Methods: In this population-based cohort study, we developed and validated a deep learning survival model using consecutive cases of newly diagnosed stage I to IV esophageal cancer between January 2004 and December 2015 in a Surveillance, Epidemiology, and End Results (SEER) database. The model was externally validated in an independent cohort from Fujian Provincial Hospital. The C statistic was used to compare the performance of the deep learning survival model and TNM staging system. Two other deep learning risk prediction models were trained for treatment recommendations. A Kaplan– Meier survival curve was used to compare survival between the population that followed the recommended therapy and those who did not. CITATION Huang C, Dai Y, Chen Q, Chen H, Lin Y, Wu J, Xu X and Chen X (2022) Development and validation of a deep learning model to predict survival of patients with esophageal cancer. Front. Oncol. 12:971190. doi: 10.3389/fonc.2022.971190 COPYRIGHT © 2022 Huang, Dai, Chen, Chen, Lin, Wu, Xu and Chen. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). TYPE Original Research PUBLISHED 10 August 2022 DOI 10.3389/fonc.2022.971190 TYPE Original Research PUBLISHED 10 August 2022 DOI 10.3389/fonc.2022.971190 TYPE Original Research PUBLISHED 10 August 2022 DOI 10.3389/fonc.2022.971190 OPEN ACCESS The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: A total of 9069 patients were included in this study. The deep learning network showed more promising results in predicting esophageal cancer- speciﬁc survival than the TNM stage in the internal test dataset (C-index=0.753 vs. 0.638) and external validation dataset (C-index=0.687 vs. 0.643). The population who received the recommended treatments had superior survival compared to those who did not, based on the internal test dataset (hazard ratio, 0.753; 95% CI, 0.556-0.987; P=0.042) and the external validation dataset (hazard ratio, 0.633; 95% CI, 0.459-0.834; P=0.0003). 01 Frontiers in Oncology frontiersin.org frontiersin.org Huang et al. 10.3389/fonc.2022.971190 Conclusion: Deep learning neural networks have potential advantages over traditional linear models in prognostic assessment and treatment recommendations. This novel analytical approach may provide reliable information on individual survival and treatment recommendations for patients with esophageal cancer. KEYWORDS deep learning, esophageal cancer, DeepSurv, survival prediction, treatment recommendation deep learning, esophageal cancer, DeepSurv, survival prediction, treatment recommendation deep learning, esophageal cancer, DeepSurv, survival prediction, treatment recommendation Introduction performed better than other state-of-the-art survival methods for modeling the interactions between a patient’s covariates and treatment effectiveness. She et al. found that DeepSurv has a potential beneﬁt in prognostic evaluation and treatment recommendations with respect to lung cancer-speciﬁc survival (15). Esophageal cancer (EC) is the most common gastrointestinal tumor globally and ranked seventh in terms of incidence and sixth in terms of overall mortality in 2018 (1). Despite progress in the treatment and management of EC in recent years, the long-term survival of patients undergoing esophagectomy remains poor (17.1-55%) (2). The beneﬁts of adjuvant therapy have been debated and inconclusive. Therefore, it is very important to stratify the risk and make individualized treatment recommendations for newly diagnosed patients. In this study, we ﬁrst explored the performance of the DeepSurv model in analyzing the real-world clinical data of patients with esophageal cancer. Second, we evaluated the reliability of the DeepSurv model in providing treatment recommendations based on individual characteristics. The American Joint Committee on Cancer (AJCC) tumor, node, and metastasis (TNM) staging system is widely used to stratify disease risk, predict patient survival outcomes, and make decisions regarding cancer treatment (3). However, the AJCC staging system is not accurate enough to predict the survival of patients with esophageal cancer who received multimodality treatment (4). Moreover, survival rates in stage-matched cohorts varies widely (5). To improve the precision of EC survival estimations, nomograms have gained popularity as a method for predicting outcomes (6–9). A nomogram is a Cox proportional hazard (CPH) model designed to allow straightforward graphical calculation of the probability of a speciﬁc outcome, such as esophageal cancer-speciﬁc survival (ECSS). However, these models have some limitations in time–event prediction for the clinical management of patients with cancer, including an accurate assessment of overall survival (OS) and progression time (10). Moreover, it is not sufﬁcient to consider only the linear relationship of clinical features when making treatment decisions (11). Therefore, a better model that focuses on nonlinear variables is required. Frontiers in Oncology Eligibility criteria and clinical information For the training cohort, we selected patients from the Surveillance, Epidemiology, and End Results (SEER) database: SEER 18 Regs Custom Data [with additional treatment ﬁelds], Nov 2018 Sub [1975-2016 varying]. We obtained permission to access the database by signing the SEER Research Data Agreement form and submitting it via email. The inclusion criteria were as follows: (1) pathologically conﬁrmed primary stage I to IV EC (only adenocarcinoma and squamous cell carcinoma) between January 2004 and December 2015 and (2) the presence of one malignant primary lesion. The exclusion criteria were as follows: (1) missing clinicopathological data and (2) patients with perioperative mortality (mortality within 30 days after operation). Baseline patient information (sex, age, race, and marital status), tumor characteristics (primary site, histologic grade, histologic type, and TNM stage), SEER code (CS extension, CS mets at DX, regional nodes examined, regional nodes positive, and CS tumor size), and treatment details were collected (Table S1). The outcome of interest in this study was ECSS, which was deﬁned as the interval from diagnosis to death as a result of EC. These cases were randomly divided into training and test cohorts at a ratio of 8 to 2. Another test cohort from our database was provided to externally validate the DeepSurv model. After obtaining institutional review board Deep learning networks provide insights into the highly complex linear/nonlinear associations between prognostic clinical features and the individual risk of death (12). Matsuo et al. developed a deep learning neural network model that exhibited superior performance compared to the CPH model for survival prediction in women with cervical cancer (13). Katzman et al. developed a CPH deep neural network called DeepSurv (14), which can be used to predict the effects of patient covariates on patient survival. The authors demonstrated that DeepSurv Frontiers in Oncology 02 frontiersin.org Huang et al. 10.3389/fonc.2022.971190 l q ð Þ = −1 NE o i,Ei=1 ^h q x ð Þ −log o j∈R Ti ð Þ e ^h q x ð Þ ! + a ∥q ∥2 2 approval from Fujian Provincial Hospital, we selected patients received esophagectomy from January 2011 to December 2016 in Fujian Provincial Hospital (CHINA dataset), and these patients were completely distinct from those in the SEER database. The requirement for informed consent was waived owing to the retrospective nature of this study. Deep learning model design We performed survival analysis based on the deep learning model DeepSurv described by Katzman el al. (14) to predict individual patient outcomes. DeepSurv is a multilayer fully connected network composed of input, hidden, and output layers. Nonlinear features are introduced through the hidden layer of the neural network to ﬁt the proportional hazard function under nonlinear conditions. The expression for the hidden layer is f(X) =Relu(qX+b), where Relu is a nonlinear activation function, q is the parameter matrix, X is the input feature, and b is the bias term. The deep learning model learns the complex relationship between individual covariates and treatment effects by replacing the linear combination of features hb (x) = bTx with the output hq(x) of the nonlinear network layer. This model simulates the actual clinical treatment risk of the population and has a strong generalization ability. The model uses weight decay regularization, batch normalization, and dropout to prevent overﬁtting. The loss function of deep learning is set as the Cox partial likelihood with constraints and is deﬁned as Eligibility criteria and clinical information The inclusion criteria speciﬁed patients with pathologic stage I to IV; and complete resection of microscopic tumors. Patients with secondary malignancies, perioperative mortality, and missing of clinical records were excluded from the study. A ﬂow chart of dataset construction is shown in Figure S1. where q is a parameter of the neural network, a is the regular coefﬁcient, and ^h q(x) is the output of the model. NE represents the number of patients who eventually died (14). The loss function is used to estimate the degree of inconsistency between the predicted value and the real value of the model. The smaller the loss function is, the better the robustness of the model is. The model uses the Adam optimization algorithm to optimize the loss function and update the parameters (16). Random search was used to optimize the hyper-parameters of the network because it is more efﬁcient than grid search when dealing with high-dimensional data (17). Random search ﬁnds optimal model hyper-parameters by selecting a random combination of parameters from the search space. In this study, we performed a randomized hyper-parameter search over the number of layers in [2, 7], the number of neurons in each layer in [4, 100], the learning rate in [0.00001, 0.01]. The model structure was optimized using 500 iterations of random search on the training set for predicting the survival of patients with EC. Hyper-parameters search showed increasing the number of hidden layers can lead to improved model performance until the number of hidden layers exceeded three. So a 5-layer network with three hidden layers was a good choice. Similarly, the number of neurons in each layer was optimized according to random search results. Training curves Figure 2A shows the training loss curves of the survival network. The accuracy of the model during the training process was represented by the loss function. The loss function continues to decrease with an increase in the number of iterations. Within 200 epochs, the curve is relatively smooth, indicating that the model has a strong ﬁtting ability and can quickly learn effective discriminant feature information from the training samples. While the model has a good ﬁtting ability on the training set, it also maintains a good generalization ability on the test and validation sets. Figures 2B, C show the training loss curves for the two treatment recommendation models. Owing to the decrease in data diversity in the recommended training set, the decrease in the loss function was smoother than that of the survival model. Next, patients who underwent esophagectomy were screened from the three datasets and divided into the surgery alone and adjuvant therapy groups according to whether they received adjuvant radiotherapy and/or chemotherapy (Figure S1). The data in the surgery alone (n=939) and adjuvant therapy (n=568) training sets were used to train two DeepSurv risk prediction models. The survival risk for each patient on different treatment regimens in the test set (SEER: n=387; CHINA: n=383) was predicted, and treatment options with a lower risk were recommended (Figure 1). Finally, patients in the test set were divided into two groups based on whether the recommended treatment was used. We used the Kaplan–Meier method to analyze the ECSS between different groups, and the log-rank test was used to compare survival curves. Statistical Analysis First, a calibration plot was used to test the consistency between the 3-year and 5-year ECSS predicted by the DeepSurv model and the actual survival of each case in the test dataset. The calibration plot (Figure 3) shows that most points are arranged around a straight line at an angle of 45° to the x-axis, indicating that the predicted value is very close to the actual value. A two-sided P value less than 0.05 was considered statistically signiﬁcant. The Akaike information criterion (AIC) value was calculated to assess the risk of overﬁtting (18). The deep learning model was developed using Python (version 3.6.7). The CPH regression model and the C statistical were determined by survival, survminer, and rms packages with R statistical software (Version 4.2.0), and the survival curves were plotted using GraphPad Prism 7 (GraphPad Software). In the SEER test set, the prediction performance of DeepSurv was better than that of TNM staging (C-index=0.753 vs. 0.638), and similar results were obtained using the CHINA test set (C- index=0.687 vs. 0.643). The C-index of the surgery alone model in the SEER and CHINA test sets was 0.734 and 0.689, respectively. The C-index of the adjuvant therapy model in the SEER and CHINA test sets was 0.721 and 0.634, respectively. The feature component weightings in the DeepSurv model are listed in Table S2. Baseline characteristics and survival The performances of the CPH and DeepSurv models in predicting the ECSS were also compared. Table 2 lists the factors included in the CPH model. The DeepSurv model performed better than the CPH model in the SEER test set (C-index=0.753 vs. 0.728) and CHINA test set (C-index=0.687 vs. 0.655). The AIC values of the TNM stage, CPH, and DeepSurv model were 70521, 69331 and 69262, respectively. According to the inclusion criteria, 9069 patients with EC were included in this study. The baseline clinical characteristics of the patients are shown in Table 1. A total of 8,569 patients from the SEER database were included. The median (interquartile range) age was 65 (23-101), and the major race was white (7293[95.1%]). The majority of tumors were in the lower third of the esophagus (6091 [71.1%]), stage IV disease (1753[20.4%]), and adenocarcinoma (5883[68.6%]). The median (interquartile range) follow-up time was 15 (0-155) months. During the follow-up time, 5469 patients (63.8%) died with their cause of death attributed to EC. There were 500 patients diagnosed with EC in the CHINA database. In that dataset, 227 patients died with their cause of death attributed to EC Data analysis First, a 5-layer neural network was trained to predict the ECSS of the patients in the training dataset (n=6855) (Figure 1). To validate the prediction performance, we used Harrell’s C FIGURE 1 Diagram of the study procedure. FIGURE 1 Diagram of the study procedure. 03 Frontiers in Oncology frontiersin.org Huang et al. 10.3389/fonc.2022.971190 over a median (interquartile range) follow-up time of 47 (2- 155) months. over a median (interquartile range) follow-up time of 47 (2- 155) months. statistic and calibration plots to evaluate network discrimination and calibration in the SEER (n=1714) and CHINA (n=500) test datasets. An additional CPH regression model was performed following the backward stepwise approach, using all variables included in the DeepSurv model. The CPH model is a classic model for clinical survival analysis that uses a linear function hb (x) = bTx to estimate the true risk function h(x). The prediction performances of the DeepSurv, CPH, and TNM staging models were compared using the C statistic. Frontiers in Oncology Treatment recommender The baseline clinical characteristics of the patients included in the treatment recommendation study are presented in Table 3. By plotting the Kaplan–Meier survival curve, the Frontiers in Oncology 04 frontiersin.org Huang et al. 10.3389/fonc.2022.971190 TABLE 1 Characteristics of patients in the whole data sets of survival analysis. TABLE 1 Characteristics of patients in the whole data sets of survival analysis. Characteristic Data set, No. (%) Training Test1 (SEER) Test2 (CHINA) Age at diagnosis, median (range), y 65 (23,101) 65 (23,97) 62 (34,88) Race White 5854 (85.4) 1439 (84.0) 0 (0) Black 648 (9.5) 176 (10.2) 0 (0) Others1 353 (5.1) 99 (5.8) 500 (100) Sex Male 5505 (80.3) 1386 (80.9) 350 (70.0) Female 1350 (19.7) 328 (19.1) 150 (30.0) Marital status Widowed 673 (9.8) 206 (12.0) 18 (3.6) Married 4150 (60.5) 1008 (58.8) 404 (80.8) Single 1168 (17.0) 290 (16.9) 47 (9.4) Divorced 780 (11.5) 182 (10.6) 28 (5.6) Separated 70 (1.0) 25 (1.5) 3 (0.6) Unmarried or domestic partner 14 (0.2) 3 (0.2) 0 (0) Primary site Upper third of esophagus 367 (5.4) 76 (4.4) 53 (10.6) Middle third of esophagus 1133 (16.5) 307 (17.8) 251 (50.2) Lower third of esophagus 4883 (71.2) 1208 (70.5) 169 (33.8) Overlapping lesion of esophagus 301 (4.4) 78 (4.6) 8 (1.6) Cervical esophagus 115 (1.7) 25 (1.5) 1 (0.2) Abdominal esophagus 56 (0.8) 20 (1.2) 18 (3.6) Histologic type Adenocarcinoma 4690 (68.4) 1193 (69.6) 14 (2.8) Squamous cell carcinoma 2165 (31.6) 521 (30.4) 486 (97.2) Grade Grade III, Poorly differentiated 3277 (47.8) 803 (46.8) 120 (24.0) Grade II, Moderately differentiated 3110 (45.4) 807 (47.1) 347 (69.4) Grade I, Well differentiated 468 (6.8) 104 (6.1) 33 (6.6) Stage (AJCC 7th) ` IA 657 (9.6) 168 (9.7) 25 (5.0) IB 869 (12.7) 228 (13.3) 70 (14.0) IIA 321 (4.7) 63 (3.7) 60 (12.0) IIB 1316 (19.2) 327 (19.1) 155 (31.0) IIIA 1342 (19.6) 332 (19.4) 111 (22.2) IIIB 454 (6.6) 123 (7.2) 50 (10.0) IIIC 496 (7.2) 120 (7.0) 28 (5.6) IV 1400 (20.4) 353 (20.6) 1 (0.2) T stage T1a 558 (8.1) 130 (7.5) 27 (5.4) T1b 506 (7.4) 124 (7.2) 64 (12.8) T1 NOS 1026 (15.0) 284 (16.6) 0 (0) T2 962 (14.0) 210 (12.3) 95 (19.0) T3 2908 (42.4) 743 (43.3) 302 (60.4) T4a 307 (4.5) 70 (4.1) 4 (0.8) T4b 246 (3.6) 61 (3.6) 8 (1.6) (Continued) 05 Frontiers in Oncology frontiersin.org Huang et al. 10.3389/fonc.2022.971190 Characteristic Data set, No. Model visualization clinical prognosis of the two groups of patients (those who followed the treatment recommendation vs. those who did not follow the treatment recommendation) were compared (Figure 4). The survival rate of patients who followed the treatment recommendations was signiﬁcantly higher than that of patients who did not (SEER: hazard ratio [HR], 0.753; 95% CI, 0.556-0.987; P=0.042 vs. CHINA: HR, 0.633; 95% CI, 0.459- 0.834; P=0.0003). ECSS favored surgery alone compared with surgery combined with adjuvant therapy in the subgroup with surgery alone recommendation (SEER: HR, 0.745; 95% CI, 0.543-0.983; P=0.044 vs. CHINA: HR, 0.643; 95% CI, 0.412- 0.967; P=0.035). In the subgroup with adjuvant therapy recommendation, patients who only received surgical treatment experienced signiﬁcantly worse ECSS than those who received surgery combined with adjuvant therapy in the CHINA dataset (HR, 1.657; 95% CI, 1.138-2.639; P=0.012). No signiﬁcant difference in ECSS was observed between the two treatment opinions in the SEER dataset (HR, 1.782; 95% CI, 0.670-6.252; P=0.225). clinical prognosis of the two groups of patients (those who followed the treatment recommendation vs. those who did not follow the treatment recommendation) were compared (Figure 4). The survival rate of patients who followed the treatment recommendations was signiﬁcantly higher than that of patients who did not (SEER: hazard ratio [HR], 0.753; 95% CI, 0.556-0.987; P=0.042 vs. CHINA: HR, 0.633; 95% CI, 0.459- 0.834; P=0.0003). ECSS favored surgery alone compared with surgery combined with adjuvant therapy in the subgroup with surgery alone recommendation (SEER: HR, 0.745; 95% CI, 0.543-0.983; P=0.044 vs. CHINA: HR, 0.643; 95% CI, 0.412- 0.967; P=0.035). In the subgroup with adjuvant therapy recommendation, patients who only received surgical treatment experienced signiﬁcantly worse ECSS than those who received surgery combined with adjuvant therapy in the CHINA dataset (HR, 1.657; 95% CI, 1.138-2.639; P=0.012). No signiﬁcant difference in ECSS was observed between the two treatment opinions in the SEER dataset (HR, 1.782; 95% CI, 0.670-6.252; P=0.225). We have designed an interactive interface to more intuitively display the treatment recommendations provided by the DeepSurv model. The interface includes user input area on the left and treatment recommendation area on the right (Figure 5). Surgeons can input the patient’s prognostic information in the user input area, and click the treatment recommendation button to see the survival risk of different treatment methods in the treatment recommendation area (Supplement Video). This interactive interface helps surgeons to choose treatment options with lower survival risk. Treatment recommender (%) Training Test1 (SEER) Test2 (CHINA) T4 NOS 342 (5.0) 92 (5.4) 0 (0) N stage N0 3373 (49.2) 859 (50.1) 279 (55.8) N1 2361 (34.4) 554 (32.3) 140 (28.0) N2 788 (11.5) 223 (13.0) 63 (12.6) N3 333 (4.9) 78 (4.6) 18 (3.6) M stage M0 5455 (79.6) 1361 (79.4) 499 (99.8) M1 1400 (20.4) 353 (20.6) 1 (0.2) Therapy to primary site None 3553 (51.8) 925 (54.0) 0 (0) Esophagectomy 3095 (45.1) 729 (42.5) 500 (100) Local tumor destruction 207 (3.1) 60 (3.5) 0 (0) Radiation sequence No radiation 4724 (68.9) 1243 (72.5) 314 (62.8) Radiation prior to surgery 1529 (22.3) 331 (19.3) 69 (13.8) Radiation after surgery 513 (7.5) 120 (7.0) 117 (23.4) Others2 89 (1.3) 20 (1.2) 0 (0) Chemotherapy Yes 2177 (31.8) 1143 (66.7) 282 (56.4) No 4678 (68.2) 571 (33.3) 218 (43.6) ECSS Alive 2486 (36.3) 614 (35.8) 273 (54.6) Dead 4369 (63.7) 1100 (64.2) 227 (45.4) 1American Indian/AK Native, Asian/Paciﬁc Islander. 2Radiation before and after surgery/Surgery both before and after radiation/Sequence unknown, but both were given/Intraoperative radiation with other radiation before/after surgery. ECSS, Esophageal Cancer-Speciﬁc Survival; AJCC, American Joint Committee on Cancer; NOS, Not otherwise speciﬁc. American Indian/AK Native, Asian/Paciﬁc Islander. 2Radiation before and after surgery/Surgery both before and after radiation/Sequence unknown, but both were given/Intraoperative radiation with other radiation before/after surgery. ECSS, Esophageal Cancer-Speciﬁc Survival; AJCC, American Joint Committee on Cancer; NOS, Not otherwise speciﬁc. Discussion This study demonstrated the performance of the DeepSurv model in predicting the prognosis of EC patients, providing treatment recommendations, and found that the performance of Frontiers in Oncology 06 frontiersin.org frontiersin.org Huang et al. 10.3389/fonc.2022.971190 A B C FIGURE 2 Training loss curves of networks in the survival network (A), treatment recommendation network of surgery alone (B), and treatment recommendation network of adjuvant therapy (C). The x-axis represents the number of iterations, and the y-axis represents the loss function. A B B C FIGURE 2 Training loss curves of networks in the survival network (A), treatment recommendation network of surgery alone (B), and treatment recommendation network of adjuvant therapy (C). The x-axis represents the number of iterations, and the y-axis represents the loss function. 07 Frontiers in Oncology frontiersin.org frontiersin.org Huang et al. 10.3389/fonc.2022.971190 A B D C FIGURE 3 Calibration plots for Esophageal Cancer-Speciﬁc Survival (ECSS) for the DeepSurv model. (A) 3-year and (B) 5-year ECSS of Surveillance, Epidemiology, and End Results (SEER) dataset and (C) 3-year and (D) 5-year ECSS of CHINA dataset. A B A B B D D C C D FIGURE 3 Calibration plots for Esophageal Cancer-Speciﬁc Survival (ECSS) for the DeepSurv model. (A) 3-year and (B) 5-year ECSS of Surveillan Epidemiology, and End Results (SEER) dataset and (C) 3-year and (D) 5-year ECSS of CHINA dataset. response prediction (25), and in other ﬁelds. To date, there have been few studies on the application of deep learning neural networks to survival prediction in patients with EC. Moﬁdi et al. (26) used artiﬁcial neural networks to predict the 1-year and 3- year survival rates of postoperative EC patients, with accuracy rates reaching 88% and 91.5%, respectively, while the accuracy rates of TNM staging were only 71.6% and 74.7%, respectively. Sun et al. developed a survival risk prediction model for EC based on nine blood indices (27). Lin et al. developed a 3D attention autoencoder-based survival prediction network for esophageal cancer using pretreatment CT images (28). Rahman and his colleges demonstrated that the Random Survival Forest model performed better than TNM stage for survival prediction of patients after esophagectomy (29). However, these studies generally have the disadvantages of small sample size and lack of external validation. the deep learning neural network in predicting ECSS was better than that of the CPH model and TNM staging. Frontiers in Oncology Discussion Additionally, this study found that patients who followed the treatment plan recommended by the DeepSurv model experienced signiﬁcantly better ECSS than those who did not. A series of nomograms have been reported to predict the survival of patients with EC (6–9). Shao et al. (8) established a nomogram to predict the survival of patients with EC undergoing radical resection which included seven variables with the C-index of internal and external validation set as 0.66 and 0.65, respectively. Nomogram is a CPH-based risk prediction model that assumes that the risk of death is a linear combination of its covariates. However, in a real clinical scenario, the assumption that the risk function is linear may be oversimpliﬁed. Therefore, a more complex survival model is required to better ﬁt survival data to the nonlinear risk function. Neural networks are widely used in the diagnosis of endoscopic and radiological imaging of EC (19–22), evaluation of the depth of tumor invasion and lymph node metastasis (23, 24), treatment DeepSurv, ﬁrst proposed by Katzman et al., is a multilayer perceptron similar to the Faraggi–Simon network (14). The Faraggi–Simon network is a feed-forward neural network, and Frontiers in Oncology 08 frontiersin.org frontiersin.org Huang et al. 10.3389/fonc.2022.971190 TABLE 2 The variables included in the Cox proportional hazard model. Variable Univariable Analysis Multivariable Analysis P HR 95% CI b P Age <0.001 1.0119 1.0090-1.0147 0.0118 <0.001 Sex 0.77 – – – – Race <0.001 1.0714 1.0099-1.1367 0.0689 0.022 Marital status <0.001 1.0453 1.0234-1.0676 0.0443 <0.001 Primary site 0.043 1.0706 1.0297-1.1131 0.0682 <0.001 Histologic <0.001 0.8748 0.8117-0.9428 -0.1338 <0.001 Grade <0.001 1.1706 1.1116-1.2328 0.1576 <0.001 Therapy to primary site <0.001 0.5273 0.4779-0.5818 -0.64 <0.001 Sequence of radiation <0.001 0.983 0.9499-1.0173 -0.0171 0.328 Chemotherapy 0.28 – – – – Regional nodes examined <0.001 0.9813 0.9764-0.9862 -0.0189 <0.001 Regional nodes positive <0.001 1.0865 1.0720-1.1013 0.0835 <0.001 Stage (AJCC 7th) <0.001 1.156 1.1170-1.1964 0.145 <0.001 T stage <0.001 0.936 0.8974-0.9763 -0.0661 0.002 N stage <0.001 0.9604 0.9130-1.0103 -0.0404 0.118 M stage <0.001 0.7126 0.5885-0.8628 -0.3389 <0.001 CS tumor size <0.001 1.0023 1.0016-1.0029 0.0023 <0.001 CS extension1 <0.001 1.0011 1.0007-1.0015 0.0011 <0.001 CS mets at DX2 <0.001 1.0156 1.0118-1.0194 0.0155 <0.001 1Details are available from: https://web2.facs.org/cstage0205/esophagus/Esophagus_bbb.html. 2Details are available from: https://web2.facs.org/cstage0205/esophagus/Esophagus_hbg.html. HR, Hazard Ratio; CI, Conﬁdence Interval; AJCC, American Joint Committee on Cancer; NOS, Not otherwise speciﬁc. model is superior to the TNM and CPH models in predicting the ECSS. Frontiers in Oncology 1Details are available from: https://web2.facs.org/cstage0205/esophagus/Esophagus_bbb.html. 2Details are available from: https://web2.facs.org/cstage0205/esophagus/Esophagus_hbg.html. HR, Hazard Ratio; CI, Conﬁdence Interval; AJCC, American Joint Committee on Cancer; NOS, Not otherwise speciﬁc. Discussion its advantage is that it can achieve prognostic prediction without performing feature selection on multiple variables. She et al. found that DeepSurv was superior to the traditional linear model for predicting lung cancer-speciﬁc survival (15). In this study, for the ﬁrst time, the DeepSurv model was used to analyze large- scale EC clinical data, and the model was validated using independent external data, which helped address deﬁciencies in previous studies. This model includes 19 factors and 96 features, whereas the CPH model constructed with the same data includes only 15 variables. The C statistic of the DeepSurv model was better than that of the CPH model and TNM staging in both the SEER and CHINA test datasets, indicating that the DeepSurv model has better discrimination ability. Meanwhile, the DeepSurv model with a low AIC indicated a better model ﬁt. Baseline clinical characteristics revealed that the training cohort was dominated by white adenocarcinoma patients with stage IV disease, and more than half of the patients did not receive surgical treatment. The external validation cohort included all Asian patients; the pathological type was mostly squamous cell carcinoma and the stage was mostly stage IIB. A better C statistic can still be obtained in the validation data that are completely different from the training set, indicating that the DeepSurv Currently, there is no consensus regarding the use of adjuvant treatment after radical esophagectomy. Studies have shown that postoperative adjuvant radiotherapy can improve the survival rate of patients with lymph node metastasis (30, 31). For pT2-3N0M0 patients without lymph node metastasis, studies have shown that the use of conformal radiotherapy as postoperative radiotherapy may improve overall survival (OS) and disease-free survival rate (32). A retrospective study reported that postoperative adjuvant chemotherapy could improve the survival rate of patients with esophageal squamous cell carcinoma with lymph node metastasis (33). Postoperative adjuvant chemotherapy is recommended for patients with adenocarcinoma of the esophagus and esophagogastric junction (34). However, no large randomized controlled clinical study has conﬁrmed these conclusions. Deng et al. used the nomogram total score as a reference for postoperative adjuvant treatment in EC (7), and found that for patients with scores between 72 and 227, the 5-year OS rate could be improved by at least 10% through postoperative adjuvant therapy. The advantage of the deep learning model Frontiers in Oncology 09 frontiersin.org Data set, No. Discussion for treatment recommendation is that if the clinical features that affect the prognosis are input into the model, the risk of different treatment plans can be immediately obtained, which is conducive to clinical decision making. In our study, the treatment plan recommended by the deep learning model conferred survival beneﬁts to patients. In subgroup analysis, postoperative adjuvant therapy cannot improve the prognosis of patients with recommendations for surgery alone using the deep learning model. This result is similar to that of previous studies, indicating that very low-risk and very high-risk patients have limited beneﬁts from postoperative adjuvant therapy (7). On the other hand, surgery combined with adjuvant therapy signiﬁcantly improved ECSS in patients with adjuvant therapy recommendations in the CHINA dataset. Unfortunately, no signiﬁcant difference in the ECSS was observed between the two treatment opinions in the SEER dataset, which may be related to the lack of samples in the adjuvant therapy recommendation subgroup. Our ﬁndings show the potential of the deep learning model as a clinical decision-making tool to help guide patient management. for treatment recommendation is that if the clinical features that affect the prognosis are input into the model, the risk of different treatment plans can be immediately obtained, which is conducive to clinical decision making. In our study, the treatment plan recommended by the deep learning model conferred survival beneﬁts to patients. In subgroup analysis, postoperative adjuvant therapy cannot improve the prognosis of patients with recommendations for surgery alone using the deep learning model. This result is similar to that of previous studies, indicating that very low-risk and very high-risk patients have limited beneﬁts from postoperative adjuvant therapy (7). On the other hand, surgery combined with adjuvant therapy signiﬁcantly improved ECSS in patients with adjuvant therapy recommendations in the CHINA dataset. Unfortunately, no signiﬁcant difference in the ECSS was observed between the two treatment opinions in the SEER dataset, which may be related to the lack of samples in the adjuvant therapy recommendation subgroup. Our ﬁndings show the potential of the deep learning model as a clinical decision-making tool to help guide patient management. Clearly, deep learning has advantages in analyzing the nonlinear relationship between clinical features and clinical outcomes; however, there are still shortcomings. First, the function of a deep learning network is similar to a black box, which makes the prediction process difﬁcult to interpret. Discussion Second, the deep learning model based on a fully connected neural network is more sensitive to noise, and its feature expression ability and robustness still need to be improved. Although the sample of SEER database is large, however, the SEER database has drawbacks: (1) lack of key pathological features that are closely associated with prognosis, such as marginal status, vessel invasion, resection status (R0/R1/R2); (2) there was no information regarding chemotherapy regimen, drugs, dosage, and toxicities; (3) although there is information on the anatomical target ﬁeld of radiation, further information on speciﬁc radiation type is lacking. These data points are incredibly important for prognosticating survival. Therefore, this model needs to be further improved. In addition, because of the single-center design and insufﬁcient number of cases, the external validation of this model is insufﬁcient, and further studies are needed to verify the advantages of the deep learning network in survival prediction. Discussion (%) Training Test1 (SEER) Test2 (CHINA) (26,92) 63 (29,90) 60 (34,88) 0 (88.9) 334 (86.3) 0 (0) (5.7) 27 (7.0) 0 (0) (5.4) 26 (6.7) 383 (100) 2 (83.1) 324 (83.7) 269 (70.2) 5 (16.9) 63 (16.3) 114 (29.8) 5 (7.6) 27 (7.0) 10 (2.6) 0 (65.7) 248 (64.1) 335 (87.5) 4 (15.5) 68 (17.6) 23 (6) 3 (10.2) 39 (10.1) 14 (3.7) (0.9) 4 (1.0) 1 (0.3) (0.1) 1 (0.3) 0 (0) (2.1) 12 (3.1) 47 (12.3) 0 (13.3) 49 (12.7) 194 (50.7) 5 (79.2) 302 (78.0) 117 (30.5) (3.4) 12 (3.1) 17 (4.4) (0.7) 2 (0.5) 1 (0.3) (1.3) 10 (2.6) 7 (1.8) 2 (78.4) 304 (78.6) 14 (3.7) 5 (21.6) 83 (21.4) 369 (96.3) 7 (42.9) 168 (43.4) 84 (21.9) 0 (46.4) 180 (46.5) 278 (72.6) 0 (10.7) 39 (10.1) 21 (5.5) 15.10 ±11.40 13.99±11.20 25.10 ±14.10 1.67±3.49 1.81±3.67 1.36±2.65 329 (21.8) 76 (19.6) 23 (6) (Continued) Data set, No. (%) Training Test1 (SEER) Test2 (CHINA) 252 (16.7) 60 (15.5) 61 (15.9) 83 (5.5) 22 (5.7) 37 (9.7) 310 (20.6) 75 (19.4) 119 (31.1) 220 (14.6) 55 (14.2) 75 (19.6) 119 (7.9) 38 (9.8) 46 (12) 119 (7.9) 33 (8.5) 21 (5.5) 75 (5.0) 28 (7.2) 1 (0.3) 214 (14.2) 52 (13.4) 23 (6) 350 (23.2) 79 (20.4) 57 (14.9) 35 (2.3) 10 (2.6) 0 (0) 251 (16.7) 54 (14.0) 66 (17.2) 592 (39.3) 165 (42.6) 229 (59.8) 9 (0.6) 5 (1.3) 2 (0.5) 6 (0.4) 3 (0.8) 6 (1.6) 50 (3.3) 19 (4.9) 0 (0) 843 (55.9) 218 (56.3) 219 (57.2) 354 (23.5) 83 (21.4) 94 (24.5) 192 (12.8) 55 (14.2) 55 (14.4) 118 (7.8) 31 (8.0) 15 (3.9) 1432 (95) 359 (92.8) 382 (99.7) 75 (5.0) 28 (7.2) 1 (0.3) 1173 (77.8) 312 (80.6) 314 (82) 334 (22.2) 75 (19.4) 69 (18) 522 (34.6) 145 (37.5) 167 (43.6) 985 (65.4) 242 (62.5) 216 (56.4) 939 (62.3) 234 (60.5) 205 (53.5) 568 (37.7) 153 (39.5) 178 (46.5) (Continued) .5) .5) ed) Huang et al. 10.3389/fonc.2022.971190 10.3389/fonc.2022.971190 TABLE 3 Continued Characteristic Data set, No. (%) Training Test1 (SEER) Test2 (CHINA) ECSS Alive 765 (50.8) 184 (47.5) 201 (52.5) Dead 742 (49.2) 203 (52.5) 182 (47.5) 1 American Indian/AK Native, Asian/Paciﬁc Islander. ECSS, Esophageal Cancer-Speciﬁc Survival; AJCC, American Joint Committee on Cancer; NOS, Not otherwise speciﬁc. Conclusions In this study, for the ﬁrst time, a neural network-based CPH model was used to analyze the relationship between various clinical features and survival outcomes of patients with EC in a real clinical scenario, and satisfactory results were achieved. As a new analytical tool, the DeepSurv model will likely become more widely applied in outcome prediction and treatment recommendations for patients with EC. 12 frontiersin.org frontiersin.org Huang et al. 10.3389/fonc.2022.971190 A B D E F C FIGURE 4 Esophageal cancer-speciﬁc survival comparisons of Surveillance, Epidemiology, and End Results (SEER) test dataset (A), SEER surgery alone recommendation test dataset (B), and SEER adjuvant therapy recommendation test dataset (C). Esophageal cancer-speciﬁc survival comparisons of CHINA test dataset (D), CHINA surgery alone recommendation test dataset (E), and CHINA adjuvant therapy recommendation test dataset (F). A B C B C E F D FIGURE 4 Esophageal cancer-speciﬁc survival comparisons of Surveillance, Epidemiology, and End Results (SEER) test dataset (A), SEER surgery alone recommendation test dataset (B), and SEER adjuvant therapy recommendation test dataset (C). Esophageal cancer-speciﬁc survival comparisons of CHINA test dataset (D), CHINA surgery alone recommendation test dataset (E), and CHINA adjuvant therapy recommendation test dataset (F). FIGURE 5 User interface to display the treatment recommendations provided by the DeepSurv model. FIGURE 5 User interface to display the treatment recommendations provided by the DeepSurv model. Frontiers in Oncology References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Fujian Provincial Hospital (protocol code k2021-12-042). Patient consent was waived due to the retrospective nature of this study. Data availability statement form via email. The data that support the ﬁndings of this study are available from the SEER database but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Requests to access these datasets should be directed to xunyuxu@sina.com. The data analyzed in this study is subject to the following licenses/restrictions: We obtained permission to access the database after signing and submitting the SEER Research Data Agreement 13 Frontiers in Oncology frontiersin.org frontiersin.org Huang et al. 10.3389/fonc.2022.971190 10.3389/fonc.2022.971190 Funding The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fonc.2022.971190/full#supplementary-material This research was funded by the Special Fund of Fujian Provincial Finance Department [grant number (2021)917] and Publisher’s note CH: Validation, Writing - Original Draft, Writing - Review & Editing, Visualization. YD: Resources, Writing - Original Draft, Writing - Review & Editing, Visualization. QC: Resources. HC: Methodology, Software. YL: Investigation, Data Curation. JW: Investigation, Data Curation. XX: Conceptualization, Project administration, Funding acquisition. XC: Conceptualization, Methodology, Software, Formal analysis, Supervision. All authors contributed to the article and approved the submitted version. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Frontiers in Oncology frontiersin.org References Survival outcome prediction in cervical cancer: Cox models vs deep-learning model. Am J Obstet Gynecol (2019) 220(4):381.e1–.e14. doi: 10.1016/ j.ajog.2018.12.030 5. Rice T, Ishwaran H, Hofstetter W, Kelsen D, Apperson-Hansen C, Blackstone E. 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https://openalex.org/W2096843949	https://europepmc.org/articles/pmc3259321?pdf=render	English	null	The skeleton and musculature on foetal MRI	Insights into imaging	2,011	cc-by	5,914	Abstract Background Magnetic resonance imaging (MRI) is used as an adjunct to ultrasound in prenatal imaging, the latter being the standard technique in obstetrical medicine. Methods Initial results demonstrate the ability to visualise the foetal skeleton and muscles on MRI, and highlight the potentially useful applications for foetal MRI, which has significantly profited from innovations in sequence tech- nology. Echoplanar imaging, thick-slab T2-weighted (w) imaging, and dynamic sequences are techniques comple- mentary to classical T2-w imaging. Background Magnetic resonance imaging (MRI) is used as an adjunct to ultrasound in prenatal imaging, the latter being the standard technique in obstetrical medicine. Methods Initial results demonstrate the ability to visualise the foetal skeleton and muscles on MRI, and highlight the potentially useful applications for foetal MRI, which has significantly profited from innovations in sequence tech- nology. Echoplanar imaging, thick-slab T2-weighted (w) imaging, and dynamic sequences are techniques comple- mentary to classical T2-w imaging. Conclusion More research and technical refinement will be necessary to investigate normal human skeletal develop- ment and to identify MR imaging characteristics of skeletal abnormalities. Keywords Foetal MRI . Skeleton . Muscles . Congenital abnormalities U. Nemec: D. Prayer Department of Radiology, Division of Neuroradiology and Musculoskeletal Radiology, Medical University Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria Insights Imaging (2011) 2:309–318 DOI 10.1007/s13244-011-0075-6 Insights Imaging (2011) 2:309–318 DOI 10.1007/s13244-011-0075-6 REVIEW REVIEW The skeleton and musculature on foetal MRI Ursula Nemec & Stefan F. Nemec & Deborah Krakow & Peter C. Brugger & Gustavo Malinger & John M. Graham Jr. & David L. Rimoin & Daniela Prayer Received: 19 October 2010 /Revised: 21 November 2010 /Accepted: 27 January 2011 /Published online: 19 February 2011 # European Society of Radiology 2011 Results Recent study data indicate that foetal MRI may be useful in the imaging of spinal dysraphism and in differenti- ating between isolated and complex skeletal deformities with associated congenital malformations, which might have an impact on pre- and postnatal management. Introduction Foetal magnetic resonance imaging (MRI) has become an increasingly used imaging technique, serving as an adjunct to ultrasound in prenatal diagnosis [1–4]. Numerous recent studies have emphasised the benefits of MRI for the demonstration of congenital abnormalities of the brain and lungs, or complex syndromes [1–7]. S. F. Nemec (*): J. M. Graham Jr.: D. L. Rimoin Medical Genetics Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, PACT Suite 400, Los Angeles, CA 90048, USA e-mail: stefan.nemec@cshs.org However, there are almost no reports in the literature describing MRI for the prenatal visualisation and diagnosis of skeletal and muscular abnormalities, with the exception of spinal dysraphism [8–10]. Therefore, the authors sought to demonstrate and summarise their preliminary results for the visualisation of the skeleton in utero on MRI. The imaging techniques are described in detail, and the potential impact of a prenatal MRI diagnosis is discussed. D. Krakow Department of Orthopedic Surgery, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA P. C. Brugger Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, Waehringerstrasse 13, 1090 Vienna, Austria P. C. Brugger Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, Waehringerstrasse 13, 1090 Vienna, Austria Table 1 Application of T2-w single-shot (SSh) turbo- spin-echo (TSE) sequences Imaging G. Malinger Fetal Neurology Clinic, Prenatal Diagnosis Unit, Department of Obstetrics and Gynecology, Tel Aviv University, Tel Aviv, Israel In addition to commonly used T2-weighted (w) imaging to depict foetal anatomy and anomalies at all gestational ages, 310 Insights Imaging (2011) 2:309–318 Parameters Axial, coronal, and sagittal planes; repetition time (TR): shortest; echo time (TE): 100 ms; TSE factor: 92; field of view: 200–230 mm; matrix: 256×153; slice thickness: 3–4 mm; slices: 18; flip angle: 90°; duration: 18.7 s Assessment Whole body evaluation Clinical application Head size and shape (microcephaly; encephaloceles) Brain (associated malformations; acquired anomalies) (TE: 140 ms in GW 18–29) Face and foetal profile (facial clefts) Orbits (content; interorbital distance) Teeth buds and hard palate (cleft palate) External ear size, shape, and position; and fluid-filled inner ears Thorax shape and lung volume (lung hypoplasia in skeletal dysplasias) Spine (content/extent of spinal dysraphism) Arms and legs; hands and feet (size, shape, number of fingers/toes) Musculature (decreased thickness; fatty atrophy after GW 30) Table 1 Application of T2-w single-shot (SSh) turbo- spin-echo (TSE) sequences Table 1 Application of T2-w single-shot (SSh) turbo- spin-echo (TSE) sequences which is the case with conventional T2-w imaging, is changing. Recent developments in sequence technology have overcome these previous deficiencies. EP sequences now provide the delineation of bony structures, depending on the gestational age. Currently, EPI represents the only technique by which to image the skeleton before 27 gestational weeks (GW) on MRI, as the bones are demonstrated as hypointense structures and the cartilaginous epiphyses as hyperintense structures (Fig. 1, Table 2) [1, 9, 11]. EPI shows a pronounced distinction between bone and adjacent muscula- ture, but low spatial resolution limits the use of this technique [17]. Sequence parameters must be modified in older foetuses, as the distinction between bone and muscles becomes less conspicuous with advancing gestational age. MRI protocols should include imaging techniques such as echo planar imaging (EPI), thick-slab T2-w sequences, and dynamic steady-state free precession (SSFP) sequences to visualise the developing musculoskeletal system [1, 11, 12]. Tables 1, 2, 3 and 4 show the areas of application of these MRI sequences and feature the sequence parameters used in our institution. Table 3 Application of thick-slab T2-w imaging EPI Currently, ultrasound provides information about bone ossifi- cation in prenatal standard imaging. Ossification centres, visualised as areas of increased echogenicity, are evaluated with ultrasound from 9 gestational weeks onwards [13–15]. The paradigm of MRI as “virtually blind to bones” [16], Insights Imaging (2011) 2:309–318 Table 4 Application of dynamic steady-state free precession (SSFP) sequences Parameters Repetition time (TR): 3.14 ms; echo time (TE): 1.57 ms; field of view: 320 mm; matrix: 176 × 110; slice thickness: 30 mm; gap: 0; flip angle: 60°; 4–6 frames per second; up to 8 repetitions; duration: 34 s Assessment Foetal movements Clinical application Imaging of movement patterns of extremities, head, and body; swallowing; and diaphragm excursions Contractures/arthrogryposis Akinesia (foetal akinesia deformation sequence; neuromuscular disorders) Foetal bulk motion Parameters Repetition time (TR): 3.14 ms; echo time (TE): 1.57 ms; field of view: 320 mm; matrix: 176 × 110; slice thickness: 30 mm; gap: 0; flip angle: 60°; 4–6 frames per second; up to 8 repetitions; duration: 34 s Assessment Foetal movements Clinical application Imaging of movement patterns of extremities, head, and body; swallowing; and diaphragm excursions Contractures/arthrogryposis Repetition time (TR): 3.14 ms; echo time (TE): 1.57 ms; field of view: 320 mm; matrix: 176 × 110; slice thickness: 30 mm; gap: 0; flip angle: 60°; 4–6 frames per second; up to 8 repetitions; duration: 34 s p p Assessment Foetal movements Clinical application Imaging of movement patterns of extremities, head, and body; swallowing; and diaphragm excursions Contractures/arthrogryposis Akinesia (foetal akinesia deformation sequence; neuromuscular disorders) Foetal bulk motion Dynamic sequences Thick-slab T2-w sequences Thick-slab T2-w imaging generates a three-dimensional (3D) image of the foetus (Fig. 2, Table 3). The whole foetus is covered by this technique, even in the third trimester, because of a large field of view (FOV) [12], which cannot be supplied by ultrasound. Abnormal proportions and some anomalies may be more easily recognised on a single image, which provides an overview of the whole foetus [12, 18, 19]. Thus, thick-slab T2-w sequences may be useful in cases of clubfeet, focal malpositioning disorders, or arthrogryposis (Fig. 3) [12]. The visualisation of long bones and extremities on commonly used slices from multiplanar MRI sequences may be impaired by the lack of limb continuity. Thick-slab images depict the shape and position of the whole limb on one image. This information may be crucial in cases of limb deformation or deficiency for any reason. The thick-slab T2-w method provides a “shine-through” effect for the inner foetal organs and may represent an alternative to 3D US in certain cases (Fig. 2) [12, 18, 19]. Ultrasound allows real-time visualisation of any plane in foetal imaging, and is, therefore, a true dynamic imaging method, contrary to MRI [20, 21]. The presence, reduction, or absence of foetal movements of the extremities can be better delineated on ultrasound than on foetal MRI [1]. However, the introduction of dynamic SSFP sequences using four to six images per second has permitted the demonstration of movements of the foetal extremities and gross foetal motion (Fig. 4) [1, 11, 22]. MRI may not cover the extremities or even detect any foetal details if the foetus is rapidly moving, which can be overcome with dynamic sequences (Table 4) [11, 23]. Some limb abnormalities will be more obvious, in motion, on dynamic images. Therefore, dynamic MR images are instrumental for the evaluation of extremity malformations, as well as movement disorders. Quantitative movement studies are not performed with MRI [1]. Consequently, the examining physician should be cautious about diagnosing movement reduction on 30–60-s MR studies. The movement pattern of the extremities should Fig. 1 Foetus at 24+2 gestational weeks (GW) with normal skeletal development. Table 3 Application of thick-slab T2-w imaging Parameters Coronal and sagittal planes; repetition time (TR): 8,000 ms; echo time (TE): 400–800 ms; field of view: 210–320 mm; matrix: 256×205; slice thickness: 30–50 mm; flip angle: 90°; up to 15 projections (12°–15° angulation); duration: 8 s Assessment Foetal proportions and surface, whole foetus from different angles Clinical application Facial features (micrognathia; dysmorphic features) Intrauterine growth restriction (IUGR) Dwarfism, mesomelia or rizomelia (skeletal dysplasias) Extremity positioning (contractures, arthrogryposis) Extremity thickness (hydrops; subcutaneous oedema; muscle mass) Limb deformity/deficiency (amniotic bands; clubfeet; skeletal dysplasias) External masses (extensive lymphangiomas) Discontinuity of the body surface (omphalocele; spina bifida) Parameters Coronal and sagittal planes; repetition time (TR): 8,000 ms; echo time (TE): 400–800 ms; field of view: 210–320 mm; matrix: 256×205; slice thickness: 30–50 mm; flip angle: 90°; up to 15 projections (12°–15° angulation); duration: 8 s Assessment Foetal proportions and surface, whole foetus from different angles Clinical application Facial features (micrognathia; dysmorphic features) Intrauterine growth restriction (IUGR) Dwarfism, mesomelia or rizomelia (skeletal dysplasias) Extremity positioning (contractures, arthrogryposis) Extremity thickness (hydrops; subcutaneous oedema; muscle mass) Limb deformity/deficiency (amniotic bands; clubfeet; skeletal dysplasias) External masses (extensive lymphangiomas) Discontinuity of the body surface (omphalocele; spina bifida) Table 2 Application of single-shot fast-field-echo (SSh FFE) sequen- ces (echo-planar imaging) Parameters Coronal and sagittal planes; repetition time (TR): 3,000 ms; echo time (TE): shortest; field of view: 230 mm; matrix: 160 × 95; slice thickness: 4 mm; flip angle: 90°; duration: 12 s Assessment Foetal skeleton Clinical application Facial profile (after GW 28; delineation enhanced because of chemical shift by subcutaneous fat) Hard palate (cleft palate) Imaging of normal hyperintense cartilaginous epiphysis and hypointense diaphysis Bone length and shape (bent bones; skeletal dysplasias) Ossification disorders (osteogenesis imperfecta) Parameters Coronal and sagittal planes; repetition time (TR): 8,000 ms; echo time (TE): 400–800 ms; field of view: 210–320 mm; matrix: 256×205; slice thickness: 30–50 mm; flip angle: 90°; up to 15 projections (12°–15° angulation); duration: 8 s Assessment Foetal proportions and surface, whole foetus from different angles Clinical application Facial features (micrognathia; dysmorphic features) Intrauterine growth restriction (IUGR) Dwarfism, mesomelia or rizomelia (skeletal dysplasias) Extremity positioning (contractures, arthrogryposis) Extremity thickness (hydrops; subcutaneous oedema; muscle mass) Limb deformity/deficiency (amniotic bands; clubfeet; skeletal dysplasias) External masses (extensive lymphangiomas) Discontinuity of the body surface (omphalocele; spina bifida) Parameters Coronal and sagittal planes; repetition time (TR): 8,000 ms; echo time (TE): 400–800 ms; field of view: 210–320 mm; matrix: 256×205; slice thickness: 30–50 mm; flip angle: 90°; up to 15 projections (12°–15° angulation); duration: 8 s Table 2 Application of single-shot fast-field-echo (SSh FFE) sequen- ces (echo-planar imaging) Parameters Coronal and sagittal planes; repetition time (TR): 3,000 ms; echo time (TE): shortest; field of view: 230 mm; matrix: 160 × 95; slice thickness: 4 mm; flip angle: 90°; duration: 12 s Assessment Foetal skeleton Clinical application Facial profile (after GW 28; delineation enhanced because of chemical shift by subcutaneous fat) Hard palate (cleft palate) Imaging of normal hyperintense cartilaginous epiphysis and hypointense diaphysis Bone length and shape (bent bones; skeletal dysplasias) Ossification disorders (osteogenesis imperfecta) Table 2 Application of single-shot fast-field-echo (SSh FFE) sequen- ces (echo-planar imaging) 311 Insights Imaging (2011) 2:309–318 Dynamic sequences The sagittal echo planar (EP) images demonstrate the femur (a) and the humerus (b) with hypointense diaphysis (arrows) and hyperintense proximal and distal epiphysis (arrowheads); normal hypointense ossification of the spine according to the gestational age (c) W) with normal skeletal images demonstrate the e diaphysis (arrows) and hyperintense proximal and distal epiphysis (arrowheads); normal hypointense ossification of the spine according to the gestational age (c) hyperintense proximal and distal epiphysis (arrowheads); normal hypointense ossification of the spine according to the gestational age (c) Fig. 1 Foetus at 24+2 gestational weeks (GW) with normal skeletal development. The sagittal echo planar (EP) images demonstrate the femur (a) and the humerus (b) with hypointense diaphysis (arrows) and 312 Insights Imaging (2011) 2:309–318 Fig. 2 Foetus at 33+0 GW with normal anatomy in polyhydramnios. The thick-slab T2-w sequence enables the visualisation of the whole foetus on one image and shows normal positioning of the extremities ultrasound may be impaired due to acoustic bony shadow- ing [27]. In comparison, T2-w MRI demonstrates the posterior palate consistently. MRI has been used for the detection of isolated cleft palate, as well as clefts of both the primary and secondary palate (Fig. 7) [28]. Isolated cleft lip, however, may be impossible to diagnose on MRI because of partial volume averaging (Tables 1, 2). On MRI, the skull base and cranio-cervical junction should be examined in detail, particularly the width and content of the foramen magnum, as a narrow foramen magnum may occur in some skeletal dysplasias, and cerebellar herniation occurs in Chiari malformations [10]. Thorax and spine The fixed nature of an extremity contracture can be visualised as the absence of changes in the extremity’s position during the course of the examination. Dynamic MRI studies should be evaluated only in combina- tion with morphology to differentiate a possible temporary variant of malpositioning from an actual abnormality that may affect the musculoskeletal system or the nervous system, particularly the brainstem. Foetal akinesia, defined by an absence of motion, could be interpreted as a sign of (neuro) muscular disease in combination with thin muscles and polyhydramnios (Fig. 5); lung hypoplasia and certain facial abnormalities could suggest a Pena-Shokeir phenotype [24]. Fig. 3 Foetus at 31+2 GW with multiple contractures. The thick-slab T2-w image shows an abnormal inward rotation of the foot compared with the course of the calf (arrow), resulting in a clubfoot deformity Thorax and spine Although MRI can visualise the shape of the thorax, which may be essential in some syndromes or skeletal dysplasias (Fig. 8), its predominant task is the assessment of the foetal lungs [1, 5]. Concerning the foetal spine, results indicate that MRI may provide additional information beyond ultrasound in foetuses with spina bifida and subsequent neurosurgery [15]. Based upon advanced resolution, this type of spinal dysraphism can be better evaluated on MRI than on ultrasound, as MRI provides an exact delineation of the cord and the interface between cerebrospinal fluid and the extradural space (Table 1) [15]. The prognosis of spinal dysraphism is influenced by the level and the type of the lesion, the possibility of associated anomalies, ventriculo- megaly, and the type of closure. The options for a pregnant woman in the case of confirmed spina bifida include termination of pregnancy, foetal surgery, and planned Caesarean section with immediate postnatal surgical repair. Overall, MRI has been recommended for preoperative Fig. 2 Foetus at 33+0 GW with normal anatomy in polyhydramnios. The thick-slab T2-w sequence enables the visualisation of the whole foetus on one image and shows normal positioning of the extremities be assessed based on published ultrasound criteria [21] in dynamic sequences, applied five times, at about 5, 10, 15, 20, and 25 min. A modification of the ultrasound criteria [21] (reduced amplitude and speed, or number of participating body parts) may be used to evaluate generalised disorders or focal mobility problems. The fixed nature of an extremity contracture can be visualised as the absence of changes in the extremity’s position during the course of the examination. Dynamic MRI studies should be evaluated only in combina- tion with morphology to differentiate a possible temporary variant of malpositioning from an actual abnormality that may affect the musculoskeletal system or the nervous system, particularly the brainstem. Foetal akinesia, defined by an absence of motion, could be interpreted as a sign of (neuro) muscular disease in combination with thin muscles and polyhydramnios (Fig. 5); lung hypoplasia and certain facial abnormalities could suggest a Pena-Shokeir phenotype [24]. be assessed based on published ultrasound criteria [21] in dynamic sequences, applied five times, at about 5, 10, 15, 20, and 25 min. A modification of the ultrasound criteria [21] (reduced amplitude and speed, or number of participating body parts) may be used to evaluate generalised disorders or focal mobility problems. Skull The size and shape of the foetal head/skull is usually visualised on ultrasound studies [25]. In the case of asymmetrical head enlargement, MRI may be useful in excluding the presence of associated cerebral malforma- tions [1, 26]. Possible encephaloceles should be examined for their content (Fig. 6). Both 2D and 3D ultrasound provides detailed visualisation of cleft lip and the anterior palate [27]. The examination of the posterior palate on Fig. 3 Foetus at 31+2 GW with multiple contractures. The thick-slab T2-w image shows an abnormal inward rotation of the foot compared with the course of the calf (arrow), resulting in a clubfoot deformity Insights Imaging (2011) 2:309–318 313 Fig. 4 Foetus at 27+4 GW with normal motor development. The sequence of dynamic images (from left to right) shows active movement of the extremities, the body, and the head, as well as jaw movements (arrow) and swallowing Fig. 4 Foetus at 27+4 GW with normal motor development. The sequence of dynamic images (from left to right) extremities, the body, and the head, as well as jaw movements (arrow) and swallowing Fig. 4 Foetus at 27+4 GW with normal motor development. The sequence of dynamic images (from left to right) shows active movement of the extremities, the body, and the head, as well as jaw movements (arrow) and swallowing imaging with 3D ultrasound [30, 31]. However, it may be difficult or impossible to assess all extremities sonograph- ically on the dependent side of the foetus, or if the amniotic fluid is severely reduced, or late in gestation. On MRI, EPI sequences can be used to image foetal bone and skeletal development. Details of the developing epiphysis may be recognised (Fig. 11) [1, 11], and disorders of ossification may be visualised (Fig. 12, Table 2). Connolly et al. described the MR imaging appearances of the foetal pig femur at each trimester and correlated the results with histology [32]. In that study, MRI depicted contour changes of the femoral intercondylar notch, changes in the epiph- yseal ossification centre, and changes in the signal intensity and shape of the bone marrow [32]. Currently, the stages of bone development on MRI are not entirely known for human foetuses, and there is only initial experience in the visual- evaluation of spinal dysraphism, particularly for foetuses with an open spinal canal and associated cerebral or cerebellar malformations (Fig. 9) [8, 10]. Skull Congenital scoliosis, as a result of improper formation and/or segmen- tation, is the most frequent congenital deformity of the spine [29]. MRI may be helpful in the diagnosis of malformations or other anomalies often associated with this condition (Fig. 10). Fig. 5 Foetus at 27+1 GW with neuromuscular disease. The thick-slab T2-w image shows the foetus with generalised subcutaneous oedema, thin extremities, and polyhydramnios (a). The axial T2-w image of the thorax and upper extremities demonstrates, in addition to diffuse oedema, partial atrophy of the musculature (arrow) (b) Muscles Magnetic resonance imaging is the most accurate method of delineating any kind of muscular abnormalities in paediatric Fig. 7 Foetus at 30+0 GW with bilateral cleft lip and palate and otherwise normal anatomy. The coronal T2-w image of the face shows a broad fluid (hyperintense) connection between the naso- and oropharynx on both sides (arrows) Long bones and extremities Ultrasound is the method of choice for measuring long bones and observing subtle findings that involve the distal appendages, which can also be observed with foetal MRI [30, 31]. The extremities are particularly well suited to Fig. 5 Foetus at 27+1 GW with neuromuscular disease. The thick-slab T2-w image shows the foetus with generalised subcutaneous oedema, thin extremities, and polyhydramnios (a). The axial T2-w image of the thorax and upper extremities demonstrates, in addition to diffuse oedema, partial atrophy of the musculature (arrow) (b) 314 Insights Imaging (2011) 2:309–318 Fig. 6 Foetus at 32+2 GW with craniofacial maldevelopment. The axial T2-w image of the head shows a large orbitofrontal meningoen- cephalocele, with distinct protrusion of the frontal lobes Fig. 8 Foetus at 32+0 GW with thoracic deformity in unclassified syndrome. The coronal T2-w image shows abnormally shaped lungs, with narrowing of the mid-thorax due to rip deformities; the latter are not clearly visible on the image Fig. 8 Foetus at 32+0 GW with thoracic deformity in unclassified syndrome. The coronal T2-w image shows abnormally shaped lungs, with narrowing of the mid-thorax due to rip deformities; the latter are not clearly visible on the image Fig. 6 Foetus at 32+2 GW with craniofacial maldevelopment. The axial T2-w image of the head shows a large orbitofrontal meningoen- cephalocele, with distinct protrusion of the frontal lobes isation of foetal bones [33]. In growth-restricted foetuses, the MRI appearance of the placenta may provide an indication of the severity and underlying disease process [34]. populations [35], but MRI of the foetal musculature is only in the early stages of investigation. With regard to the structure, normal individual muscles (with few exceptions, e.g., the diaphragm) cannot be delineated, because muscles display a homogeneous T2-w hypointensity [1]. However, tissue composition changes with advancing gestation, as evidenced by EPI sequences. MRI can show the thickness and contours of the skeletal muscles, and atrophy (Fig. 5) [1], which may indicate the presence of a neuromuscular disorder. In addition, T1- and T2-w signals may be pathological with increased T2-w signal intensities (Table 1) [1]. It has been stated that impairment of muscle development must reach a critical stage, occurring relative- ly late in pregnancy, to result in significant changes in foetal motility and morphology [36]. Overall, abnormal muscular development may be observed with limb abnor- malities, such as arthrogryposis, spinal muscle dystrophy, or muscle dystrophy [37, 38]. Complex defects of the musculoskeletal system Abnormalities may be either isolated or may be accom- panied by other defects, particularly in genetic disorders or in chromosomal syndromes [39]. The prenatal diagno- sis of musculoskeletal anomalies should be based on information assembled from imaging combined with biochemical and genetic workups [40, 41]. Prenatal diagnosis will serve as a prognostic tool and in counselling the parents. The management of pregnancies with muscu- loskeletal disease depends mainly on three factors: the time of diagnosis, the severity of disease, and the parents' Fig. 7 Foetus at 30+0 GW with bilateral cleft lip and palate and otherwise normal anatomy. The coronal T2-w image of the face shows a broad fluid (hyperintense) connection between the naso- and oropharynx on both sides (arrows) 315 Insights Imaging (2011) 2:309–318 decision [15]. When identifying a musculoskeletal abnor- mality, the whole foetus should be assessed carefully, particularly the CNS and the lungs, to detect any abnormalities (Fig. 13) [42]. The presence or absence of specific MRI findings will help to differentiate between isolated and complex anomalies. For instance, amniotic bands should be considered in cases of isolated limb deficiency [43]. Figure 14 features a mnemonic about the most important imaging considerations when imaging the extremities. Foetal skeletal dysplasias are usually recognised by ultrasound [41, 44–46]. However, published reports have described ultrasound as only moderately accurate in the Fig. 9 Foetus at 23+3 GW with a Chiari II malformation. (a) The sagittal T2-w image of the whole foetus shows a lumbosacral neural tube defect (ellipsis) and a herniation of the cerebellum (arrow). The sagittal (b) and axial (c) T2-w images of the body and pelvis show a large spinal defect referring to a meningomyelocele that extends from the level of L2 to the end of the sacrum (ellipsis). The axial T2-w image of the brain demonstrates internal hydrocephaly with marked widening of the lateral ventricles (d) Fig. 10 Foetus at 29+1 GW with scoliosis. The sagittal T2-w image of the whole foetus demonstrates a paravertebral cystic lesion referred to as an intrathoracic neurenteric cyst (arrow) decision [15]. When identifying a musculoskeletal abnor- mality, the whole foetus should be assessed carefully, particularly the CNS and the lungs, to detect any abnormalities (Fig. 13) [42]. The presence or absence of specific MRI findings will help to differentiate between isolated and complex anomalies. For instance, amniotic bands should be considered in cases of isolated limb deficiency [43]. adjunct to ultrasound in the diagnosis of lethal skeletal dysplasias [47]. adjunct to ultrasound in the diagnosis of lethal skeletal dysplasias [47]. Fig. 11 Foetuses with normal long-bone development at 24 and 40 GW. The sagittal EP images of the thigh demonstrate hyperintense epiphyseal structures, with no distal femoral ossification centre at 24 GW (a), and a small hypointense ossification centre (arrow) in the older foetus (b) Complex defects of the musculoskeletal system Figure 14 features a mnemonic about the most important imaging considerations when imaging the extremities. Foetal skeletal dysplasias are usually recognised by ultrasound [41, 44–46]. However, published reports have described ultrasound as only moderately accurate in the detection of foetal musculoskeletal anomalies and specific skeletal dysplasias [45, 46], which may indicate a potential future role of MRI in these conditions. Foetal MR imaging may complement the use of molecular genetics to diagnose skeletal dysplasias [33]. After 30 weeks of gestation, conventional radiography of the maternal abdomen may also help to identify possible bone abnormalities [45]. Recent case series report the application of prenatal computed tomography with 3D reconstructions as an Fig. 10 Foetus at 29+1 GW with scoliosis. The sagittal T2-w image of the whole foetus demonstrates a paravertebral cystic lesion referred to as an intrathoracic neurenteric cyst (arrow) 316 Insights Imaging (2011) 2:309–318 Fig. 13 Foetus at 21+6 GW with complex musculoskeletal abnormalities and foetal akinesia in polyhydramnios. The sagittal T2-w image of the whole foetus shows a short malformed spine with severe scoliosis. The skull is also elongated, referring to dolichocephaly with internal hydrocephaly (arrow) Fig. 11 Foetuses with normal long-bone development at 24 and 40 GW. The sagittal EP images of the thigh demonstrate hyperintense epiphyseal structures, with no distal femoral ossification centre at 24 GW (a), and a small hypointense ossification centre (arrow) in the older foetus (b) Fig. 13 Foetus at 21+6 GW with complex musculoskeletal abnormalities and foetal akinesia in polyhydramnios. The sagittal T2-w image of the whole foetus shows a short malformed spine with severe scoliosis. The skull is also elongated, referring to dolichocephaly with internal hydrocephaly (arrow) References 24. Senocak EU, Oguz KK, Haliloglu G, Karcaaltincaba D, Akata D, Kandemir O (2009) Prenatal diagnosis of Pena-Shokeir syndrome phenotype by ultrasonography and MR imaging. Pediatr Radiol 39:377–380 1. 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Brunelle F (2001) Fetal imaging in a new era. Ultrasound Obstet Gynecol 18:91–95 17. Chen Q, Levine D (2001) Fast fetal magnetic resonance imaging techniques. Top Magn Reson Imaging 12:67–79 39. Lachman RS (2006) Taybi & Lachman’s radiology of syndromes, metabolic disorders and skeletal dysplasias, 5th edn. Mosby, Philadelphia 18. Huisman TA, Solopova A (2009) MR fetography using heavily T2-weighted sequences: comparison of thin- and thick-slab acquisitions. Eur J Radiol 71:557–563 40. Krakow D, Rimoin DL (2010) The skeletal dysplasias. Conflict of interest None 22. Chung R, Kasprian G, Brugger PC, Prayer D (2009) The current state and future of fetal imaging. Clin Perinatol 36:685–699 23. Pugash D, Brugger PC, Bettelheim D, Prayer D (2008) Prenatal ultrasound and fetal MRI: the comparative value of each modality in prenatal diagnosis. Eur J Radiol 68:214–226 Conclusion Initial results demonstrate the visualisation of the normal and abnormal skeleton on foetal MRI, which highlights the use of MRI as an adjunct to prenatal ultrasound. MRI may be helpful in foetal spinal imaging, and in the differentia- tion between isolated and complex abnormalities, which might have an impact on pre- and postnatal management, as complex abnormalities may be related to an unfavourable outcome. Preliminary experience suggests an improvement in diagnosis through additional MRI findings in specific cases compared with ultrasound. However, increased efforts are needed to refine MRI techniques for the visualisation of the foetal skeleton and to clarify the value of MRI compared with standard ultrasound. Fig. 12 Foetus at 22+6 GW with lethal hypophosphatasia. The coronal EP image of the upper extremity shows a short and abnormally shaped humerus (arrow) and no ossification of the bones of the forearm, which was indicative of the mineralisation disorder Fig. 12 Foetus at 22+6 GW with lethal hypophosphatasia. The coronal EP image of the upper extremity shows a short and abnormally shaped humerus (arrow) and no ossification of the bones of the forearm, which was indicative of the mineralisation disorder Fig. 14 Mnemonic for MRI Fig. 14 Mnemonic for MRI Fig. 12 Foetus at 22+6 GW with lethal hypophosphatasia. The coronal EP image of the upper extremity shows a short and abnormally shaped humerus (arrow) and no ossification of the bones of the forearm, which was indicative of the mineralisation disorder Fig. 14 Mnemonic for MRI 317 Insights Imaging (2011) 2:309–318 19. Chaumoitre K, Wikberg E, Shojai R et al (2006) Fetal magnetic resonance hydrography: evaluation of a single-shot thick-slab RARE (rapid acquisition with relaxation enhancement) sequence in fetal thoracoabdominal pathology. Ultrasound Obstet Gynecol 27:537–544 Acknowledgments U.N. and S.F.N. co-wrote the manuscript; both authors contributed equally to this work. The authors would like to thank Ms. Mary McAllister for her help in editing the manuscript. Financial disclosure The authors have indicated they have no financial relationships relevant to this article to disclose. 20. de Vries JI, Fong BF (2006) Normal fetal motility: an overview. Ultrasound Obstet Gynecol 27:701–711 21. Lüchinger AB, Hadders-Algra M, van Kan CM, de Vries JI (2008) Fetal onset of general movements. Pediatr Res 63:191–195 Conflict of interest None References Genet Med 12:327–341 318 Insights Imaging (2011) 2:309–318 retrospective and prospective analysis. Am J Med Genet A 146A:1917–1924 41. Krakow D, Lachman RS, Rimoin DL (2009) Guidelines for the prenatal diagnosis of fetal skeletal dysplasias. Genet Med 11:127– 133 retrospective and prospective analysis. Am J Med Genet A 146A:1917–1924 45. Cassart M (2010) Suspected fetal skeletal malformations or bone diseases: how to explore. Pediatr Radiol 40:1046–1051 42. Miller E, Blaser S, Shannon P, Widjaja E (2009) Brain and bone abnormalities of thanatophoric dwarfism. AJR Am J Roentgenol 192:48–51 46. Schramm T, Gloning KP, Minderer S et al (2009) Prenatal sonographic diagnosis of skeletal dysplasias. Ultrasound Obstet Gynecol 34:160–170 43. Kawamura K, Chung KC (2009) Constriction band syndrome. Hand Clin 25:257–264 47. Tsutsumi S, Sawai H, Nishimura G, Hayasaka K, Kurachi H (2008) Prenatal diagnosis of thanatophoric dysplasia by 3-D helical computed tomography and genetic analysis. Fetal Diagn Ther 24:420–424 44. Krakow D, Alanay Y, Rimoin LP et al (2008) Evaluation of prenatal-onset osteochondrodysplasias by ultrasonography: a
https://openalex.org/W2320048603	https://www.scielo.br/j/fm/a/RYmRV4g9sQFRRLyfzRdmM4j/?lang=en&format=pdf	English	null	Physical therapy in avoidable hospitalizations for primary care-sensitive conditions	Fisioterapia em Movimento	2,016	cc-by	5,266	andra Bombarda Müller[a], Nadia Cristina Valentini[b], Maria Eugênia Bresolin Pinto[ [a] Universidade Vale do Rio dos Sinos, (UNISINOS), São Leopoldo, RS, Brazil [b] Universidade Federal do Rio Grande do Sul, (UFRGS), Porto Alegre, RS, Brazil [c] Universidade Federal de Ciência da Saúde de Porto Alegre, (UFCSPA), Porto Alegre, RS, Brazil [a] Universidade Vale do Rio dos Sinos, (UNISINOS), São Leopoldo, RS, Brazil [b] Universidade Federal do Rio Grande do Sul, (UFRGS), Porto Alegre, RS, Brazil [c] Universidade Federal de Ciência da Saúde de Porto Alegre, (UFCSPA), Porto Alegre, RS, Brazil ISSN 0103-5150 Fisioter. Mov., Curitiba, v. 29, n. 1, p. 183-91, Jan./Mar. 2016 Licenciado sob uma Licença Creative Commons DOI: http://dx.doi.org.10.1590/0103-5150.029.001.AR03 ABM: MSc, e-mail: abombarda@unisinos.br NCV: PhD, e-mail: nadiacv@esef.ufrgs,br MEBP: PhD, e-mail: eugenia@ufcspa.edu.br Abstract Avoidable hospitalizations for primary care-sensitive conditions have been used as indicators of access to timely and appropriate care because hospital admissions for many conditions could be prevented by inter- ventions in primary care. Physical therapists play an important role in health promotion, disease preven- tion, and the pursuit of fairness and improvements in the effectiveness of health care services, which are the goals of the public policies proposed by the Brazilian unified health care system. We used MEDLINE and SciELO to search the literature for articles concerning the association between physical therapy and the reduction of avoidable hospitalizations for primary care-sensitive conditions. The literature on the topic is still in its infancy and confined to relatively few studies. Although the available literature associates access to quality primary care with reduced hospitalizations for primary care-sensitive conditions, there is a need for original studies investigating whether there is an association between physical therapy and decreased hospital admissions for primary care-sensitive conditions. Keywords: Avoidable hospitalization. Ambulatory care-sensitive condition. Physical therapy. ABM: MSc, e-mail: abombarda@unisinos.br NCV: PhD, e-mail: nadiacv@esef.ufrgs,br MEBP: PhD, e-mail: eugenia@ufcspa.edu.br Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Müller AB, Valentini NC, Pinto MEB. 184 Resumo As hospitalizações evitáveis por condições sensíveis à atenção primária têm sido utilizadas como indicadores de acesso à atenção oportuna e adequada, fundamentada na elevada capacidade de resolução por parte da atenção primária de determinados problemas de saúde. Nesse contexto, a atuação do fisioterapeuta faz parte deste processo, pois a promoção da saúde e a prevenção de doenças, assim como a busca de equidade e maior resolutividade dos atendimentos prestados - objetivos das políticas públicas preconizadas pelo sistema único de saúde no Brasil que definem as ações propostas para o acolhimento dos usuários - também estão compreen- didas nas ações de fisioterapia. O objetivo desta revisão foi verificar a associação da fisioterapia à redução de hospitalizações evitáveis por condições sensíveis à atenção primária, por meio de pesquisa e análise de artigos indexados na base de dados MEDLINE e na biblioteca virtual SciELO. Foi constatada escassa produção biblio- gráfica relacionada ao tema, ainda muito incipiente. Embora a revisão da literatura disponível associe o acesso à atenção primária de qualidade à redução de hospitalizações por condições sensíveis a essa modalidade de atenção, há a necessidade de estudos originais que verifiquem se existe ou não associação entre a atuação fisioterapêutica e a redução de hospitalizações por condições sensíveis à atenção primária. Palavras-chave: Hospitalizações evitáveis. Condições sensíveis à atenção primária. Fisioterapia. Introduction Hospitalizations for ACSC could be prevented by effective interventions in primary care. The reduc- tion of the risk of hospitalization can be achieved by promotion of health, prevention of diseases, early diagnosis and treatment of acute episodes, adequate control and monitoring of chronic diseases, and proper management of resources (3 - 9). For exam- ple, primary care interventions can help reduce the number of hospital admissions for preventable infec- tious diseases by implementing vaccination against measles, tetanus, diphtheria, etc.; providing imme- diate treatment for gastroenteritis and pneumonia; and reducing hospital admission, readmission and stay for acute complications of noncommunicable diseases (diabetes mellitus, hypertension and heart failure) (17). Primary health care (PHC) is the health care model proposed by the World Health Organization (WHO), which recommends the provision of universal access to health promotion, disease prevention, diagnosis, treatment, rehabilitation and health maintenance (1). PHC serves as the individual's "gateway" to the Unified Health System (SUS) in Brazil. The SUS is a hierarchical system based on principles of universal- ity, comprehensiveness and equity (2). y p q y ( ) Studies conducted in recent years have high- lighted the positive impact of health promotion and restoration activities on the Brazilian population, al- though the implementation and organization of com- prehensive and continuous primary care to meet the demands and needs of distinct population groups is still associated with access difficulties and poor qual- ity of services (3 - 6). Avoidable hospitalizations for primary care-sensitive conditions have been used as indicators of access to timely and appropriate care. They serve as a tool for the analysis of the impact and performance of PHC services, because hospital admissions for many conditions could be prevented by interventions in primary care. These interventions result in a reduction in hospital admissions, an in- crease in preventive measures, and higher quality of outpatient care (3 - 9). Thus, individuals who seek primary care services and benefits from the resolution of their problem do not experience worsening of their condition and do not require hospitalization. Nevertheless, other fac- tors may also interfere with this outcome: the man- ner in which health care teams organize their work; community's sociocultural determinants; strategic location for access to health care services; and lack of preparation for handling the situation that triggered the damage to health (8, 21 - 23). Introduction (Conclusion) Table 1 - List of Primary care-sensitive conditions (Conclusion) which risk factors are associated with those people who seek health care services. There is no global con- sensus on the choice of these diagnoses, because the list of ACSC changes according to the local reality or weather conditions of a given territory (4, 15). Because this indicator has been increasingly used in Brazil, in 2008 the Ministry of Health defined and published the Brazilian list of ACSC (Table 1) (24), taking into consideration the country's epidemiologi- cal profile and health system, and adapting the fol- lowing criteria used in previous international studies: (1) existence of scientific evidence that the cause of hospitalization is a primary care-sensitive condition; (2) being an easily diagnosed condition; (3) being a health problem that affects much of the population (i.e., not being a rare event/condition); (4) being a condition that could have been prevented or solved by interventions in primary care; (5) need for hospi- talization when the condition occurs; (6) diagnosis is not induced by financial incentives. (3) The Brazilian list comprises a larger number of infectious diseases, unlike foreign lists. This is due to the epidemiological characteristics of the country, where there is still a high prevalence of these diseases. (25) Table 1 - List of Primary care-sensitive conditions ICD-10 diagnosis Code used Diseases preventable by immunization and sensitive conditions A15.0 to A15.3; A15.4 to A15.9; A16.0 to A16.2; A16.3 to A16.9; A17.0; A17.1 to A17.9; A18; A19; A33 to A37; A51 to A53; A95; B05; B06; B16; B26; B50 to B54; G00.0; I00 to I02 Infectious gastroenteritis and complications A00 to A09; E86 Anemia D50 Nutritional deficiencies E40 to E46; E50 to E64 Ear, nose and throat infections H66; J00 to J03; J06; J31 Bacterial pneumonia J13; J14; J15.3; J15.4; J15.8; J15.9; J18.1 Asthma J45; J46 Lung diseases J20; J21; J40; J41 to J44; J47 (To be continued) Table 1 - List of Primary care-sensitive conditions (To be continued) Note: Ordinance SAS/MS No. 221, of April 17, 2008. Physical therapists play an important role in health promotion and disease prevention, as well as in the pursuit of fairness and improvements in the effectiveness of health care services - which are the goals of the public policies proposed by the Brazilian unified health care system to provide user embrace- ment (26). Introduction The purpose of this literature review was to determine whether there is association between the use of physical therapy and the reduction of pre- ventable hospitalizations for ACSC. Introduction The investigation of the causes of preventable hos- pitalizations for ACSC makes it possible to determine Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Physical therapy in avoidable hospitalizations for primary care-sensitive conditions which risk factors are associated with who seek health care services. There is n sensus on the choice of these diagnoses list of ACSC changes according to the or weather conditions of a given terri Because this indicator has been incre in Brazil, in 2008 the Ministry of Health published the Brazilian list of ACSC (T taking into consideration the country's e cal profile and health system, and adap lowing criteria used in previous internat (1) existence of scientific evidence that hospitalization is a primary care-sensiti (2) being an easily diagnosed condition health problem that affects much of th (i.e., not being a rare event/condition) condition that could have been preven by interventions in primary care; (5) ne talization when the condition occurs; (6 not induced by financial incentives. (3) T list comprises a larger number of infecti unlike foreign lists. This is due to the epi characteristics of the country, where t high prevalence of these diseases. (25) Table 1 - List of Primary care-sensitive ICD-10 diagnosis Code used Diseases preventable by immunization and sensitive conditions A15.0 to A15.3 A15.9; A16.0 t A16.3 to A16.9 A17.1 to A17.9 A33 to A37; A5 A95; B05; B06 B50 to B54; G0 I02 Infectious gastroenteritis and complications A00 to A09; E8 Anemia D50 Nutritional deficiencies E40 to E46; E5 Ear, nose and throat infections H66; J00 to J0 Bacterial pneumonia J13; J14; J15. J15.8; J15.9; J Asthma J45; J46 Lung diseases J20; J21; J40; J47 185 Table 1 - List of Primary care-sensitive conditions ICD-10 diagnosis Code used Hypertension I10; I11 Angina I20 Heart failure I50; J81 Cerebrovascular diseases G45; G46; I63 to I67; I69 Diabetes Mellitus E10.1; E10.1 to E10.9; E11.0; E11.1; E11.2 to E11.9; E12.0; E12.1; E12.2 to E12.9; E13.0; E13.1; E13.2 to E13.9; E14.0; E14.1; E14.2 to E14.9 Epilepsies G40; G41 Kidney and urinary tract infections N10 to N12; N30; N34; N39.0 Infections of the skin and subcutaneous tissues A46; L01 to L04; L08 Inflammatory disease of female pelvic organs N70 to N73; N75; N76 Gastrointestinal ulcer K25 to K28; K92.0; K92.1; K92.2 Pregnancy and childbirth- related diseases A50; O23; P35 Note: Ordinance SAS/MS No. 221, of April 17, 2008. Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Materials and Methods Literature review. We searched MEDLINE (ac- cessed through PubMed) using the following key- words: “ambulatory care-sensitive conditions”, “pri- mary care-sensitive conditions”, “primary health care”, “physical therapy modalities” and “avoidable Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Müller AB, Valentini NC, Pinto MEB. 186 hospitalizations”. In addition, we searched SciELO using following terms in Portuguese “avoidable hos- pitalizations”, “physical therapy”, “asthma”, “hiperten- sion”, “diabetes mellitus”, “cerebrovascular diseases” and “heart failure”. Searches were conducted between September and December 2012. Due to the lack of studies associating Physical Therapy and ACSC, we also analyzed the references cited in the articles found, as well as the theses and dissertations related to the terms mentioned above, in order to investi- gate the type of assistance provided to this popula- tion. The final sample of this study was composed of studies on PHC characteristics associated with the risk of hospitalization for ACSC, and on physical therapy treatment of the noncommunicable chronic diseases (NCDs) listed above. Most of the articles found on the topic of ACSC (10) were cross-sectional studies. Fourteen papers on health indicators were ecological studies. We also identified two systematic reviews and seven theses/dissertations. None of the papers analyzed the use of physical therapy in the prevention of avoidable hospitalizations for ACSC or NCDs such as asthma, hypertension, diabetes mel- litus, cerebrovascular disease and heart failure. Primary care- sensitive conditions Avoidable hospitalizations Physical Therapy MEDLINE (PubMed) Avoidable hospitalizations Physical Therapy Asthma Hypertension Diabetes Mellitus Cerebrovascular diseases Heart failure SciELO 231 citations / possibly relevant titles 127 32 full papers + 7 theses / dissertations no evidence of physical therapy use in the prevention of avoidable hospitalizations for ACSC 74 possibly relevant titles 37 abstract 28 full papers reporting the conjunct use of Physical Therapy and other therapies for the treatment of the diseases listed here No evidence of physical therapy use in the prevention of avoidable hospitalizations for NCDs Figure 1- Flowchart of the methodology adopted. Primary care- sensitive conditions Avoidable hospitalizations Physical Therapy no evidence of physical therapy use in the prevention of avoidable hospitalizations for ACSC 74 possibly relevant titles 37 abstract 28 full papers reporting the conjunct use of Physical Therapy and other therapies for the treatment of the diseases listed here Figure 1- Flowchart of the methodology adopted. Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Discussion availability of data collected with software applica- tions make it possible to use this indicator to develop studies on this topic, and implement specific actions to meet this care demand. This literature search, how- ever, was hampered by the high variability of descrip- tors/keywords used to index the papers (15, 28). Hospitalizations for ACSC serve as a useful indi- cator for the Unified Health System (SUS). The idea behind this indicator is that hospitalizations for cer- tain conditions reflect failures of the health system to provide access to quality primary care, because these conditions should have detected at a much earlier stage, which would have avoided the increase in their severity and/or prevented the onset of complications, thus eliminating the need for hospitalization (25). The definition of the Brazilian list of causes and the International studies (6, 10, 21 - 23) indicate that admissions for ACSC are influenced by many differ- ent factors related to accessibility to care, such as socioeconomic status of the patient, organizational features of primary care, hospitalization criteria and supplemental health coverage. This is in line with the Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Physical therapy in avoidable hospitalizations for primary care-sensitive conditions 187 findings of national studies involving diagnoses of pneumonia and heart failure, for example (4, 11, 17, 21-23). Physical therapy plays a purely curative role in the treatment of these conditions. It helps avoid the worsening of these conditions and improve the quality of life of patients by aiding in the reestablish- ment of adequate kinetic/functional capacity. have identified this trend in the state of Minas Gerais (38); Rehen and Egry (2009) report a reduction in the frequency of hospitalizations for ACSC in the state of São Paulo between 2000 and 2007 (8, 9); Dias da Costa et al (2010) have also identified a decrease in hospital admission rates for ACSC in the state of Rio Grande do Sul after 2001 (7, 13). Over the last years several international litera- ture reviews on the topic have concluded that access to quality primary care reduces hospitalizations for conditions sensitive to primary care (21-23, 41). The results of national studies corroborate this estimate: the degree of coverage of Family Health Strategy (FHS) programs was associated with decreased hos- pitalizations rates for ACSC in general, as well as the hospitalizations rates for congestive heart failure and acute diarrhea in children under five years of age (18, 34, 38). Discussion The increase in FHS programs was also associated with reduced length of stay of inpatients (4, 38). This corroborates the thesis that investments in PHC, in addition to other inherent benefits, have resulted in shorter hospital stays for ACSC (5, 38). There are many possibilities for the use of physical therapy in ACSC and these represent an important contribution for the different levels of health care, di- rectly and indirectly, both for the promotion of health and prevention of diseases, and in rehabilitation processes (39, 40, 44). The provision of accessible and effective primary care avoids hospitalizations. Physical therapists can help minimize uncontrolled complications caused by a chronic disease, and facili- tate access to preventive health services. Actions for the prevention of conditions listed as health care quality indicators can all be implemented by multidisciplinary teams. These actions range from the provision of information and education about: the importance of vaccination for the prevention of diseases and of keeping the vaccination status up to date; changes in behavior to avoid viral and bacterial infections; signs and symptoms for the detection of the most prevalent diseases in the community; and what to do in emergency situations As evidenced in the study conducted by Mobley and colleagues (6) in 2006, the increased availability of physical therapists in primary care was associated with lower hospital- ization rates among older adults in the United States. Teamwork was also associated with a lower prob- ability of ACSC diagnosis among inpatients in Bagé, RS (14). This implies a decentralization of health care provision from physicians to other health care provid- ers. As a consequence, there is a more comprehensive approach to the disease process, which allows its bet- ter resolution by the multidisciplinary team. According to the report of a survey conducted by the Ministry of Health until 2012, between 1999 and 2007, hospitalizations of younger and older adults for ACSC accounted for 30% of hospital admissions in Brazil (43). In the period of the study, there was a 24% reduction in the rate of hospitalizations for ACSC and a 9% reduction in the rate of hospitaliza- tions for conditions unrelated to primary care. This study shows a direct relationship between invest- ments in PHC and decreased hospitalization rates for ACSC (43). Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Final Considerations In addition to its impact on the reduction of hos- pitalizations for primary care sensitive conditions, quality primary care delivery might also influence the decrease in length of stay of inpatients and the amount of hospital readmissions. As a result of the above, another desirable effect is the reduction of public costs for hospitalizations, either because of avoided hospitalizations or of anticipated discharges. Thus, it is important to quantify the benefits that could be brought about by the expansion of primary care — both for the reduction in the length of hospi- tal stay and of the costs associated with it. The use of physical therapy in primary care requires an adjust- ment of this care modality to the reality of PHC and to the needs of the population. There is a wide range of possibilities for its qualified participation. The investi- gation of the epidemiological profile of the population, its environmental conditions, and the prevalence of morbidity in patients seen in PHC help in the planning of health education activities and in the development of appropriate physical therapy strategies. In addition, in the context of non-drug treatments in particular, physical therapists working in the FHS can create strategies to encourage and promote the practice of physical activity by patients with hyper- tension, diabetes, obesity, sedentary lifestyles, dyslip- idemia, as this is a fundamental element in the care and monitoring of patients at risk of cerebrovascular accident (42). With regard to the management of lung diseases and their progression, especially asthma, physical ther- apists can also intervene at an individual or collective level by implementing activities and programs that take into consideration social, economic, cultural and environmental aspects involved in care. They should act not only in the treatment of asthma attacks, but also be a key player in the education for the prevention of new episodes by providing proper guidance and instructions about the maintenance of an airy atmo- sphere. These measures could result in better quality of life and lower morbidity. Frequent hospitalizations for asthma are associated with elevated morbidity and represent an important public health problem. Inadequate control of the disease has led to an increase in the number of emergency room visits, hospitaliza- tions and deaths. Discussion The Mafra research (2010), which was conducted throughout the country, demonstrates that in 2007 hospitalizations for ACSC accounted for 27.44% of all hospitalizations, excluding deliveries (38). When comparing the evolution of admissions for ACSC and for other health problems, the study indicates a sharper reduction of the first. While the number of hospitalizations for ACSC decreased by around 19% in the period of the study, admissions for other reasons decreased by 8.5%. This fact is consistent with the thesis that associates the decline in hospital admissions with increase in the provision of primary care services, which effectively took place in Brazil during the period analyzed (4, 12, 14, 15, 20, 25). Working with human movement, its object of study, Physical therapy can have a positive impact on risk factors for noncommunicable diseases. This could be achieved, for example, by encouraging pa- tients to avoid sedentary behaviors, as the risk of cerebrovascular diseases and high blood pressure- and diabetes-related diseases is associated, among other factors, with the absence of physical activities. Much can be done to help spread the word about methods and means of exerting physical activity, body practices and movement therapies in PHC set- tings. It is important to involve different sectors of Other studies have indicated a downward trend in hospitalization rates for ACSC: Perpetual and Wong (2006), based on data from 1998 to 2004, Fisioter Mov. 2016 Jan/Mar;29(1):183-91 Müller AB, Valentini NC, Pinto MEB. 188 the community, such as neighborhood associations, schools, churches, NGOs and universities. Patients with risk factors for cerebrovascular diseases in particu- lar could be instructed by health professionals about all the opportunities for physical activity: at work (stretching, postural changes), during transportation (walk to work or to the supermarkt whenever pos- sible), during the performance of domestic chores and during leisure time (sports and recreation). Because chronically ill patients are at high risk for further stroke, they should receive higher priority and indi- vidual, customized interventions, for example. These individuals need to be more frequently and closely monitored, and they should also receive treatment to rehabilitate eventual cognitive, behavioral, speech, motor or sensory sequelae. The fundamental goal of a rehabilitation program (either outpatient or at home) is to help patients adapt to their disabilities, facilitate their functional recovery, and promote social, profes- sional and familiar reintegration. Discussion conjunction with drug therapy represents one of the pillars of success in these programs, and have shown to help reduce disease exacerbation, hospitalizations for asthma, and costs of disease (46). Final Considerations Despite the unfavorable scenario, health programs offering an appropriate diagnostic and therapeutic approach for patients, as well as edu- cational activities and rehabilitation measures, have succeeded in reducing hospitalizations for asthma at- tacks (44, 45). The provision of education programs in As the literature on the topic is still in its infancy and confined to relatively few studies, there is a need for original studies investigating whether there is an association between physical therapy and decreased hospital admissions for primary care-sensitive condi- tions. The disclosure of this information may help in the planning and evaluation of health care services, by providing diagnostic and therapeutic resources based on the use of physical therapy. Physical therapy can make a greater contribution to this recent and incipient topic. Physical therapists play an impor- tant role in this process, especially by helping in the development and implementantion of activities and practices that are associated with the resolution of primary care-sensitive conditions. Fisioter Mov. 2016 Jan/Mar;29(1):183-91 1. World Health Organization. [Cited in 20/10/2012]. Available from: http://www.who.int/publications. 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https://openalex.org/W2084883448	https://zenodo.org/records/1560220/files/article.pdf	English	null	V. <i>The bearing of rotation on relativity</i>	The London, Edinburgh and Dublin philosophical magazine and journal of science	1,920	public-domain	2,877	Philosophical Magazine Series 6 SSN: 1941-5982 (Print) 1941-5990 (Online) Journal homepage: http://www.tandfonline.com/loi/tphm17 Date: 14 June 2016, At: 00:18 Download by: [Universite Laval] Prof. R.A. Sampson F.R.S. To cite this article: Prof. R.A. Sampson F.R.S. (1920) V. The bearing of rotation on relativity , Philosophical Magazine Series 6, 40:235, 67-72, DOI: 10.1080/14786440708636097 To link to this article: http://dx.doi.org/10.1080/14786440708636097 Published online: 08 Apr 2009. Submit your article to this journal Article views: 4 View related articles Download by: [Universite Laval] Tl~e Bearing of l~otation on Relativity. 67 which is a product in the collateral actinium series. But the difficulties here are not inconsiderable aside from the fact that the existence of such ~n isotope is somewhat diffi- cult to imagine. If present in amounts proportional to the actinium this product would have to emit comparatively long range (7"2 cm.) ~-particles and would therefore have a very short life period. Such a conclusion does not seem at all probable in the light of our present knowledge. p g p g It is not impossible that the values accepted for the ranges of the a-partlcles from uranium are considerably in error and that this is the reas,m for the lack of agreement between theory and experiment. But until some more definite data have been obtained there seems to be little justification for abstruse speculation on the genetic relationship in the earlier stages of the nranimn series. * Communicated by the Author. Summary. The relation of the activity of radium to the activity of the uranimn with which it is in radioactive equilibrium has been redetermined. The results obtained indicate that if the activity of uranium is takeu as unity the activity of the radium is equal to approximately 0"4:9. q pp y The total activity of uranium mixed with equilibrium quantities of its disintegration products has been compared with the activity of the uranimn alone, and the former has been found to be 4"73 times the latter. A critical examination has been made of the various theories which have been proposed to explain the genesis of radium and actinium from uranimn. None of these theories appears to satisi)~ the necessary requirements. V. ]'he Bearivg of Rotation on Rdativity. By Prof. R. A. SAMPSOn, F.R.S.* C ONSIDER two concentric spheres with a very small space between them so that we need not distinguish between their radii. An observer A is placed on the outer surface of tlle inner sphere and an observer B on the inner surface of the outer. All phenomena are supposed to pass in the space between the two spheres. C b p p Regard this system and its changes first from a purely geometrical point of view. A and B will possess in common a natural unit of length, being the circumference of their * Communicated by the Author. y F2 68 Prof. R. A. Sampson on t],e sphere. Let the arc AB as it exists at any moment be determined as a fraction of this unit. :Let it be determined again in the same way at a later moment. If the two do not agree, we can say that a relative rotation of the t~vo spheres must have occurred, through a definite angle, abont an axis perpendicular to tim plane of the great circle AB. Whether any relative rotation about an axis in the plane AB has taker~ place, or whether both spheres have executed in commorL any other rotation about any axis whatever, the observers at A and B will be unable to say. We may express this position by saying that A and B are under circumstances of complete geometrical relativity. The whole description is, however, an abstraction. It is the abstraction which lies at the basis of geometry; it eliminates time from consideration and supposes figures to exist with definite distances between the points. Summary. Then according to Lodge's experiment, whatever rotations the spheres may have, the passage of this light will he un- affected by i~. Let B be fixed and diametrically opposite to A at the moment of emission. The waves or ravs will spread in a sheet over the sphere, converge upon B~at the same moment andin the same phrase, andissuing from B will return again to the point from which they were emitted. But if A has moved in the interval, this will permit the observer A to ascertain the point in space which he occupied at a given past moment, A 0 sit),. It is the point upon which the rays converge and are received all together and at once in the same phase~that is, the point diametric:ally opposite to B. the same phase that is, the point diametric:ally opposite to B. It may be said that when the globe A is in rotation, it must be'supposed to undergo the FitzGerald contraction parallel to its equator, dctbrmlng it from a sphere; and therefore the geodesics which are the paths of rays will in gener~l no longer converge upon a single point as they do for a sphere, but pass alongside it. This, however, is a second-order effect, and would not interfere with the deter- mination of AA0, which is of the first order ; moreover, it would of itself demonstrate the rotation bythe non-concurrence of the rays. We should therefore conclude that by the use of signals transmitted through the tether i~ is possible to determine the motion of A, imaginedas a rotation. There is no sign of conflict here with results of experiments with matter alone, referred to earlier; but the conclusion hears awkwardly upon the Principle of Relativity for linear motions, and it is desirable to examine whether the division is apparent only or real. Consider whether rotation is essentially connected with the &scussion. Let A o. Aj, A2, A3 be four points successively occupied by the observer. Describe a sphere through A0, A~, A~, A3, and let light pass round it as above, being emitted from A0. Adjust the rotation of this sphere so that the time taken to car,'y the obgerver from the position Ao to the position A~ is equal to the time taken by light to girdle the sphere. Summary. But i~ reality any distance assigned requires time for its deter- ruination, and the standard case would be this: A and B each have hold of a graduated measure, allowing it to slip through their hands, and as they watch its successive readings they signal them to one another; each will then only be aware of the other's reading, that is to say, of the other's distance at any moment, as complicated by the time of transmission of the signals. This aberrational allowance is an inevitable attendant upon actual physical iffeasuros. It is inseparable from motion. It would not be surprising if the ideas of motion required a complete surrender of the scheme of abstract geometrical relativity defined above. g y How much the physical theory of motion affects our notion~ of absolute and relative is very well known. Let the sphere, A be the Earth and the sphere B a complete opaque sheet of cloud rotating with it. A Foucault pendulum set up at the north pole would of itself change its plane of oscillation with respect to the maridians, pointi.ng out an absolute direction in space, and an absolute rate of rotation, of which the ob- server would be unaware without this, or other similar, appeal to dynamics. A similar pendulum set up at the equator would show no ebange of azimuth at all. By no conceivable- explanation can this familiar experiment be made consistent with complete geometrical relativity of the system, geome- trically self-contained, within the bounds of which it oecurs~ Other cases could easily be mentioned. Bnt my purpose at the moment is to pursue a little further the aberrational con- siderations introduced above. In 1893 and 1897 Sir Oliver Lodge, considering the :Miehelson-Morley experiment, then not infrequently taken, Bea~.ing of Rotation on Relativity. 69 as proving that the Earth swept with it the teflter in its vicinity, spun two large heavy metal disks, parallel and close to one another, with high angular velocity, in order to test whether they exercised any viscous drag upon the ~ether between them. Light was transmitted to and fro across the intervening space, but the effect upon it was null. Now return to the two spheres A and B. Let their surfaces be perfect reflectors. Let light sigmds be emitted from A. Summary. Then the argument runs just as he/ore, and the conclusion can be drawn that the previous position of A is 70 Prof. R. A. Sampson on the ascertainable in every case except when three of the points A0, A1, A:, A3 lie upon one straight line. The argument then has no bearing on the I~rinciple of Relativity for linear motion, even if we include accelerated linear motion. The latter stands j~,st where it did. j , It is worth remarking that the Principle of l~eiativity, owing to its generality in asserting that no experiment can be derived that will falsify it, can never be est~blished except upon trial. It suffers under the difficulty always found in provinga negative. Thus the Michelson-Mor]ey experiment is, of course, consistent with it, but the experiment is explained without its aid by adopting the FitzGerald contraction, which there is otherwise strong reason to accept. g p At each successive test of the principle there is the same possibility of escape. Thus, though the test imagine d above fails as a criticism of its application to rectilinear motion, it is easy to imagine a test case which would exclude acceleration from its domain. The annual aberration of the stars is, in fact, just an observation of the variation of the motion of the Earth in direction ; and if we could observe with sufficient refinement, it would equally reveal a variation in its amount. The only field that remains is that of uniform linear motion ; and even here I cannot see that it is free from attack. The gradual transmission of light, was first demonstrated by Roemer from the eclipses of Jupiter's first satellite. As the Earfll approaches Jupiter, immersions alone are observed, and as it recedes only emersions, the changing distance is marked by an apparent reduction of the period of revolution in the former case, and a compensating increase in the latter. The quantities have been observed, and agree with dimensions of the orbits of the Earth and Jupiter found by Euclidean triangulation and a velocity of light found from terrestrial experiments. The comparisons take place in all directions in the ecliptic. The quantity directly determined is the time taken by light to travel a distance equal to that travelled by, the Enrth hetween two eclipses. Summary. There is here no question of contraction of the measuring rod, for the phenomenon is one involving the relative velocity of light in the first order. Hence we can conclude that if there is any undiscovered drift of the solar system through the tether, it must take place in a direction perpendicular to the plane of the ecliptic; this is reliable to such a degree of accuracy as these obser, rations will bear, and that is by no means an inconsir derable one. If we are unable to draw a conclusion one way or another with regard to relative drift of the tether perpendicular to the plane of the ecliptic, that is only because it happens that we 71 Bearir, g of Rotation on Relativity. Bearir, g of Rotation on Relativity. cannot find a system that supplies a natural test, and are unable to make one. cannot find a system that supplies a natural test, and are unable to make one. In the foregoing sentences it is, of course, adopted thai when a phenomenon occurring on Jupiter is transmitted by light to the Earth, the velocity of its transmission through the intervening space is entirely independent of any drift that Jupiter may possess relative to the rather ; this drift is shared, let us say, by the whole solar system, and it becomes an observable quantity at the receiving point--the Earth, because it may change the distance over ~hich the trans- mission through pure sether must take place. g p p Returning to the imagined model of two concentric spheres~ consider t~gain this bearing upon relative time and its mea- surement, as these appear in the usual exposition of the Principle of Relativity. p y Consider the two observers A and B, imagined at the beginning, and suppose for simplicity that A, It are both on the equator of the uniform relative motion of the spheres. If A, It compare their clocks by light signals, say, it may be shown, as in the works on Relativity, that their zeroes will differ by ux/c ~, where x is the arc AB, u the velocity of B relative to A, and c the velocity of light. If the relative motion of B is in the same direction as the transmission of the signal, B's time is slow. Summary. But in our imagined case, B will receive two signals, generally at different times and always in opposite directions, and might consequently set two clocks by them, one slow and the other fast. These clocks would keep permanently different rates, one losing and the other gaining ru~/c ~ in each relative revolution or "day," where T is tt~e length of this "day." But this "day" would itself be an observable phenomenon, possessed in common by both A and B, and marked as the interval between two consecutive coincidences of It with A. In our case, then, B would be able to ascertain that the times kept by his two clocks rated by two light signals were both erroneous, nor would he require to adopt the same numerical measure of the velocity of light as A, in order to provide himself with a standard measure of time, as he is supposed r do, in the Theory of Relativity. y y It is hardly necessary to point out that oar imagined case of time-determination is not an artificial one, but is as close as an ideal construction permits to the actual practice by which the solar day and the sidereal day are found. We may go a little further. It has been the practice to observe at Greenwich the transit of the lunar crater- MSsting A. Imagine an observer in the Moon, situated in M5sting A and taldng the moment when Greenwich crossed the medial The Bearing of Rotation on Relativity. 72 line of the Earth's disk,--which would bring him into the meridian of Greenwich. If we suppose that there is a definite moment of occurrence of the geometric phenomenon, each observer would get it late by the same amount, about 1"3, being the time of transmission of light; but on repeated observations each would have exactly the same time-intervals of what may be called a lunar day at his service for common standardization of time. In conclusion, I would add a few sentences on the general bearing of these remarks. I take it there is no useful knowledge apart from repetition, including its opposite, contrast. An isolated event, with no indication of its history or relation, is meaningless--like an inscription without a clue. Summary. Consequently we find that in the field of mechanics, the cases that involve perpetual repetition are those reduced to the best order--as the rotation of the Earth, the revolution of the planets, and periodical mechanical motions of every kind. Therefore, in spite of the apparently greater simplicity of pure translational motion, I submit that itis in the simplest cases of circular motion that we should naturally look for light upon the que.stion of how far we are able to perceive our own drift through the ~ether. It is not tolerable to erect a new theory, of completely general scope, and leave on one side these familiar natural events, the best analysed that we possess. Properly regarded, the paradoxes to which a theory leads are its most promising features, for they indicate points where something can be learnt by pressing the examination. Some of the numerous paradoxes to which the Theory of Relativity has given rise may prove to be merely verbal or otherwise unessen/,ial ; those exposed above suggest, I submit, something more than this--namely, that the theory for rectilinear motion is a degenerate ease, which leads to untrue results if we attempt to embrace circular motions ; admirably compact as its mathematical form may be, it owes this coherence to a reduction that shuts off the best known fields of Nature. Yet no one can doubt that we must have a theory of relativity, limiting, and probably limiting very much, what it is possible to know. It is the theory of the interdependence of our senses of intensity or measurement in their various fields~spaee, time, and if you like, others. It is the work of pure mathematicians to give us one. Owing to the difficulty of his task, the mathematician must be allowed to make any abstraction which he finds workable. The interest thereafter is to see how far his construction may be taken as depicting Ntture, in the way that we know Nature to work.
https://openalex.org/W4386895132	https://www.mdpi.com/2072-6694/15/18/4627/pdf?version=1695115712	English	null	Neo-Adjuvant Treatment in Primary Resectable Pancreatic Cancer: A Systematic Review and PRISMA-Compliant Updated Metanalysis of Oncological Outcomes	Cancers	2,023	cc-by	13,392	Systematic Review Neo-Adjuvant Treatment in Primary Resectable Pancreatic Cancer: A Systematic Review and PRISMA-Compliant Updated Metanalysis of Oncological Outcomes Raffaello Roesel 1,† , Letizia Deantonio 2,3,†, Lorenzo Bernardi 1, Maria Luisa Garo 4 , Pietro Majno-Hurst 1,3, Alberto Vannelli 5 , Marco Cefalì 6 , Maria Celeste Palmarocchi 6, Maria Carla Valli 2, Guido Pesola 6, Alessandra Cristaudi 1,‡ and Sara De Dosso 3,6,,‡ 1 Department of Visceral Surgery, Hospital of Lugano (EOC), 6900 Lugano, Switzerla pietro.majno-hurst@eoc.ch (P.M.-H.); alessandra.cristaudi@eoc.ch (A.C.) 1 Department of Visceral Surgery, Hospital of Lugano (EOC), 6900 Lugano, Switz pietro.majno-hurst@eoc.ch (P.M.-H.); alessandra.cristaudi@eoc.ch (A.C.) 1 Department of Visceral Surgery, Hospital of Lugano (EOC), 6900 Lugano, Switzerland; 2 Radiation Oncology Department, Oncology Institute of Southern Switzerland (IOSI), EOC, 6500 Bellinzona, Switzerland; letizia.deantonio@eoc.ch (L.D.); mariacarla.valli@eoc.ch (M.C.V.) y , ( ), g , 4 Independent Researcher, 00199 Rome, Italy; marilu.garo@gmail.com p g y, p , , y; 6 Medical Oncology Department, Oncology Institute of Southern Switzerland (IOSI), EOC, 6500 Bellinzona, Switzerland; marco.cefali@eoc.ch (M.C.); mariaceleste.palmarocchi@eoc.ch (M.C.P.); guido.pesola@eoc.ch (G.P.) g Correspondence: sara.dedosso@eoc.ch p † These authors contributed equally to this work and share co-ﬁrst Authorship. q y contributed equally to this work and share co-last Authorsh ‡ These authors contributed equally to this work and share co-last Authorship. Simple Summary: Pancreatic adenocarcinoma is the fourth leading cause of cancer-related death in industrialized countries. In locally advanced and borderline resectable pancreatic cancer, neoadjuvant therapy (NAT) has been shown to be effective in eliminating potentially circulating tumor cells and distant micrometastases, shrinking local tumors, and identifying high-grade malignancies that do not beneﬁt from surgery. However, in patients with resectable pancreatic adenocarcinoma, who represent 20% of new diagnoses and for whom US followed by adjuvant chemotherapy is the standard of care, NAT is controversial because it carries several potential drawbacks that may prevent surgery and increase the risk of clinical deterioration. Randomized clinical trials, retrospective studies, and a few systematic reviews and meta-analyses reported controversial results, and although the safety and feasibility of such an approach are supported, a wider implementation is still a matter of debate. Considering the different methodological approaches (RCTs vs. retrospective studies), the difﬁculty in providing high-quality evidence due to small patient numbers, and the emergence of new evidence, an update of the current evidence seems essential to help clinicians and researchers understand the role of NAT and offer a new potentially beneﬁcial treatment approach. cancers cancers Systematic Review Neo-Adjuvant Treatment in Primary Resectable Pancreatic Cancer: A Systematic Review and PRISMA-Compliant Updated Metanalysis of Oncological Outcomes Thus, the aim of this systematic review and meta-analysis is to evaluate the role of NAT in prolonging overall survival and disease-free survival and improving R0 and N0 rates compared with upfront resection in patients with resectable pancreatic cancer. cancers cancers Citation: Roesel, R.; Deantonio, L.; Bernardi, L.; Garo, M.L.; Majno-Hurst, P.; Vannelli, A.; Cefalì, M.; Palmarocchi, M.C.; Valli, M.C.; Pesola, G.; et al. Neo-Adjuvant Treatment in Primary Resectable Pancreatic Cancer: A Systematic Review and PRISMA-Compliant Updated Metanalysis of Oncological Outcomes. Cancers 2023, 15, 4627. https://doi.org/10.3390/ cancers15184627 Received: 7 September 2023 Accepted: 14 September 2023 Published: 19 September 2023 Abstract: Background: Despite advances in treatment, the prognosis of resectable pancreatic adeno- carcinoma remains poor. Neoadjuvant therapy (NAT) has gained great interest in hopes of improving survival. However, the results of available studies based on different treatment approaches, such as chemotherapy and chemoradiotherapy, showed contrasting results. The aim of this systematic review and meta-analysis is to clarify the beneﬁt of NAT compared to upfront surgery (US) in primarily resectable pancreatic adenocarcinoma. Methods: A PRISMA literature review identiﬁed 139 studies, of which 15 were ﬁnally included in the systematic review and meta-analysis. All data from eligible articles was summarized in a systematic summary and then used for the meta-analysis. Speciﬁcally, we used HR for OS and DFS and risk estimates (odds ratios) for the R0 resection rate and the N+ rate. The risk of bias was correctly assessed according to the nature of the studies included. Results: From the pooled HRs, OS for NAT patients was better, with an HR for death of 0.80 (95% CI: 0.72–0.90) 1. Introduction Globally, in 2020, pancreatic adenocarcinoma will be the 14th most common malig- nancy, with 495,773 new worldwide cases [1] and an average lifetime cumulative risk of occurrence of 1 in 64 [2]. By 2030, pancreatic cancer is expected to become the second cause of cancer-related death, despite survival improvements with current treatments [3]. At the time of diagnosis, pancreatic cancer is considered primarily resectable in 10–20% of cases based on the absence of metastases and loco-regional arterial inﬁltration [4]. Tumor contact with either the portal or superior mesenteric vein of less than 180◦without deformation of the vessel is not considered a contraindication to surgery [5]. In this context, a radical (R0) surgery with standard D2 lymphadenectomy is the cornerstone of treatment [6]. The introduction of adjuvant chemotherapy has improved prognosis and is one of the factors mainly associated with long-term survival [7]. In this regard, FOLFIRINOX is currently the best adjuvant treatment regimen, according to the results of the PRODIGE24 trial, which showed a signiﬁcantly better median overall survival (OS) of 54 months with FOLFIRI- NOX compared to 37 months with gemcitabine in a highly selected population [8]. As an alternative, gemcitabine-based regimens can be considered in less ﬁt patients, according to the positive results of the ESPAC-4 trial [9], and gemcitabine alone should be used only in frail patients [10,11]. Nonetheless, only patients with a good performance status and a good recovery after surgery can receive adjuvant chemotherapy, corresponding to half of the overall population of patients with resectable pancreatic cancer. Thus, a consider- able proportion of patients are unable to start adjuvant treatment due to the morbidity of pancreatic surgery, and an even greater proportion of resected patients fail to complete all planned cycles of adjuvant therapy [12–14]. Despite the current available treatments, the 5-year OS rate is still poor, close to 20% [15,16], with a 3-year disease-free survival (DFS) rate between 24% and 39% [10]. Therefore, the development of new strategies to improve clinical results in this setting is currently a priority. In this regard, considerable interest has been focused on the role of neoadjuvant therapy (NAT), which has been demonstrated to improve OS without necessarily increasing the resection rate in patients with borderline resectable tumors [17,18]. The potential advantages of a neoadjuvant strategy are the eradication of micro-metastases and a higher chemotherapy completion rate compared to the adjuvant setting. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/cancers Cancers 2023, 15, 4627. https://doi.org/10.3390/cancers15184627 Cancers 2023, 15, 4627 2 of 17 at a signiﬁcance level of less than 1%. In the sub-group analysis, no difference was found between patients treated with chemoradiotherapy or chemotherapy exclusively. The meta-analysis of seven studies that reported DFS for NAT resulted in a pooled HR for progression of 0.66 (95% CI: 0.56–0.79) with a signiﬁcance level of less than 1%. A signiﬁcantly lower risk of positive lymph nodes (OR: 0.45; 95% CI: 0.32–0.63) and an improved R0 resection rate (OR: 1.70; 95% CI: 1.23–2.36) were also found in patients treated with NAT, despite high heterogeneity. Conclusions: NAT is associated with improved survival for patients with resectable pancreatic adenocarcinoma; however, the optimal treatment strategy has yet to be deﬁned, and further studies are required. Keywords: resectable pancreatic cancer; pancreatic adenocarcinoma; neoadjuvant chemotherapy; neoadjuvant chemoradiotherapy; neo-adjuvant treatment 2. Materials and Methods This work was conducted in accordance with PRISMA guidelines [19] and registered on PROSPERO (CRD42022382272). The components of the PICO questions were: (pop- ulation) patients with resectable pancreatic cancer; (intervention) neoadjuvant treatment (NAT); (comparator) upfront surgery; (outcome): overall survival, disease-free survival, R0 resection rate, and positive lymph node rate. 1. Introduction Furthermore, NAT may increase the microscopically margin-negative resection rate (R0 rate), nodal negative disease (N0), and may select patients with rapidly progressive tumors, thus sparing ineffective surgery. On the other hand, NAT-related adverse events could worsen the patient’s performance status, thereby delaying surgery. These potential conﬂicts can only be clariﬁed by evidence from randomized trials comparing neoadjuvant and adjuvant treatment strategies. Currently, in the setting of resectable pancreatic adenocarcinoma, there are only a few clinical studies comparing NAT versus upfront surgery (US), reporting controversial results. The aim of the present systematic review and meta-analysis is to Cancers 2023, 15, 4627 3 of 17 3 of 17 evaluate the role of NAT in prolonging OS and DFS and improving R0 and N0 rates compared with upfront resection. evaluate the role of NAT in prolonging OS and DFS and improving R0 and N0 rates compared with upfront resection. 2.2. Search Strategy A systematic search strategy was carried out on PubMed, EMBASE, and the Cochrane Register of Controlled Trials, irrespective of language or publication date, from November 2022 to February 2023. The search strategy included the following terms: (“pancreas” OR “pancreatic”) AND (“cancer” OR “carcinoma” OR “adenocarcinoma” OR “tumour” OR “neoplasm”) AND (“chemotherapy” OR “irradiation” OR “radiotherapy” OR “radiation therapy” OR “chemoradiotherapy”) AND (“surgery” OR “resectable” OR “up-front resectable”) AND (“neoadjuvant” OR “preoperative”). Removal of duplicates and screening of titles/abstracts were carried out by two independent reviewers (RR and LB). The full texts of the remaining potentially relevant articles that met the inclusion and exclusion criteria were retrieved and reviewed by two further independent reviewers (MG and AC). Any disagreement was discussed until a decision was reached by consensus. The ﬁnal eligibility of each study was checked, and the reasons for exclusion were recorded. Two authors (RR and LB) made the ﬁnal selection of articles. In cases of disagreement, other authors were consulted to reach consensus. 2.1. Eligibility Criteria Peer-reviewed research articles were considered. Eligible studies were selected ac- cording to the following criteria: (1) RCTs, prospective or retrospective studies comparing the effects of NAT vs. US; (2) studies including resectable patients with pancreatic can- cer; (3) studies reporting primarily overall survival (OS) and disease-free survival (DFS); (4) studies using chemotherapy or chemoradiotherapy as a preoperative neoadjuvant strat- egy. We considered only papers where the deﬁnition of resectability was reported and based on: (1) no extra-pancreatic disease; (2) no tumor extension to the superior mesenteric artery or celiac axis; and (3) limited (<180◦) and/or no occlusion of the superior mesenteric vein (SMV) or the SMV-portal vein (PV). Furthermore, any deﬁnition of residual disease (R-status) after surgery was accepted. Studies with insufficient information, overlapping samples, reviews, meta-analyses, studies without complete data from the resectable disease cohort, or studies that included only preoperative radiotherapy as a neoadjuvant strategy were excluded. For overlapping samples, we used the most recent publication or the one with complete information; for studies, one of which was an update of the previous ones, we used the most updated information. The data collected included: 2. Sample size: total, NAT, and US sample size, respectively 4. Primary outcome: survival data (OS and DFS). Primary outcome: survival data (OS and DFS). y 5. Secondary outcome: R0 resection rate and positive lymph node rate (N+). For the primary outcomes, we obtained hazard ratio estimates (HR) and 95% CI directly from the individual studies if they were provided by the authors. Otherwise, survival data were extracted from Kaplan–Meier curves with Digitizelt software 2.5.10 (DigitizeIt, Braunschweig, Germany) and subsequently reported in STATA17 (StataCorp., College Station, TX, USA) to determine the HR estimate and its variants. For RCTs, only intention-to-treat analyses were used. 2.4. Risk of Bias—Quality Assessment The quality of RCTs was independently assessed by two authors using the Cochrane Risk of Bias Tool (RoB2). Five domains of bias (i.e., randomization process, deviations from planned interventions, missing outcome data, outcome measurement, and selection of reported outcomes) were assessed and reported according to the Cochrane Handbook for Systematic Reviews of Interventions [22]. A judgment of "high" indicated a high risk of bias, "low" indicated a low risk of bias, and "some concerns" indicated the presence of bias due to a lack of information or uncertainty about the potential for bias. Studies were thus categorized as having a low or high risk of bias or some concerns. For non-RCT studies, the risk of bias was assessed using the Newcastle-Ottawa Quality Scale (NOS) [23]. The instrument consists of eight items, four of which relate to selection, one to comparability, and three to exposure. A score of less than 4 points indicates a high risk of bias; a score of 5 indicates a moderate risk of bias; and a score of more than 5 indicates a low risk of bias. The risk of bias was assessed by three independent authors (RR, LB, and AC). Possible disagreements about the quality score were resolved through discussion and consensus among all authors. 2.5. Statistical Analysis All data from eligible articles were summarized in a systematic summary and then used for the meta-analysis. Speciﬁcally, we used HR for OS and DFS and risk estimates (odds ratios) for the R0 resection rate and the N+ rate. A random-effects approach (DerSimonian-Laird) was used, hypothesizing a possible heterogeneity between studies due to differences in study designs, observation periods, patient characteristics, neoad- juvant and adjuvant therapies, and follow-up duration. In addition, heterogeneity was assessed using the Cochrane Q-test and the I2 statistic according to the Cochrane Handbook for Systematic Reviews of Interventions [24]. Outliers as a possible source of heterogeneity were investigated using the Galbraith plot for OS and DFS and the L’Abbè plot for R0 resection rate and N+ rate. Using the Galbraith plot, we analyzed which studies fell outside the 95% CI range, while using the L’Abbè plot, we assessed which studies deviated signiﬁ- cantly from the effect size line. For the sample size subgroup analysis, we set a cut-off of 150 patients, which was chosen after determining the median of the total sample size of the included studies (167) and rounding up to the nearest multiple of 50. We also performed a cumulative meta-analysis to track the accumulation of evidence per year. Finally, we performed a “leave-one-out” analysis (one study removed) as a sensitive analysis to examine the impact of individual studies on the overall prevalence results. Publication bias was assessed according to Cochrane guidelines if the total number of studies was ≥10. All analyses were performed using STATA18 (StataCorp., College Station, TX, USA). 2.3. Data Extraction Two authors (RR and LB) independently extracted data from the full texts of the studies that met the inclusion criteria. Discrepancies were resolved through team discussion. The authors of the included studies were not contacted to obtain unpublished data. In the case of incomplete information from primary studies, data were extracted from two previous meta-analyses [20,21]. The data collected included: 1. Study characteristics: ﬁrst author, year, country, observation period, and study design. 1. Study characteristics: ﬁrst author, year, country, observation period, and study design 2. Sample size: total, NAT, and US sample size, respectively. 3. Patients’ demographic characteristics: gender and age. Cancers 2023, 15, 4627 4 of 17 4. Primary outcome: survival data (OS and DFS). 3.1. Search Results and Studies Characteristics The search strategy identiﬁed 11,083 articles from databases (EMBASE: 7751; PubMed: 2780; Cochrane Library: 522) (Figure 1). After excluding duplicates (n = 2510), 8573 articles were screened by title, of which 8434 were excluded by title and a further 101 by title and Cancers 2023, 15, 4627 5 of 17 5 of 17 abstract. Thirty-eight articles met the inclusion criteria and were subsequently screened in full text. Of these, 23 were excluded (14 did not meet the inclusion criteria, 7 were found to be protocols of unpublished data, and 2 were updated in included studies). Finally, 15 studies from databases were included in this work [17,25–38]. In the study by Birrier et al., we considered only the results from their RCT, excluding those from Casadei et al. and Golcher et al., whereas the results from Casadei et al. and Golcher et al. were retrieved from the original publication [27,28,30]. IEW 3 of 11 Figure 1. PRISMA Flow-chart. Identification of studies via databases and registers Identification Screening Included Records identified from databases search (n = 11083) EMBASE (n = 7751) PubMed (n = 2780) Cochrane Library (n = 552) Records removed before screening: Duplicate records removed (n = 2510) Records screened (n = 8573) Records sought for retrieval (n = 139) Records assessed for eligibility (n = 38) Records excluded after title screening (n = 8434) Records excluded after Title and Abstract screening (n = 101) Records excluded: Did not meet inclusion criteria (n = 14) Protocol publication of unpublished studies (n = 7) Results updated in included studies (n = 2) Studies included in review (n = 15) Reports of included studies (n = 15) Figure 1. PRISMA Flow-chart. R REVIEW 4 o Identification of studies via databases and registers Records excluded after Title and Abstract screening (n = 101) Records sought for retrieval (n = 139) Figure 1. PRISMA Flow-chart. Figure 1. PRISMA Flow-chart. The quality assessment did not reveal any speciﬁc concerns in terms of bias (Figure 2 and Table 1). The quality assessment did not reveal any speciﬁc concerns in terms of bias (Figure 2 and Table 1). Figure 2. Risk of Bias—RCT [18,26,27,29,32,34]. T bl 1 Ri k f Bi N tl Ott l t (NOS) Authors Experimental Comparator Main Outcome Secondary Outcomes Weight D1 D2 D3 D4 D5 Overall Birrer et al. 3.1. Search Results and Studies Characteristics (2021) N A T U S O S, D FS N one 1 Low risk C asadei et al. (2015) N A T U S O S, D FS R0 Resection Rate and N + Ra 1 Som e concerns G olcher et al. (2015) N A T U S O S, D FS R0 Resection Rate and N + Ra 1 H igh risk Reni et al. (2018) N A T U S O S, D FS R0 Resection Rate and N + Ra 1 Seufferlein et al. (2023) N A T U S O S, D FS R0 Resection Rate 1 D 1 Random isation process V ersteijne et al. (2022) N A T U S O S, D FS R0 Resection Rate 1 D 2 D eviations from the intended intervention D 3 M issing outcom e data D 4 M easurem ent of the outcom e D 5 Selection of the reported result + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ! - Figure 2. Risk of Bias—RCT [18,26,27,29,32,34]. Figure 2. Risk of Bias—RCT [18,26,27,29,32,34]. T bl 1 Ri k f Bi N tl Ott l t (NOS) Authors Experimental Comparator Main Outcome Secondary Outcomes Weight D1 D2 D3 D4 D5 Overall Birrer et al. (2021) N A T U S O S, D FS N one 1 Low risk C asadei et al. (2015) N A T U S O S, D FS R0 Resection Rate and N + Ra 1 Som e concerns G olcher et al. (2015) N A T U S O S, D FS R0 Resection Rate and N + Ra 1 H igh risk Reni et al. (2018) N A T U S O S, D FS R0 Resection Rate and N + Ra 1 Seufferlein et al. (2023) N A T U S O S, D FS R0 Resection Rate 1 D 1 Random isation process V ersteijne et al. 3.1. Search Results and Studies Characteristics Five studies were conducted in the USA [30,31,33,36,37], two in Italy [27,32], Japan [28,35] and Germany [29,34] respectively, and one each in France [25], Switzerland [26], the Nether- lands [18] and Korea [38]. The longest study—a retrospective study—examined an ob- servation period of 32 years from 1989 to 2021 [37], while the shortest—one RCT—had a follow-up of 4 years from 2015 to 2019 [34]. Seven studies had a total sample size of more than 150 patients, with two studies including 6802 [37] and 8020 [30] patients, respectively. The median OS for NAT ranged from 15 months [25] to 50.2 months [35], while for the US it ranged from 14.3 months [18] to 32.7 months [35]. Almost all studies used gemcitabine as neoadjuvant therapy (as monotherapy or in combination with other drugs) and as adju- vant chemotherapy after surgery; nine studies used a combination of chemotherapy and radiotherapy as neoadjuvant therapy. More active regimens with modern chemotherapy combinations (PEXG: cisplatin, epirubicin, gemcitabine and capecitabine; Gemcitabine plus Nab-paclitaxel; FOLFIRINOX) were used only in three recent studies [32,34,38]. Table 2. Study Characteristics. Author Country Period Multicenter Study Study Design Sample Median OS Total NAT US Barbier et al. (2011) [25] France 1997–2006 No Retrospective Study 173 88 85 NAT: 15 mo (3–72) US: 17 mo (1–109) Birrer et al. (2021) [26] Switzerland 2009–2018 No RCT 34 16 18 NR Casadei et al. (2015) [27] Italy 2007–2014 No RCT 38 18 20 NAT: 22.4 mo (10.2–34.6) US: 19.5 mo (7.5–31.5) Author Is the Case Deﬁnition Adequate? Representativeness of the Cases Selection of Controls Deﬁnition of Controls Comparability of Cases and Controls on the Basis of the Design or Analysis Ascertainment of Exposure Same Method of Ascertainment for Cases and Controls Non-Response Rate Total Score Barbier et al. (2011) [25] 1 1 0 1 1 1 1 1 7 Fujii et al. (2017) [28] 1 1 0 1 2 1 1 0 7 Mokdad et al. (2017) [30] 1 1 0 1 2 1 1 0 7 Papalezova et al. (2012) [31] 1 1 0 1 2 1 1 1 8 Roland et al. (2015) [33] 1 1 0 1 1 1 1 0 6 Sho et al. (2015) [35] 1 1 0 1 2 1 1 0 7 Tzeng et al. (2014) [36] 1 1 0 1 2 1 1 1 8 Vidri et al. 3.1. Search Results and Studies Characteristics (2021) [37] 1 1 0 1 2 1 1 1 8 Yoon et al. (2022) [38] 1 1 0 1 2 1 1 1 8 A score of less than 4 points indicates a high risk of bias; a score of 5 indicates a moderate risk of bias; and a score of more than 5 indicates a low risk of bias. A score of less than 4 points indicates a high risk of bias; a score of 5 indicates a moderate risk of bias; and a score of more than 5 indicates a low risk of bias. 3.1. Search Results and Studies Characteristics (2022) N A T U S O S, D FS R0 Resection Rate 1 D 2 D eviations from the intended intervention D 3 M issing outcom e data D 4 M easurem ent of the outcom e D 5 Selection of the reported result + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ! - Figure 2. Risk of Bias—RCT [18,26,27,29,32,34]. Figure 2. Risk of Bias—RCT [18,26,27,29,32,34]. Figure 2. Risk of Bias—RCT [18,26,27,29,32,34]. Cancers 2023, 15, 4627 6 of 17 Table 1. Risk of Bias—Newcastle-Ottawa scale assessment (NOS). Author Is the Case Deﬁnition Adequate? Representativeness of the Cases Selection of Controls Deﬁnition of Controls Comparability of Cases and Controls on the Basis of the Design or Analysis Ascertainment of Exposure Same Method of Ascertainment for Cases and Controls Non-Response Rate Total Score Barbier et al. (2011) [25] 1 1 0 1 1 1 1 1 7 Fujii et al. (2017) [28] 1 1 0 1 2 1 1 0 7 Mokdad et al. (2017) [30] 1 1 0 1 2 1 1 0 7 Papalezova et al. (2012) [31] 1 1 0 1 2 1 1 1 8 Roland et al. (2015) [33] 1 1 0 1 1 1 1 0 6 Sho et al. (2015) [35] 1 1 0 1 2 1 1 0 7 Tzeng et al. (2014) [36] 1 1 0 1 2 1 1 1 8 Vidri et al. (2021) [37] 1 1 0 1 2 1 1 1 8 Yoon et al. (2022) [38] 1 1 0 1 2 1 1 1 8 A score of less than 4 points indicates a high risk of bias; a score of 5 indicates a moderate risk of bias; and a score of more than 5 indicates a low risk of bias. 3.2. Studies Characteristics Sixteen thousand seven hundred and thirty-one (16,731) patients with a mean male-to- female ratio of 1.32 were included (Table 2 and Supplementary Materials Table S1), of whom 4166 underwent NAT and 12,565 US were included in this meta-analysis. Seven studies were retrospective studies, six were RCTs, and in two, the study design was not reported. 3.2. Studies Characteristics Sixteen thousand seven hundred and thirty-one (16,731) patients with a mean male-to- female ratio of 1.32 were included (Table 2 and Supplementary Materials Table S1), of whom 4166 underwent NAT and 12,565 US were included in this meta-analysis. Seven studies were retrospective studies, six were RCTs, and in two, the study design was not reported. Five studies were conducted in the USA [30,31,33,36,37], two in Italy [27,32], Japan [28,35] and Germany [29,34] respectively, and one each in France [25], Switzerland [26], the Nether- lands [18] and Korea [38]. The longest study—a retrospective study—examined an ob- servation period of 32 years from 1989 to 2021 [37], while the shortest—one RCT—had a follow-up of 4 years from 2015 to 2019 [34]. Seven studies had a total sample size of more than 150 patients, with two studies including 6802 [37] and 8020 [30] patients, respectively. The median OS for NAT ranged from 15 months [25] to 50.2 months [35], while for the US it ranged from 14.3 months [18] to 32.7 months [35]. Almost all studies used gemcitabine as neoadjuvant therapy (as monotherapy or in combination with other drugs) and as adju- vant chemotherapy after surgery; nine studies used a combination of chemotherapy and radiotherapy as neoadjuvant therapy. More active regimens with modern chemotherapy combinations (PEXG: cisplatin, epirubicin, gemcitabine and capecitabine; Gemcitabine plus Nab-paclitaxel; FOLFIRINOX) were used only in three recent studies [32,34,38]. Table 2. Study Characteristics. Author Country Period Multicenter Study Study Design Sample Median OS Total NAT US Barbier et al. (2011) [25] France 1997–2006 No Retrospective Study 173 88 85 NAT: 15 mo (3–72) US: 17 mo (1–109) Birrer et al. (2021) [26] Switzerland 2009–2018 No RCT 34 16 18 NR Casadei et al. (2015) [27] Italy 2007–2014 No RCT 38 18 20 NAT: 22.4 mo (10.2–34.6) US: 19.5 mo (7.5–31.5) Table 2. Study Characteristics. 7 of 17 Cancers 2023, 15, 4627 Table 2. Cont. Author Country Period Multicenter Study Study Design Sample Median OS Total NAT US Fujii et al. (2017) [28] Japan 2001–2013 Yes NR 273 40 233 NAT: 24.9 mo US: 23.5 mo Golcher et al. (2015) [29] Germany 2003–2009 Yes RCT 66 33 33 NAT: 17.4 mo US: 17.4 mo Mokdad et al. (2017) [30] USA 2006–2012 No NR 8020 2005 6015 NAT: 26 mo US: 21 mo Papalezova et al. 3.2. Studies Characteristics (2012) [31] USA 1999–2007 No Retrospective Study 236 144 92 NAT: 15 mo US: 13 mo Reni et al. (2018) [32] Italy 2010–2015 No RCT 62 32 30 NAT: 38.2 mo (27.3–49.1) US: 26.4 mo (15.8–26.7) Roland et al. (2015) [33] USA 1990–2008 No Retrospective Study 307 222 85 NR NR Seufferlein et al. (2023) [34] Germany 2015–2019 Yes RCT 118 59 59 NAT: 25.5 mo (19.7–29.7) US: 16.7 mo (11.6–22.2) Sho et al. (2015) [35] Japan 2006–2013 No Retrospective Study 100 44 56 NAT: 50.2 mo US: 32.7 mo Tzeng et al. (2014) [36] USA 2002–2007 No Retrospective Study 167 115 52 NAT: 28 mo (21.7–34.3) US: 25.3 mo (19.9–30.7) Versteijne et al. (2022) [18] Netherlands 2013–2017 Yes RCT 133 65 68 NAT: 15.7 mo (12.9–20.6) US: 14.3 mo (12.7–17.9) Vidri et al. (2021) [37] USA 1989–2021 Yes Retrospective Study 6802 1118 5684 NAT: 27.6 mo (IQR: 38.8) US: 25.6 mo (IQR: 40.9) Yoon et al. (2022) [38] Korea 2012–2019 No Retrospective Study 202 167 35 NR NR NAT: Neoadjuvant Therapy; US: Upfront Surgery; NR: Not reported; RCT: Randomized Controlled Trial. Table 2. Cont. 3.3. Overall Survival The overall trend for HR was conﬁrmed in the subgroup analysis of RCTs, from which HR for death was 0.74 (95% CI: 0.61–0.90, z = −3.06, p < 0.001, 𝜏2 = 0.01, I2 = 0.00%), while no signiﬁcant HR emerged from retrospective study analysis (HR: 0.90, 95% CI: 0.76–1.06, z = −1.24, p = 0.21, 𝜏2 = 0.01, I2 = 21.98%) (Supplementary Materials—Figure S2). The cumulative analysis revealed a clear trend from 2011 to 2022, showing how HR stabilized at 0.80 (95% CI: 0.72–0.91) (Supplementary Materials—Figure S3). In the leave- one-out analysis, one study deviated signiﬁcantly from the overall eﬀect [25], as already h i th G lb ith l t (S l t M t i l Fi S4) Th ti Figure 4. Overall Survival: (A) Subgroup analysis related to the use of chemotherapy vs. chemoradiotherapy—Mixed refers to 3 studies that included patients treated both with and without radiotherapy; (B) Subgroup analysis related to sample size (<150 pz. vs. ≥150 pz.) [18,25–38]. The overall trend for HR was conﬁrmed in the subgroup analysis of RCTs, from which HR for death was 0.74 (95% CI: 0.61–0.90, z = −3.06, p < 0.001, τ2 = 0.01, I2 = 0.00%), while no signiﬁcant HR emerged from retrospective study analysis (HR: 0.90, 95% CI: 0.76–1.06, z = −1.24, p = 0.21, τ2 = 0.01, I2 = 21.98%) (Supplementary Materials—Figure S2). The cumulative analysis revealed a clear trend from 2011 to 2022, showing how HR stabilized at 0.80 (95% CI: 0.72–0.91) (Supplementary Materials—Figure S3). In the atients treated with chemoradiotherapy as neoadjuva 0.86 (95% CI: 0.75–0.99, z = −2.07, p = 0.04, 𝜏2 = 0.01, I2 d from studies that included only patients treated wi 0.74, 95% CI: 0.57–0.96, z = −2.24, p = 0.02, 𝜏2 = 0.00, I2 er for studies that included patients who received eith emoradiotherapy (HR: 0.72, 95% CI: 0.68–0.76, z = −11.5 (Figure 4A). A signiﬁcantly lower HR was observed 150 patients (HR: 0.72, 95% CI: 0.60–0.87, z = −3.50, p e studies that included more than 150 patients showed a moderate degree of heterogeneity (HR: 0.86, 95% C 0.03, I2 = 58.96%) (Figure 4B). (B) For studies that inc treatment, the HR for de 19.96%), similar to that neoadjuvant chemothera 0.31%). 3.3. Overall Survival 2011 [25], Mokdad et al. 2017 [30]). Figure 3. Overall Survival: (A) Forest Plot [18,25–38]; (B) Overall Survival without out- liers [18,26–29,31–38] (n = 13) (outlier: Barbier et al. 2011 [25], Mokdad et al. 2017 [30]). ncers 2023, 15, x FOR PEER REVIEW 6 of 11 tients showed a non-signiﬁcantly lower HR and a moderate degree of heterogeneity (HR: 0.86, 95% CI: 0.73–1.02, z = −1.77, p = 0.08, τ2 = 0.03, I2 = 58.96%) (Figure 4B). p pp y g p excluding this outlier was 0.81 (95% CI: 0.72–0.90; z = −3.67, p < 0.001, 𝜏2 = 0.00, I2 = 0.00%) Figure 3B). (B) (A) (A) (B) Figure 3. Overall Survival: (A) Forest Plot [18,25–38]; (B) Overall Survival without outliers [18,26– 29,31–38] (n = 13) (outlier: Barbier et al. 2011 [25], Mokdad et al. 2017 [30]). Figure 3. Overall Survival: (A) Forest Plot [18,25–38]; (B) Overall Survival without out- liers [18,26–29,31–38] (n = 13) (outlier: Barbier et al. 2011 [25], Mokdad et al. 2017 [30]). W 6 of 11 For studies that included patients treated with chemoradiotherapy as neoadjuvant treatment, the HR for death was 0.86 (95% CI: 0.75–0.99, z = −2.07, p = 0.04, 𝜏2 = 0.01, I2 = 19.96%), similar to that observed from studies that included only patients treated with neoadjuvant chemotherapy (HR: 0.74, 95% CI: 0.57–0.96, z = −2.24, p = 0.02, 𝜏2 = 0.00, I2 = 0.31%). HR resulted slightly lower for studies that included patients who received either neoadjuvant chemotherapy or chemoradiotherapy (HR: 0.72, 95% CI: 0.68–0.76, z = −11.58, p < 0.001, 𝜏2 = 0.00, I2 = 0.00%) (Figure 4A). A signiﬁcantly lower HR was observed in studies that included fewer than 150 patients (HR: 0.72, 95% CI: 0.60–0.87, z = −3.50, p < 0.001, 𝜏2 = 0.01, I2 = 0.00%), while studies that included more than 150 patients showed a non-signiﬁcantly lower HR and a moderate degree of heterogeneity (HR: 0.86, 95% CI: 0.73–1.02, z = −1.77, p = 0.08, 𝜏2 = 0.03, I2 = 58.96%) (Figure 4B). (A) (B) Figure 4. Overall Survival: (A) Subgroup analysis related to the use of chemotherapy vs. chemora- diotherapy—Mixed refers to 3 studies that included patients treated both with and without radio- therapy; (B) Subgroup analysis related to sample size (<150 pz. vs. ≥150 pz.) [18,25–38]. 3.3. Overall Survival All ﬁfteen studies with a total of 16,371 patients were included to investigate the role of NAT on OS. From the pooled HRs, OS for NAT patients was better, with an HR for death of 0.80 (95% CI: 0.72–0.90) with a signiﬁcance level of less than 1% (z = −3.95, p < 0.001, τ2 = 0.01, I2 = 31.09%) (Figure 3A). Two studies [25,30] were detected outside the shaded area in the Galbraith plot (Supplementary Materials: Figure S1). The pooled HR excluding this outlier was 0.81 (95% CI: 0.72–0.90; z = −3.67, p < 0.001, τ2 = 0.00, I2 = 0.00%) (Figure 3B). For studies that included patients treated with chemoradiotherapy as neoadjuvant treatment, the HR for death was 0.86 (95% CI: 0.75–0.99, z = −2.07, p = 0.04, τ2 = 0.01, I2 = 19.96%), similar to that observed from studies that included only patients treated with neoadjuvant chemotherapy (HR: 0.74, 95% CI: 0.57–0.96, z = −2.24, p = 0.02, τ2 = 0.00, I2 = 0.31%). HR resulted slightly lower for studies that included patients who received either neoadjuvant chemotherapy or chemoradiotherapy (HR: 0.72, 95% CI: 0.68–0.76, z = −11.58, p < 0.001, τ2 = 0.00, I2 = 0.00%) (Figure 4A). A signiﬁcantly lower HR was observed in studies that included fewer than 150 patients (HR: 0.72, 95% CI: 0.60–0.87, z = −3.50, p < 0.001, τ2 = 0.01, I2 = 0.00%), while studies that included more than 150 pa- Cancers 2023, 15, 4627 8 of 17 R for 5, p < tients showed a non-signiﬁcantly lower HR and a moderate degree of heterogeneity (HR: 0.86, 95% CI: 0.73–1.02, z = −1.77, p = 0.08, τ2 = 0.03, I2 = 58.96%) (Figure 4B). p ( pp y g ) p excluding this outlier was 0.81 (95% CI: 0.72–0.90; z = −3.67, p < 0.001, 𝜏2 = 0.00, I2 = 0.00%) (Figure 3B). tients showed a non-signiﬁcantly lower HR and a moderate degree of heterogeneity (HR: 0.86, 95% CI: 0.73–1.02, z = −1.77, p = 0.08, τ2 = 0.03, I2 = 58.96%) (Figure 4B). p ( pp y g ) p excluding this outlier was 0.81 (95% CI: 0.72–0.90; z = −3.67, p < 0.001, 𝜏2 = 0.00, I2 = 0.00%) (Figure 3B). (A) (B) Figure 3. Overall Survival: (A) Forest Plot [18,25–38]; (B) Overall Survival without outliers [18,26– 29,31–38] (n = 13) (outlier: Barbier et al. 3.3. Overall Survival HR resulted slig neoadjuvant chemothera p < 0.001, 𝜏2 = 0.00, I2 = studies that included fe 0.001, 𝜏2 = 0.01, I2 = 0.00 non-signiﬁcantly lower 0.73–1.02, z = −1.77, p = 0 (A) (A) (B) Figure 4. Overall Survival: (A) Subgroup analysis related to the use of chemotherapy vs. chemora- diotherapy—Mixed refers to 3 studies that included patients treated both with and without radio- therapy; (B) Subgroup analysis related to sample size (<150 pz. vs. ≥150 pz.) [18,25–38]. Figure 4. Overall Survival: (A) Subgroup analysis related to the use of chemotherapy vs. chemoradiotherapy—Mixed refers to 3 studies that included patients treated both with and without radiotherapy; (B) Subgroup analysis related to sample size (<150 pz. vs. ≥150 pz.) [18,25–38]. The overall trend for HR was conﬁrmed in the subgroup analysis of RCTs, from which HR for death was 0.74 (95% CI: 0.61–0.90, z = −3.06, p < 0.001, 𝜏2 = 0.01, I2 = 0.00%), while no signiﬁcant HR emerged from retrospective study analysis (HR: 0.90, 95% CI: 0.76–1.06, z = −1.24, p = 0.21, 𝜏2 = 0.01, I2 = 21.98%) (Supplementary Materials—Figure S2). Th l ti l i l d l t d f 2011 t 2022 h i h HR The overall trend for HR was conﬁrmed in the subgroup analysis of RCTs, from which HR for death was 0.74 (95% CI: 0.61–0.90, z = −3.06, p < 0.001, τ2 = 0.01, I2 = 0.00%), while no signiﬁcant HR emerged from retrospective study analysis (HR: 0.90, 95% CI: 0.76–1.06, z = −1.24, p = 0.21, τ2 = 0.01, I2 = 21.98%) (Supplementary Materials—Figure S2). The overall trend for HR was conﬁrmed in the subgroup analysis of RCTs, from which HR for death was 0.74 (95% CI: 0.61–0.90, z = −3.06, p < 0.001, 𝜏2 = 0.01, I2 = 0.00%), while no signiﬁcant HR emerged from retrospective study analysis (HR: 0.90, 95% CI: 0.76–1.06, z = −1.24, p = 0.21, 𝜏2 = 0.01, I2 = 21.98%) (Supplementary Materials—Figure S2). 3.4. Disease-Free Survival Seven studies with a total of 653 patients were included in the pooled HR for DFS. From the pooled HRs, the DFS for NAT was better, with an HFR for progression of 0.66 (95% CI: 0.56–0.79) and a signiﬁcance level of less than 1% (z = −4.74, p < 0.001, τ2 = 0.00, I2 = 0.00%) (Figure 5). The Galbraith plot showed no outliers (Supplementary Materials—Figure S6). EW 7 of 11 Figure 5. Disease Free Survival (n = 7): Forest Plot [18,26,27,29,32,34,38]. Figure 5. Disease Free Survival (n = 7): Forest Plot [18,26,27,29,32,34,38]. Figure 5. Disease Free Survival (n = 7): Forest Plot [18,26,27,29,32,34,38]. Figure 5. Disease Free Survival (n = 7): Forest Plot [18,26,27,29,32,34,38]. Subgroup analysis showed that patients treated with neoadjuvant chemoradiother- apy had a HR for progression of 0.65 (95% CI: 0.52, 0.82, z = −3.65, p < 0.001, 𝜏2 = 0.00, I2 = 0.00%), which was slightly lower than that for patients treated with neoadjuvant chemo- therapy (HR: 0.73, 95% CI: 0.54–0.97, z = −2.20, p = 0.03, 𝜏2 = 0.00, I2 = 0.00%), while no conclusions could be drawn for the studies that included patients treated with both neo- adjuvant chemotherapy and chemoradiotherapy, as only one study was included (Sup- plementary Materials—Figure S7). Analysis of only RCTs conﬁrmed the signiﬁcant HR for progression (HR: 0.68, 95% CI: 0.57–0.81, z = −4.22, p < 0.001, 𝜏2 = 0.00, I2 = 0.00%) (Supple- mentary Materials—Figure S8). Subgroup analysis showed that patients treated with neoadjuvant chemoradiotherapy had a HR for progression of 0.65 (95% CI: 0.52, 0.82, z = −3.65, p < 0.001, τ2 = 0.00, I2 = 0.00%), which was slightly lower than that for patients treated with neoadjuvant chemotherapy (HR: 0.73, 95% CI: 0.54–0.97, z = −2.20, p = 0.03, τ2 = 0.00, I2 = 0.00%), while no conclusions could be drawn for the studies that included patients treated with both neoadjuvant chemotherapy and chemoradiotherapy, as only one study was included (Supplementary Materials—Figure S7). Analysis of only RCTs conﬁrmed the signiﬁcant HR for progression (HR: 0.68, 95% CI: 0.57–0.81, z = −4.22, p < 0.001, τ2 = 0.00, I2 = 0.00%) (Supplementary Materials—Figure S8). y g ) In the cumulative analysis, an increase in HR was found over the years (Supplemen- tary Materials—Figure S9), while no signiﬁcant changes were found in HR from the leave- one-out analysis (Supplementary Materials—Figure S10). 3.4. Disease-Free Survival Publication bias was not de- tected because the number of studies was less than 10. In the cumulative analysis, an increase in HR was found over the years (Supplementary Materials—Figure S9), while no signiﬁcant changes were found in HR from the leave-one- out analysis (Supplementary Materials—Figure S10). Publication bias was not detected because the number of studies was less than 10. 3.3. Overall Survival Th l ti l i l d l t d f 2011 t 2022 h i h HR The overall trend for HR was conﬁrmed in the subgroup analysis of RCTs, from which HR for death was 0.74 (95% CI: 0.61–0.90, z = −3.06, p < 0.001, τ2 = 0.01, I2 = 0.00%), while no signiﬁcant HR emerged from retrospective study analysis (HR: 0.90, 95% CI: 0.76–1.06, z = −1.24, p = 0.21, τ2 = 0.01, I2 = 21.98%) (Supplementary Materials—Figure S2). The cumulative analysis revealed a clear trend from 2011 to 2022, showing how HR stabilized at 0.80 (95% CI: 0.72–0.91) (Supplementary Materials—Figure S3). In the leave- one-out analysis one study deviated signiﬁcantly from the overall eﬀect [25] as already p ) ( pp y g ) The cumulative analysis revealed a clear trend from 2011 to 2022, showing how HR stabilized at 0.80 (95% CI: 0.72–0.91) (Supplementary Materials—Figure S3). In the Cancers 2023, 15, 4627 9 of 17 9 of 17 leave-one-out analysis, one study deviated signiﬁcantly from the overall effect [25], as already shown in the Galbraith plot (Supplementary Materials—Figure S4). There was no particular publication bias (Supplementary Materials—Figure S5), which was also conﬁrmed by Egger’s test for small study effects (p = 0.698). 3.4. Disease-Free Survival 3.4. Disease-Free Survival 3.5. R0 Resection Rate 3.5. R0 Resection Rate Thirteen studies with a total of 16,461 patients were included. In the pooled HR, NAT showed a better R0 resection rate than US (OR: 1.70, 95% CI: 1.23–2.36, z = 3.19, p < 0.001, 𝜏2 = 0.13, I2 = 46.56%) (Figure 6A). After analyzing possible outliers through the L’Abbè plot (Supplementary Materials—Figure S11), the OR recalculated without the three iden- tiﬁed outliers s Thirteen studies with a total of 16,461 patients were included. In the pooled HR, NAT showed a better R0 resection rate than US (OR: 1.70, 95% CI: 1.23–2.36, z = 3.19, p < 0.001, τ2 = 0.13, I2 = 46.56%) (Figure 6A). After analyzing possible outliers through the L’Abbè plot (Supplementary Materials—Figure S11), the OR recalculated without the three identiﬁed outliers showed a slight decrease (OR = 1.62, 95% CI: 1.33–1.99, z = 4.68, p < 0.001, τ2 = 0.01, I2 = 7.97%), but without losing its signiﬁcance level (Figure 6B). tiﬁed outliers s g τ2 = 0.01, I2 = 7.97%), but without losing its signiﬁcance level (Figure 6B). Subgroup analysis showed signiﬁcantly higher HR from retrospective studies (HR: 1.89, 95% CI: 1.09–3.29, z = 2.27, p = 0.02, τ2 = 0.20, I2 = 44.71%), while such a signif- icant trend was not conﬁrmed pooling results from RCTs (HR: 1.70, 95% CI: 0.70–4.08, z = 1.18, p = 0.24, τ2 = 0.61, I2 = 63.91%), among which a signiﬁcant heterogeneity occurred (Supplementary Materials—Figure S12). Cancers 2023, 15, 4627 10 of 17 0.01, I2 (A) (B) Figure 6. R0 Rate (n = 13): (A) Forest Plot [18,25,27–36,38]; (B) Forest Plot without outliers [27– 34,36,38] (three outliers: Barbier et al. 2011 [25], Sho et al. 2015 [35], Verstejine et al. 2022 [18])—Note: for Verstejine et al. 2022 [18] data were extracted from the Verstejine et al. 2020 [17]. Figure 6. R0 Rate (n = 13): (A) Forest Plot [18,25,27–36,38]; (B) Forest Plot without out- liers [27–34,36,38] (three outliers: Barbier et al. 2011 [25], Sho et al. 2015 [35], Verstejine et al. 2022 [18])—Note: for Verstejine et al. 2022 [18] data were extracted from the Verstejine et al. 2020 [17]. (A) (B) (B) Figure 6. R0 Rate (n = 13): (A) Forest Plot [18,25,27–36,38]; (B) Forest Plot without outliers [27– 34,36,38] (three outliers: Barbier et al. 2011 [25], Sho et al. 2015 [35], Verstejine et al. 2022 [18])—Note: for Verstejine et al. 3.5. R0 Resection Rate 3.5. R0 Resection Rate 2022 [18] data were extracted from the Verstejine et al. 2020 [17]. Figure 6. R0 Rate (n = 13): (A) Forest Plot [18,25,27–36,38]; (B) Forest Plot without out- liers [27–34,36,38] (three outliers: Barbier et al. 2011 [25], Sho et al. 2015 [35], Verstejine et al. 2022 [18])—Note: for Verstejine et al. 2022 [18] data were extracted from the Verstejine et al. 2020 [17]. Subgroup analysis showed signiﬁcantly higher HR from retrospective studies (HR: 1.89, 95% CI: 1.09–3.29, z = 2.27, p = 0.02, 𝜏2 = 0.20, I2 = 44.71%), while such a signiﬁcant trend was not conﬁrmed pooling results from RCTs (HR: 1.70, 95% CI: 0.70–4.08, z = 1.18, p = 0.24, 𝜏2 = 0.61, I2 = 63.91%), among which a signiﬁcant heterogeneity occurred (Sup- plementary Materials—Figure S12). No particular trend emerged from the cumulative meta-analysis (Supplementary Materials Figure S13) and no signiﬁcant deviation from the eﬀect size was found in the No particular trend emerged from the cumulative meta-analysis (Supplementary Materials—Figure S13), and no signiﬁcant deviation from the effect size was found in the leave-one-out analysis (Supplementary Materials—Figure S14). In the publication bias, a slight asymmetry was observed in the funnel plot (Supplementary Materials—Figure S15), although Egger’s test for small study effects did not reveal a statistically signiﬁcant indica- tion (p = 0.096). leave-one-out 3.6. N+ Rate slight asymmetry was observed in the funnel plot (Supplementary Materials—Figure S15), although Egger’s test for small study eﬀects did not reveal a statistically signiﬁcant indication (p = 0.096). 3.6. N+ Rate A meta-analysis of positive lymph nodes performed on a total of eight studies (n = 15,799 patients) showed a lower risk of positive lymph nodes in patients treated with NAT (OR: 0.45, 95% CI: 0.32–0.63, z = −4.56, 𝜏2 = 0.13, I2 = 76.43%) (Figure 7). In the quantitative A meta-analysis of positive lymph nodes performed on a total of eight studies (n = 15,799 patients) showed a lower risk of positive lymph nodes in patients treated with NAT (OR: 0.45, 95% CI: 0.32–0.63, z = −4.56, τ2 = 0.13, I2 = 76.43%) (Figure 7). In the quantitative investigation of the possible causes for the large heterogeneity, no outliers occurred (Supplementary Materials—Figure S16), and there was also no signiﬁcant element of bias in a particular study (sensitive analysis) (Supplementary Materials—Figure S17), while a clear trend towards stabilization of risk over the years emerged (Supplementary Materials—Figure S18). IEW 9 o investigation of the possible causes for the large heterogeneity, no outliers occurred (Sup- plementary Materials—Figure S16), and there was also no signiﬁcant element of bias in a particular study (sensitive analysis) (Supplementary Materials—Figure S17), while a clear trend towards stabilization of risk over the years emerged (Supplementary Materials— Figure S18). The overall trend was also conﬁrmed in the subgroup analysis performed, including RCTs (HR: 0.37, 95% CI: 0.16–0.86, z = −2.31, p = 0.02, 𝜏2 = 0.00, I2 = 0.00%) and retrospective studies (HR: 0.52, 95% CI: 0.32–0.85, z = −2.61, p = 0.01, 𝜏2 = 0.20, I2 = 68.39%), although in the latter subgroup a moderate level of heterogeneity emerged (Supplementary Materi- als—Figure S19). Publication bias was not determined. Figure 7. N+ Rate (n = 8): Forest Plot [25,29,30,32,36–38]. 4. Discussion Upfront resection with adjuvant chemotherapy still represents the standard of c for patients with resectable pancreatic cancer. Although a highly selected population (i Figure 7. N+ Rate (n = 8): Forest Plot [25,29,30,32,36–38]. The overall trend was also confirmed in the subgroup analysis performed, including RCTs (HR: 0.37, 95% CI: 0.16–0.86, z = −2.31, p = 0.02, τ2 = 0.00, I2 = 0.00%) and retrospective studies (HR: 0.52, 95% CI: 0.32–0.85, z = −2.61, p = 0.01, τ2 = 0.20, I2 = 68.39%), although in Figure 7. 4. Discussion Upfront resection with adjuvant chemotherapy still represents the standard of care for patients with resectable pancreatic cancer. Although a highly selected population (i.e., patients younger than 79 years, bilirubin <1.5 ULN, R0 or R1 resection within 12 weeks before randomization, post-surgical CT or MRI, post-operative CA 19-9 < 180 U/mL) was suitable for an intensive regimen (FOLFIRINOX), the prognosis is still poor, with a median OS of up to 53.5 months [8]. In addition, only patients with a good performance status and a good recovery after surgery can be treated with adjuvant chemotherapy, corresponding to half of the overall population of patients with resectable pancreatic cancer. In such a scenario, novel strategies are required to improve the outcome. Neoadjuvant/perioperative treatments have been shown to improve DFS and OS in other gastrointestinal cancers, such as rectal, oesophageal, and gastroesophageal cancer, in which such treatment strategies have long since been established as the standard of care. NAT has gained interest given the systemic nature of pancreatic adenocarcinoma and the difﬁculty of delivering planned adjuvant treatment to a limited number of patients. In addition, NAT may improve R0 and N0 resections, a well-known prognostic factor, and exclude from futile surgery those patients with rapidly progressive tumors. Since 2019, the National Comprehensive Cancer Network (NCCN) guidelines recommend US followed by adjuvant treatment for resectable pancreatic cancer, but advise considering NAT in those patients with high-risk features (large primary tumors, regional lymph nodes, elevated Ca19.9) [4]. g p y g y p Currently, there are a few clinical studies comparing NAT versus US in resectable pancreatic adenocarcinoma, reporting controversial results. Furthermore, considering the different methodological approaches (RCTs and retrospective studies), the difﬁculty in providing high-quality evidence due to the small sample size, and the emergence of new evidence, an update of the current evidence seems essential to help clinicians and researchers understand the potential role of NAT. Thus, the aim of this systematic review and meta-analysis was to evaluate the role of NAT for patients with resectable pancreatic cancer in improving OS and DFS and improving R0 and N0 rates compared with upfront resection. p This meta-analysis of 15 studies, including RCTs and retrospective studies published between 2011 and 2023, showed that NAT in resectable pancreatic adenocarcinoma signiﬁ- cantly improved OS, DFS, N0, and R0 resection rates. leave-one-out 3.6. N+ Rate N+ Rate (n = 8): Forest Plot [25,29,30,32,36–38 Figure 7. N+ Rate (n = 8): Forest Plot [25,29,30,32,36–38]. 4. Discussion Upfront resection with adjuvant chemotherapy still represents the standard of c for patients with resectable pancreatic cancer. Although a highly selected population ( The overall trend was also confirmed in the subgroup analysis performed, including RCTs (HR: 0.37, 95% CI: 0.16–0.86, z = −2.31, p = 0.02, τ2 = 0.00, I2 = 0.00%) and retrospective studies (HR: 0.52, 95% CI: 0.32–0.85, z = −2.61, p = 0.01, τ2 = 0.20, I2 = 68.39%), although in Cancers 2023, 15, 4627 11 of 17 11 of 17 the latter subgroup a moderate level of heterogeneity emerged (Supplementary Materials— Figure S19). the latter subgroup a moderate level of heterogeneity emerged (Supplementary Materials— Figure S19). g Publication bias was not determined. 4. Discussion Almost all studies used gemcitabine as neoadjuvant (monotherapy or in combination with other drugs) and adjuvant chemother- apy after surgery; nine studies used a combination of chemotherapy and radiotherapy as NAT. More active regimens with modern chemotherapy combinations were used only in three recent studies included in our analysis [32,34,38]. In 2018, Reni et al. demon- strated promising efﬁcacy with a multiagent combination (PEXG: cisplatin, epirubicin, gemcitabine, and capecitabine) as a neoadjuvant chemotherapy strategy in a small Phase II trial on 88 patients [32]. A median survival of 38.2 months and 3- and 5-year OS of 55% and 49%, respectively, were observed in patients who received neoadjuvant PEXG, meeting the predeﬁned criteria for success. Notably, the results of this trial suggested that the upfront use of a more effective chemotherapy regimen might reduce the risk of progression during the preoperative phase or during the ﬁrst 3 months of the adjuvant phase. Nonetheless, since the trial began, the standard of care for adjuvant therapy has changed, and other chemotherapy regimens have been developed. Accordingly, the authors decided to interrupt the planned Phase III trial. p p In the randomized phase II NEONAX trial, a perioperative or only adjuvant chemother- apy strategy with Gemcitabine plus Nab-Paclitaxel was compared in patients with re- sectable pancreatic adenocarcinoma. The primary endpoint of DFS > 55% at 18 months, based on data from CONKO-001 [11], was not reached in both arms [34]. However, a major Cancers 2023, 15, 4627 12 of 17 12 of 17 difference was demonstrated in median DFS (5.9 versus 17.9 months) in the adjuvant arm for the intention to treat (ITT) population versus modiﬁed ITT (deﬁned excluding those trial participants in the ITT population that did not receive the intended study interventions), due to several patients not being able to start adjuvant treatment for different reasons. This ﬁnding may be explained by the difference in chemotherapy exposure, with 90% of patients in the NAT arm completing pre-operative chemotherapy and 58% of patients starting adjuvant chemotherapy in the other arm. Thus, this trial demonstrates that chemotherapy delivery is most likely the most important non-surgical factor in improving the survival of patients with resectable pancreatic cancer. 4. Discussion p p In a retrospective analysis published in 2022, 202 patients from a single Korean hospital who underwent curative-intent pancreatic surgery for resectable pancreatic adenocarci- noma were divided into two groups: those undergoing US (n = 167, 82.7%) and those receiving neoadjuvant therapy followed by surgery (n = 35, 17.3%) [38]. Among this latest group, chemoradiotherapy was delivered in 43% of cases and chemotherapy alone in 57%. In most cases (n = 17), the regimen of choice was FOLFIRINOX, while Gemcitabine plus Nab-Paclitaxel, CDDP, or Erlotinib were chosen in the remaining 3 patients. As a result, a signiﬁcantly better DFS was observed with the addition of neoadjuvant therapy to the management of resectable pancreatic adenocarcinoma. g p Additionally, the Phase II trial SWOG1505 explored the potential beneﬁt of either mFOLFIRINOX or gemcitabine/nab-paclitaxel as NAT in resectable pancreatic adenocarci- noma [39]. Both perioperative regimens did not show a signiﬁcant survival beneﬁt over historical data from adjuvant trials; however, a formal comparison should be made with caution. In fact, in adjuvant trials, patients were randomized postoperatively, and therefore only patients with a complete recovery after surgery and who were ﬁt for chemotherapy were considered, whereas in SWOG1505 patients were randomized at the time of diagno- sis. On the other hand, encouraging signs of a beneﬁcial role of NAT were highlighted, with a high R0 resection rate (>80% vs. 40–60% in adjuvant trials), high N0 resections (40% vs. 20–30%), high chemotherapy exposure (90%), and a low incidence of grade 3–5 post-operative complications (16%). p p p Recently, negative results of a randomized phase 2 study were presented at ASCO 2023. The NORPACT-1 trial randomized 140 patients with resectable pancreatic cancer to receive upfront surgery and adjuvant FOLFIRINOX for six months or a short-course neoadjuvant FOLFIRINOX for two months, followed by surgery and the rest of the chemotherapy postoperatively [40]. According to the preliminary results, the upfront surgery group seems to be doing better than the group who received neoadjuvant therapy, with a median OS in the ITT population of 38.5 months (upfront surgery) versus 25.1 months (neoadjuvant) and HR 1.52 (95% CI, 0.94–2.46), p = 0.096. In the neoadjuvant cohort, a considerable drop-out of patients was observed, as only 40 of 77 completed the planned preoperative program. Furthermore, a R0 rate of 56% with neoadjuvant therapy and 39% with upfront surgery were found, ﬁgures that are unusual by modern surgical standards. 4. Discussion The study notably showed that median survival time was signiﬁcantly better in patients who com- pleted adjuvant chemotherapy than in those not able to complete the treatment. This data pointed out the signiﬁcant impact of adjuvant chemotherapy. The importance of the overall duration of systemic treatment was also shown by Reni et al. [32], and despite the not signiﬁcantly different results between NAT and US because of poor accrual, Golcher et al. also demonstrated higher multimodality therapy completion in the NAT group (58% versus 30%) [29]. Similarly, Versteijne et al. stated that the total cumulative dose of chemotherapy was signiﬁcantly higher in the neoadjuvant chemoradiotherapy group [18], supporting the hypothesis of better tolerability with neoadjuvant administration. The matched cohort series by Mokdad et al. [30] further supported that there was a signiﬁcant dropout for patients after pancreatic resection that precludes completion of adjuvant therapy, which is known to increase survival in this biologically aggressive disease. Interestingly, in our analysis, a clear trend for OS in favor of NAT from 2011 to 2023 has been demonstrated, showing how the hazard ratio stabilized at 0.80 (95% CI: 0.72–0.91). A possible explanation can be found in the improvement in chemotherapy protocols, with the above-mentioned modern and more active multiagent regimens; in the prospective and randomized nature of more recent studies with well-deﬁned inclusion criteria; and maybe in the better quality of the radiotherapy schedule; however, a quality assurance procedure was performed only in the Versteijne trial, and therefore it is difﬁcult to derive stronger conclusions. The present meta-analysis demonstrated that the R0 resection rate was higher in NAT than in the US. The increasing rate of free margin resection is an important prognostic factor, reducing the risk of local recurrence and having a positive impact on survival rates. Moreover, all studies stated that neoadjuvant treatment allows a better selection of patients that do have an advantage from surgery and prevents patients with rapid disease progression from having an unnecessary major intra-abdominal operation. p g g y j p Although our meta-analysis included both randomized clinical trials and retrospective studies comparing neoadjuvant strategies (e.g., chemotherapy and chemoradiotherapy) versus US in a setting of resectable pancreatic adenocarcinoma, OS, DFS, and N+ rates were also conﬁrmed by the subgroup analysis, which included only RCTs. 4. Discussion Also, the higher R0 and N0 resections in the neoadjuvant arm surprisingly did not translate into a survival advantage. Given all these limitations, the study does not seem to allow for any conclusion g y y Among the nine studies included in our research analyzing radiotherapy mainly concomitant to gemcitabine-based chemotherapy, three were randomized clinical trials reporting interesting ﬁndings deserving some consideration. The phase III randomized PREOPANC trial evaluated a strategy with three cycles of neoadjuvant gemcitabine com- bined with radiotherapy (36 Gy in 15 fractions) in the second cycle versus US and adjuvant gemcitabine in resectable and borderline resectable pancreatic adenocarcinoma. In the long term, updated results showed that neoadjuvant treatment improved OS in the overall cohort, but in the subgroup of resectable pancreatic adenocarcinoma, OS was not signif- icantly improved (p = 0.23). However, the 5-year OS rate showed a clinically relevant improvement of 14%, including resectable pancreatic adenocarcinoma. These data seem to conﬁrm the importance of long-term follow-up to detect a survival difference with a clinical impact. This result was in line, for example, with the CONKO-001 trial, in which survival Cancers 2023, 15, 4627 13 of 17 13 of 17 differences were found after longer follow-up. In this regard, a high rate of progression and death events has been seen in the ﬁrst year in both the NAT and US groups. We can argue that such a NAT schedule is not able to prevent early progression and that more effective schedules are required. Moreover, this recent trial showed that large RCTs exploring NAT for pancreatic adenocarcinoma can be conducted with satisfactory and rapid accrual. This was in contrast with two previous RCTs by Casadei et al. and Golcher et al. [27,29] that explored gemcitabine-based chemotherapy and conventionally fractionated radiotherapy compared to the US. Both trials experienced poor accrual and were terminated early. In Golcher’s trial, neither the median OS nor the R0 resection rate were signiﬁcantly different between the two groups. Similarly, in Casadei’s trial, R0 resection and survival rates were not different between the groups of patients. The low power of the studies suggested that the results were most likely due to unpowered data caused by premature study termination. The retrospective study by Sho et al. reported the ﬁndings of 100 patients treated with neoadjuvant gemcitabine-based chemotherapy and radiotherapy or US [35]. 4. Discussion Our positive results in favor of NAT are in contrast with a recent meta-analysis in which neoadjuvant chemotherapy or chemoradiation did not improve either DFS or OS compared to US followed by adjuvant treatment [40]. This discrepancy can be explained mainly by three reasons: First, our study followed a different logical-statistical approach. Second, in the case of incomplete results, we determined the hazard ratio using the methodological approach described in the “materials and methods” section. Third, due to different eligibility criteria, two RCT studies published only as proceeding abstracts [41,42] were included in Uson Junior were not included in our study. Our results provide evidence in favor of NAT in patients with resectable pancreatic adenocarcinoma. The promising results and feasibility of the PREOPANC trial and the Cancers 2023, 15, 4627 14 of 17 established advantage of multiagent chemotherapy schedules, such as FOLFIRINOX, have led to the conduct of several ongoing trials in the setting of resectable pancreatic adeno- carcinoma: the PREOPANC-2 trial is comparing neoadjuvant FOLFIRINOX with neoadju- vant gemcitabine-based chemoradiotherapy [43], and two randomized trials are investi- gating neoadjuvant FOLFIRINOX (ALLIANCE A021806 [clinicaltrial.govNCT04340141], PREOPANC-3 [clinicaltrial.gov NCT04927780]). The deﬁnitive results from these trials will further clarify whether or not NAT is supported in resectable pancreatic cancers. However, some limitations should be addressed. Most studies were designed and enrolled patients, or were retrospectively retrieved, when gemcitabine represented the standard of care. This monotherapy regimen is nowadays considered obsolete and used in fragile patients. Only three studies included in our analysis used more active chemotherapy regimens. In addition, among trials with a chemoradiotherapy neoadjuvant schedule, poor details were reported on radiotherapy technique and treatment volume deﬁnition, in particular concerning prophylactic irradiation or omission of locoregional negative lymph nodes, which is an important factor for N0 rates. Furthermore, some studies were prematurely closed due to poor accrual. Lastly, the published studies did not stratify patients considering potential prognostic biomarkers such as circulating tumor DNA (ctDNA), which is recently gaining interest in pancreatic cancer [44]. Despite these limitations, the reported advantage of NAT could lead to a change from the traditional approach for resectable pancreatic cancer with US, and NAT may be part of the therapeutic algorithm during multidisciplinary discussion with the chance of therapeutic beneﬁt and eventual cure. 5. Conclusions and Future Directions The present meta-analysis evaluating the survival beneﬁt of NAT sustains its imple- mentation in the context of primarily resectable pancreatic adenocarcinoma. However, standardization of therapy regimen, duration, and amount of therapy, along with the integration of radiation therapy, require further evaluation. The ongoing trials may answer these questions. As future perspectives, molecular or radiomic biomarkers, as well as liquid biopsy based on ctDNA analysis and other molecular biomarkers, may help to predict recurrence and survival and could guide the selection of patients who most beneﬁt from a certain treatment or therapeutic strategy according to response assessment before surgery. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers15184627/s1, Figure S1: Overall Survival: Galbraith Plot; Figure S2: Overall Survival: Subgroup analysis related to study design—(RCT = 6, retrospective studies = 7, Study design Not Reported = 2); Figure S3: Overall Survival: Cumulative analysis per year; Figure S4: Overall Survival: Leave-one-out analysis; Figure S5: Overall Survival: Funnel plot for publication bias; Figure S6: Disease Free-Survival: Galbraith Plot; Figure S7: Disease Free Survival (n = 7): Forest plot—Subgroup analysis related to use of neoadjuvant chemotherapy or neoadjuvant chemoradiotherapy; Figure S8: Disease Free Survival—Subgroup analysis related to study design (RCT = 6, retrospective studies = 1); Figure S9: Disease Free Survival: Cumulative Meta-Analysis; Figure S10: Disease Free Survival: Leave-one-out analysis; Figure S11: R0 Rate: L’Abbè Plot for R0 Rate Studies; Figure S12: R0 Rate—Subgroup analysis related to study design (RCT = 5, retrospective studies = 6, Study design Not Reported = 2); Figure S13: R0 Rate (n = 13): Cumulative Meta-Analysis; Figure S14: R0 Rate (n = 13): Leave-one-out Analysis; Figure S15: R0 Rate (n = 13): Funnel Plot—Publication Bias; Figure S16: N+ Rate (n = 8): L’Abbé Plot—N+ Rate; Figure S17: N+ Rate (n = 8): Sensitive Analysis; Figure S18: N+ Rate (n = 8): Cumulative meta-analysis; Figure S19: N+ Rate—Subgroup analysis related to study design (RCT = 2, retrospective studies = 5, Study design Not Reported = 1); Table S1: Studies Characteristics; Author Contributions: Conception and design of the work: S.D.D., A.C., P.M.-H., L.D., M.L.G. and A.V.; Acquisition of data: R.R. and L.B.; Data analysis: M.L.G. and R.R.; Data interpretation: S.D.D., M.L.G., A.C., L.D. and A.V.; Drafting the article: R.R., A.C., L.D., M.L.G., A.V. and S.D.D.; Critical revision of the article and ﬁnal approval of the version to be published: all authors. 5. 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[CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4286685286	https://www.frontiersin.org/articles/10.3389/fmicb.2022.917117/pdf	English	null	Microbial degradation of aristolochic acid I by endophytic fungus A.h-Fs-1 of Asarum heterotropoides	Frontiers in microbiology	2,022	cc-by	9,136	Microbial degradation of aristolochic acid I by endophytic fungus A.h-Fs-1 of Asarum heterotropoides OPEN ACCESS EDITED BY Wen-Ming Wang, Sichuan Agricultural University, China REVIEWED BY Jiang Chen, Chengdu University of Traditional Chinese Medicine, China Zhixue Zhao, Sichuan Agricultural University, China Xiaohan Wang , Dan Jiang , Qijin Shi , Guangxi Ren and Chunsheng Liu * Xiaohan Wang , Dan Jiang , Qijin Shi , Guangxi Ren and Chunsheng Liu * School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China Asari Radix et Rhizoma is commonly used in classic prescriptions of herbal medicine in several Asian countries for resuscitation, pain relief, and sore treatment, and Asarum heterotropoides (A. heterotropoides) is an important source material of Asari Radix et Rhizoma. However, the plants of the Asari Radix et Rhizoma and some plants in Asarum spp. contain aristolochic acid I (AAI), which is considered as a carcinogen. The objective of the current study is to detoxify Asarum spp. through microbial degradation of AAI in order to ensure drug safety. Based on the observation of the close correlation between endophytic fungi of A. heterotropoides and AAI, we identified an AAI-degrading fungus and screened for candidate genes involved in AAI degradation. Full- length O-demethylase genes (ODMs) were cloned including A.h-ODM-5, Fs-ODM-4, and Fs-ODM-1, and their ability to degrade AAI was tested in vitro. The results showed that the AAI-degrading fungus was identified as Neocosmospora solani (A.h-Fs-1, endophytic fungi of A. heterotropoides), and verified the capability of specific O-demethylation to modify the structure of AAI. We further identified the functional ODMs in A.h-Fs-1 capable of degrading AAI and uncovered the AAI degradation mechanism of A.h-Fs-1. The microbial degradation of AAI demonstrated in the present study offers a new method to detoxify plant materials used for herbal medicine, and would enhance the regulation of toxic ingredients content in herbal medicine source materials. Wang X, Jiang D, Shi Q, Ren G and Liu C (2022) Microbial degradation of aristolochic acid I by endophytic fungus A.h-Fs-1 of Asarum heterotropoides. Front. Microbiol. 13:917117. doi: 10.3389/fmicb.2022.917117 © 2022 Wang, Jiang, Shi, Ren and Liu. 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. Microbial degradation of aristolochic acid I by endophytic fungus A.h-Fs-1 of Asarum heterotropoides No use, distribution or reproduction is permitted which does not comply with these terms. aristolochic acid I, Asarum heterotropoides, endophytic fungi, Neocosmospora solani, o-demethylation TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fmicb.2022.917117 TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fmicb.2022.917117 TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fmicb.2022.917117 Plant materials AAs exhibit nephrotoxicity, liver toxicity, carcinogenesis, and mutagenesis and are classified as grade I carcinogens (Wang et al., 2018). Further studies indicate that AAs have multiple types of molecular structure, with aristolochic acid I (AAI) being the most toxic (Balachandran et al., 2005; Supplementary Figure S2). The toxicity of AAI is closely related to the substituent nitro, methoxy, and hydroxyl groups in its molecular structure. AAI is rapidly absorbed after entering the human body and shows an organ- specific distribution that is mainly concentrated in liver and kidney tissues (Gao et al., 2017). The content of AAI in Asarum spp. source plants, which are commonly used in herbal medicine, can reach an alarming level as high as 3,243 μg·g−1, as reported by Gao et al. (2006). Therefore, to guarantee medicinal and clinical safety, it is imperative to remove the content of AAI in Asarum spp. used as raw material for the pharmaceutical industry.i Plant materials [A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.] were sampled on April 28, 2019 from Gaojiagou (42°12′N, 125°07′E), Qingyuan Manchu Autonomous County, Fushun City, Liaoning province, Northern China, as shown in the map in Figure 1A. Five plants were collected from each of the 10 sampling points (Figure 1B), and each sample was composed of the same niche (root, rhizome, leaf, and petiole; Figure 1C) from the five plants of the same sampling point, making a total of 40 samples. Three-quarters of each sample material were directly used for the screening of endophytes in this study and the remaining one-quarter of each sample was transported to a company (Novogene, Beijing) for high- throughput sequencing and analysis (Supplementary Material 1). Detoxification of Asarum spp. by removing the AAI compound remains challenging and is still to be developed yet. Microorganisms may shed some light on this challenge because they are capable of decomposing and transforming a wide variety of substances, including some herbal endophytes (Ai et al., 2019). Microorganisms have been used to ferment traditional Chinese medicines to make their active substances more readily available or metabolizable to obtain more active substances (Wang et al., 2010; Wu, 2015; Chen, 2016). Cao et al. (2015) used 10 types of microorganisms to biotransform and detoxify 12 medicinal materials via liquid fermentation technology. Introduction The medicinal Asari Radix et Rhizoma source plants specified in the Chinese Pharmacopoeia include the dry roots and rhizomes of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., A. sieboldii Miq. var. seoulense Nakai., and A. sieboldii Miq (Chinese Pharmacopoeia Commission, 2020), which are commonly used for Frontiers in Microbiology 01 frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 resuscitation, pain relief, and sore treatment (Luo, 2017). The genus Asarum L. is typically distributed in temperate regions in the Northern Hemisphere, with the center of diversity in Eastern Asia (Lim et al., 2017). Since ancient times, Asarum spp. has been widely used as herbal medicine to treat aphthous stomatitis, toothache, and gingivitis (Yamaki et al., 1996) in traditional medical practices in China, Korea, and Japan. However, Asarum spp. source plants contain nitrophenanthrene organic acids called aristolochic acids (AAs) that were verified to be toxic. The toxicity of AAs was first reported in 1993 by Vanherweghem et al. (1993), who found that Belgian women suffered renal interstitial fibrosis and renal failure after taking weight-loss drugs containing AAs. The discovery triggered worldwide research on the toxicity mechanisms of AAs and safe and rational use of drugs containing AAs. In 2017, Ng et al. (2017) suggested that AAs are related to liver cancer in Asians and questioned the safety of traditional Chinese medicines containing AAs. The toxicity of AAs leads to the exclusion of Aristolochiaceae juss. Medicinal materials from the 2020 edition of the Chinese Pharmacopoeia, except for Asari Radix et Rhizoma which has low AAs content in its roots and rhizomes and has no alternatives yet to achieve the same medicinal effect (Chinese Pharmacopoeia Commission, 2020; Supplementary Figure S1). of Fusarium sambucinum to degrade 2-hydroxy-1, 4-benzoxazin- 3-one into small molecular compounds. Interestingly, Liu et al. (2010) added 20 fungal species to Radix aristolochiae (dry root of Aristolochia debilis) as a substrate for solid-state fermentation, and 13 fungal species were able to degrade AAI to various extent. These studies collectively provide new perspectives to degrade AAI compound in Asarum spp. through microorganism decomposition and transformation. In the present study, we aim to detoxify Asarum spp. by identifying endophytic fungus that is able to decompose its toxic compound AAI, and elucidate the mechanism of AAI degradation by endophytes, which has not been reported yet. Plant materials Studies (Yang, 2009; Pan et al., 2013; Bao et al., 2017) have shown that the endophytic fungus Aspergillus C1-Y7-4 and its spore suspension from Stellera Chamaejasme L naturally degrade flavonoids and coumarins in the original plant. Zikmundov et al. (2002) isolated four strains of endophytic fungi from Acanthaceae spp. and analyzed the ability Introduction More specifically, we applied and validated the approach of demethylation, through which AAI is naturally decomposed in human body, to allow the endophytic fungus to effectively degrade AAI compound. Our study not only advances the understanding of the molecular mechanism of endophyte detoxification, but also provides a new method to detoxify herbal medicine for improved drug safety. Screening endophytic fungi for degrading AAI in Asarum heterotropoides The 40 fresh plant samples of A. heterotropoides were rinsed and divided into groups of roots, rhizomes, laminae, and petioles (Figure 1C). After that, the samples were first treated with 0.1% mercury chloride for 12 min (the laminae were treated for 8 min) and then with 75% ethanol for 1 min. The samples were further rinsed with sterile water and then dried using sterile filter paper (Shu et al., 2021). Under aseptic conditions, the laminae were cut into 1 × 1 cm2 pieces, and the roots, rhizomes, and petioles were cut into 1 cm lengths. The sample surface was verified to be completely disinfected using tissue blotting and rinse solution detection methods (Schulz et al., 1993). After cutting, the samples were placed on a Frontiers in Microbiology 02 frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 C A B C FIGURE 1 Sample collection of Asarum heterotropoides. (A) Location of A. heterotropoides sampling points on map. (B) Distribution of all the sampling points. (C) Different niches from which A. heterotropoides was sampled. A B B A A C B FIGURE 1 Sample collection of Asarum heterotropoides. (A) Location of A. heterotropoides sampling points on map. (B) Distribution of all the sampling points. (C) Different niches from which A. heterotropoides was sampled. calculated for 2,000 replicates. The edited ITS and tef-1α sequences were compared with other available Fusarium species sequences in GenBank. Furthermore, the sequences of some known species were downloaded from GenBank and Fusarium-ID, and used to reconstruct a combined ITS region and tef-1α phylogenetic trees. For phylogenetic analysis, 12 (ITS) and 4 (tef-1α) taxa were included in the combined dataset, and Glacial ice basidiomycete (ITS, AF261656.1) and Fusarium mori (tef-1α, NRRL 52773) were used as an outgroup.h phosphate-buffered saline (PBS, pH 5.8) solid medium plate and cultured at a constant temperature of 28°C for 5 days in the dark (Bhojwani and Razdan, 1983; Liu and Ji, 2009). After 5 days of culture, the plant tissues were placed in 50 ml of an AAI basic inorganic salt liquid medium (AAI standard solution: 0.515 mg/ml AAI) and cultured at 28°C in the dark (roots and rhizomes were peeled to avoid contamination; Xiang, 2019). The obtained endophytic fungi were transferred to AAI-coated inorganic salt solid medium plates and incubated for at least 7 days. Screening endophytic fungi for degrading AAI in Asarum heterotropoides Next, according to the color and morphology of the colonies, we selected hyphae that grow vigorously on the surface for further experiments. A single colony was selected, cultured, and purified repeatedly (Most of the mycelium will grow at the bottom of the medium, and a small part of the mycelium will grow on the surface of the medium. We selected the mycelium with vigorous surface growth for purification). The fungal spores were collected by scraping the hyphae with sterile water and counted using a hemocytometer under a microscope. The spore suspension was diluted to a concentration of 108 cells/mL, and then 4.5 ml of the diluted suspension was added to 25.5 ml of (15% V/V inoculum) liquid fermentation medium [basic inorganic salt liquid medium: 300 μl AAI standard solution (0.515 mg/ml) per 50 ml of medium; Pan et al., 2013]. The solution was then cultured at 28°C (200 rpm) for 5 and 14 days in a shaker to obtain A.h-Fs-1 fermentation broth, which was centrifuged at 10,625 × g for 10 min. After centrifugation, 10 ml of the supernatant was collected and freeze-dried. Next, the pellets were reconstituted in 1 ml methanol, filtered through a 0.22-μm microporous membrane, and transferred to a sample vial for later use (Wei, 1979; Consilio Florarum Cryptogamarum Sinicarum Academiae Sinicae Edita, 2004). Identification of the screened endophytic fungus Fungal hyphae were placed on a slide, stained with a lactic acid phenol cotton blue staining solution, and covered with a cover glass for observation under a microscope (using cedar oil for immersion under the oil lens, BX53; Olympus, Tokyo, Japan). Amplification of the tef-1α gene and ITS regions was conducted using the primer pair ef1 and ef2 for tef-1α (O’Donnell et al., 1998) and ITS1 and ITS4 for the ITS regions (White et al., 1990; Supplementary Table S1). The target genes were amplified using PCR (see Supplementary Table S2 for PCR amplification system), after which the amplicons were detected and recovered using 1% agarose gel electrophoresis. PCR products were sent for sequencing to a service provider. The aligned sequences were blasted in two genome databases, GenBank and Fusarium-ID, to identify the strain. In this study, a phylogenetic tree was generated using maximum parsimony in MEGA7.0 (Kumar et al., 2016). Bootstrap values for the maximum parsimony tree (MPT) were Cloning of FS-ODM-1, FS-ODM-4, and A.h-ODM-5 Cloning of FS-ODM-1, FS-ODM-4, and A.h-ODM-5 Target bands were recovered using an OMEGA Gel Extraction Kit (Omega Bio-tek, Norcross, GA, United States), and the purified PCR products were cloned into the pEASY®-Blunt cloning vector (TransGen Biotech, Beijing, China). Ligation reactions were transformed into Escherichia coli Trans1-T1 competent cells, spread onto LB plates supplemented with ampicillin, and incubated overnight at 37°C. Colony PCR was conducted to identify positive colonies for inoculation and subsequent plasmid isolation. Finally, the isolated plasmids were sequenced by Sangon Biotech (Shanghai, China). Premier 5.0. Primers were subsequently synthesized by Sangon Biotech (Shanghai, China). RNA was extracted from A. heterotropoides and A.h-Fs-1 with TRIZOL (Takara Bio, Beijing, China) and used for complementary DNA (cDNA) synthesis. The target genes were amplified using PCR (see Supplementary Table S2 for PCR amplification system), after which the amplicons were detected and recovered using 1% agarose gel electrophoresis. Target bands were recovered using an OMEGA Gel Extraction Kit (Omega Bio-tek, Norcross, GA, United States), and the purified PCR products were cloned into the pEASY®-Blunt cloning vector (TransGen Biotech, Beijing, China). Ligation reactions were transformed into Escherichia coli Trans1-T1 competent cells, spread onto LB plates supplemented with ampicillin, and incubated overnight at 37°C. Colony PCR was conducted to identify positive colonies for inoculation and subsequent plasmid isolation. Finally, the isolated plasmids were sequenced by Sangon Biotech (Shanghai, China). Cloning of FS-ODM-1, FS-ODM-4, and A.h-ODM-5 Based on the molecular structure (Figure 2; Pfau et al., 1990; Shibutani et al., 2010; Levová et al., 2011; Stiborová et al., 2015; Chang et al., 2017; Anger et al., 2020) and carcinogenic properties of AAI, candidate genes that can degrade AAI were screened. The N. solani genome database (PRJNA368786) and A. heterotropoides transcriptome database (PRJNA477885) on NCBI were used to design gene- specific primers (Supplementary Table S1) using Primer Frontiers in Microbiology 03 frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 FIGURE 2 Degradation pathway and metabolites of AAI. The red dotted box represents the metabolic pathway and metabolites of AAI under anaerobic condition, and the blue box represents the metabolic pathway and metabolites of AAI under aerobic condition (information summarized and adapted from Pfau et al., 1990; Shibutani et al., 2010; Levová et al., 2011; Stiborová et al., 2015 Chang et al., 2017; Anger et al., 2020). FIGURE 2 Degradation pathway and metabolites of AAI. The red dotted box represents the metabolic pathway and metabolites of AAI under anaerobic condition, and the blue box represents the metabolic pathway and metabolites of AAI under aerobic condition (information summarized and adapted from Pfau et al., 1990; Shibutani et al., 2010; Levová et al., 2011; Stiborová et al., 2015 Chang et al., 2017; Anger et al., 2020). we used a series of publicly available toolkits to perform bioinformatic analysis, including ORF Finder2 to find open reading frames, ExPASy3 to predict the physical and chemical properties of the translated proteins, TMHMM Server v.2.04 for protein transmembrane domain analysis, SinalP-5.0 Server5 for protein sequence signal peptide prediction analysis, NCBI-CDD6 for protein domain analysis, SOPMA7 for protein secondary structure analysis, I-TASSER and Swiss Model8,9for 3D structural modeling, and PyMOL software to predict the tertiary structure of the protein. MEME10 and TBtools (Chen et al., 2020) were used to analyze the conserved motifs of ODMs, and DNAMAN 9 software was used for comparison with the amino acid sequences of other plants. The maximum likelihood phylogenetic tree was constructed using MEGA 7.0 software with 1,000 bootstrap repetitions. Premier 5.0. Primers were subsequently synthesized by Sangon Biotech (Shanghai, China). RNA was extracted from A. heterotropoides and A.h-Fs-1 with TRIZOL (Takara Bio, Beijing, China) and used for complementary DNA (cDNA) synthesis. The target genes were amplified using PCR (see Supplementary Table S2 for PCR amplification system), after which the amplicons were detected and recovered using 1% agarose gel electrophoresis. 2 https://www.ncbi.nlm.nih.gov/orffinder/ 3 https://web.expasy.org/protparam/ 4 http://www.cbs.dtu.dk/services/TMHMM/ 5 http://www.cbs.dtu.dk/services/SignalP/ 6 https://www.ncbi.nlm.nih.gov/cdd 7 https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html 8 http://zhanggroup.org//I-TASSER/ 9 http://swissmodel.expasy.org/ 10 http://meme-suite.org/tools/meme frontiersin.org Bioinformatic analysis of FS-ODM-1, FS-ODM-4, and A.h-ODM-5 The similarity of nucleotide and amino acid sequences was analyzed using NCBI1 BLAST tool. More specifically, 1 http://www.ncbi.nlm.nih.gov/ 04 Frontiers in Microbiology Wang et al. 10.3389/fmicb.2022.917117 Heterologous expression of FS-ODM-1, FS-ODM-4, and A.h-ODM-5 and AAI degradation assays (35, 65); column temperature, 27°C; flow rate, 0.4 ml/min; detection wavelength, 254 nm; and injection volume, 2 μl. The retention time was determined using standard samples, and the AAI content of each tissue sample was determined using a linear regression equation. Each sample was analyzed in triplicate to ensure data repeatability. The sequenced pEASY®-Blunt E1 expression vector (Ampicillin resistance, 100 mg/ml, addition amount is 0.1%V/V) harboring FS-ODM-1, FS-ODM-4, and A.h-ODM-5 was transformed into E. coli TransB (DE3) cells for heterologous protein expression. When the OD600 of the bacterial cultures reached approximately 0.6, isopropyl-beta-D- thiogalactopyranoside was added to form a solution at medium concentration of 0.1 mmol/l. The solution was divided into two portions and they were induced for protein expression under different conditions, with one portion at 16°C for 16 h and the other at 37°C for 4 h (Gao and Sun, 2021). The controls were formed by uninduced and blank strains (NC, negative control, pEASY®-Blunt E1 expression vector), and protein expression was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot (WB). High-resolution mass spectrometric conditions: Electric spray ion source (ESI); Scanning mode: Positive ion mode; Dry gas: nitrogen; Capillary voltage:3.2 kV; Nitrogen flow rate:800 l/h; Ion source temperature:150°C; Dry gas temperature: 550°C; Impact voltage: 30 V. Model of mass spectrometer: Q-Exactive Plus (Thermo Scientific, United States; Yuan et al., 2007). Statistical analysis One-way analysis of variance (ANOVA) was conducted to detect significant differences among treatments. Separation of means was tested using the least means square difference with a significance level of p < 0.05. All the data were presented as means of three replicates with a standard error unless stated otherwise. Following culture at 16°C for 16 h, a large amount of fermentation broth was obtained. The bacterial solution was centrifuged at 9380 × g for 10 min at 4°C, after which the pellet was washed twice with 1.5 ml of Phosphate-Buffered Saline (PBS, pH 7.4) in an ice bath (4°C). The pellets were resuspended in 3 ml PBS and subjected to ultrasonication to break the cells. The sonicated cell suspension was centrifuged (9,380 × g for 10 min at 4°C), and 2.8 ml of the crude enzyme extracts was added to 200 μl of AAI (0.515 mg/ml), along with NC and PBS control (the same reaction system as the crude enzyme solution). Because the suitable temperature for plant growth is 25°C and the optimal temperature for general enzyme activity is 37°C, we selected these two temperatures to compare and evaluate the AAI degradation activities of ODMs. After incubating some samples at 37°C and others at 25°C for 48 h, an equal volume of methanol was added to each sample to terminate the reaction. The samples were filtered through a 0.22 μm organic membrane and the active substances were determined using ultra-high-performance liquid chromatography (UPLC; Yin et al., 2020). Identification and verification of AAI degradation by endophytic fungus The chromatographic settings for determining the AAI (Cas:313–67-7, purity ≥98%, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) contents were as follows: mobile phase, acetonitrile: 0.1% aqueous formic acid Frontiers in Microbiology 05 frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 FIGURE 3 Interaction network of differential functional groups in different niches of A. heterotropoides. Pearson correlation between the contents of the three active ingredients and the relative contents of the endophytic fungi in different niches of A. heterotropoides. Endophytic fungi with significant correlation (p < 0.05) were used as nodes to construct the interaction network and visualization in Cytoscape. Red lines indicate a positive correlation, whereas blue lines indicate a negative correlation. Line width is proportional to the values of Pearson correlation coefficient. Raw data is provided in the EXCEL 1-ITS.xlsx. FIGURE 3 Interaction network of differential functional groups in different niches of A. heterotropoides. Pearson correlation between the contents of the three active ingredients and the relative contents of the endophytic fungi in different niches of A. heterotropoides. Endophytic fungi with significant correlation (p < 0.05) were used as nodes to construct the interaction network and visualization in Cytoscape. Red lines indicate a positive correlation, whereas blue lines indicate a negative correlation. Line width is proportional to the values of Pearson correlation coefficient. Raw data is provided in the EXCEL 1-ITS.xlsx. 1725 bp in length (Supplementary Figure S3A), encoding 296, 424, and 574 amino acids, respectively. The nucleotide and amino acid sequences of the ODMs were compared using BLASTX (GenBank), and the amino acid sequences of other strains and plants were analyzed using DNAMAN software (Supplementary Figures S3B,C). We selected the amino acid sequences of ODMs from 37 species of plants and 39 species of Fusarium strains recorded in GenBank, and constructed a phylogenetic tree using the maximum likelihood method in MEGA7.0 (Figure 6). medium plates, the colony of this degrading fungus grew slowly with loose aerial mycelia (Figure 5A) and a bit of sharp macroconidia ends (Figure 5B), and the microconidia and chlamydospore morphologies were the same as those on the potato dextrose agar medium. Comparison of the sequencing results of this AAI-degrading fungus using BLAST and ITS phylogenetic tree analysis indicated 100% similarity to N. solani (Figure 5F). The tef-1α phylogenetic tree showed a strong relationship between A.h-Fs-1 and N. solani (Figure 5G). Therefore, both the 100% similarity and the strong relationship helped to identify the endophytic fungus as N. Identification and verification of AAI degradation by endophytic fungus solani. Identification and verification of AAI degradation by endophytic fungus The changes in the content of AAI in all the niches were found similar to those of some endophytic fungi of A. heterotropoides plants. Thus, endophytic fungi of A. heterotropoides may affect the synthesis and decomposition of active ingredients, including AAI. Further, we determined the diversity of endophytic fungi and contents of active ingredients in A. heterotropoides from different ecological niches. By analyzing the differences among active ingredient contents and endophytic fungus distributions, we identified differential fungal species that exhibited significant correlations with active ingredients (Figure 3). One endophytic fungus, namely A.h-Fs-1, was identified upon screening for a strain that could degrade AAI. Next, we tested the AAI degradation function of A.h-Fs-1 using UPLC. The 14-day culture of AAI degradation showed a high and stable degradation efficiency, at 28.85% on day 5 and 64.42% on day 14 (Figure 4). We performed morphological, microscopic, and internal transcribed spacer molecular analyses to evaluate A.h-Fs-1. On the potato dextrose agar medium plates, the colony of degrading endophytic fungus showed flat and villus shape with smooth edges, and it grew rapidly (Figure 5A). The hyphae were hyaline, septate, and branched under a microscope. The microconidia had 1–2 compartments and appeared as spindle or ovoid (Figures 5B–D); the macroconidia had 3–5 compartments and appeared as prismatic or crescent with a little blunt end (Figures 5B,D). Chlamydospores grew at the hyphal apex and appeared round (Figure 5E). On AAI-coated inorganic salt solid The various niches of A. heterotropoides were homogenized in a mortar and pestle using liquid nitrogen. 0.4 g of the homogenate was mixed with 5 ml of 70% methanol (chromatography grade) to form a solution that was then vortexed for 30 s, left standing for 1 h, and weighed. After ultrasonication for 1 h, the solution was weighed again and the weight loss was compensated by 70% methanol. The supernatant of the solution was filtered through a 0.22 μm organic membrane into a vial and stored airtight at 4°C (Yin et al., 2020). To determine the contents of AAI, we performed UPLC on a Waters e2695/ Acquity H-class system with a Waters AcQUITY UPLC® BEH C18 column (100 × 2.1 mm, 1.7 μm; Waters, Milford, MA, United States). ODM cloning and homology sequence analysis (B) Minimal medium containing AAI for a degradation time of 14 days. (C) Minimal medium containing AAI and A.h-Fs-1 for a degradation time of 5 days. (D) Minimal medium containing AAI and A.h-Fs-1 for a degradation time of 14 days. (E) Minimal medium. the highest degradation efficiency, and Fs-ODM-4 showed the lowest efficiency at both temperatures (Figure 8; Supplementary Table S3). The temperature decrease from 37°C to 25°C promoted the efficiency of AAI degradation by Fs-ODM-1 and Fs-ODM-4, with the latter corresponding to a significant efficiency leap from 29.60 to 48.79%. The results indicated that the activity of degradation by Fs-ODM-1 and Fs-ODM-4 is temperature-sensitive and high temperature would restrain degradation. According to the comprehensive analysis of UPLC degradation results and gene annotation, we speculate that the degradation product may be 8-hydroxyaristolochic acid I. Through mass spectrometry detection and analysis, we can see that the substrate AAI parent ion [M + NH4] + has good stability after entering the secondary mass spectrometry, but the response value is low. LC–MS showed that the EIC response values of AAI in CK, NC, Fs-ODM-1, Fs-ODM-4, and A.h-ODM-5 were 1.21e8, 1.09e8, 1.65e7, 1.60e7, and 2.26e7, respectively (Supplementary Figure S6). absorbed by the human body. Therefore, it is imperative to develop effective methods to degrade and metabolize AAI. Through bioinformatic analysis and sole carbon source experiments, we identified an AAI-degrading strain, namely A.h-Fs-1 (N. solani). As a fungus commonly available in soil, animals, and plants (Schroers et al., 2016), N. solani is both pathogenic and beneficial to the host (Karpouzas et al., 2011; Chehri et al., 2015). For example, N. solani can produce a variety of toxins after infestation, leading to plant root rot and reduced plant production (Postma and Scholte, 1981; Tamura et al., 2015; Bryła et al., 2020; Wang et al., 2020; Phasha et al., 2021). Additionally, the endophytic strains of N. solani can produce beneficial compounds, such as emodin and rhein (Yu et al., 2015). Furthermore, N. solani isolated by Meng et al. (2016) from the ocean can produce various secondary metabolites, such as 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors. We explored the mechanism of AAI degradation by N. solani, and observed that the strain A.h-Fs-1 degraded 65% of the supplied AAI substrate in 14 days. Based on the structural characteristics of AAI and the availability of the genome of N. ODM cloning and homology sequence analysis solani, demethylase was identified as the most likely candidate for AAI degradation. In vitro assays demonstrated that all three demethylases, one from A. heterotropoides and two from N. solani, can degrade AAI. Moreover, ODM cloning and homology sequence analysis Under different induction conditions of 16°C and 37°C, we used SDS-PAGE to evaluate the expression levels of three ODMs in E. coli (Figures 7A,B). In E. coli cultures expressing ODM-TransB variants, recombinant protein was detected in the supernatants. The expression levels of three ODMs at 16°C in E. coli were higher than that at 37°C, so we took 16°C as the induction condition of bacterial solution. At the same time, we used Western blot (WB) to detect the size and expression of the three enzymes (Figure 7C). The recombinant protein sizes were determined as 68.88 kDa for Fs-ODM-1, 50.88 kDa for Fs-ODM-4, and 35.52 kDa for A.h- ODM-5. Recombinant protein extracted from E. coli was used for in vitro enzyme assays to simulate the bio-transformation of AAI at different temperatures. The UPLC (Figure 8) results showed that the crude enzyme extract could significantly reduce the content of AAI, and there was an independent peak different from the control and blank at 2.534 min. The reaction cultured at 25°C and 37°C showed different degrees of degradation. At 25°C, ODMs showed The candidate protein involved in AAI degradation was found to be demethylase by analyzing the molecular structure and carcinogenic properties of AAI. We performed bioinformatic and structural analysis (Supplementary Material 2) of the A. heterotropoides transcriptome and N. solani genome, and screened nine ODMs (five ODMs from the plant and four ODMs from endophytic fungi). However, after numerous trials of adjusting primers and cloning, only three genes were successfully cloned, including one from plant (A.h-ODM-5) and two from endophytic fungi (Fs-ODM-1 and Fs-ODM-4). Therefore, these three genes were further analyzed in subsequent experiments. A.h- ODM-5 (A. heterotropoides), Fs-ODM-1(N. solani), and Fs-ODM- 4(N. solani) were identified for cloning and in vitro verification. Bioinformatic analysis revealed that the open reading frames of A.h-ODM-5, Fs-ODM-4, and Fs-ODM-1 were 891, 1,275, and Frontiers in Microbiology 06 frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 A B C D E FIGURE 4 UPLC spectrum analysis of A.h-Fs-1 degradation of AAI. (A) Minimal medium containing AAI (0.515 mg/ml). (B) Minimal medium containing AAI for a degradation time of 14 days. (C) Minimal medium containing AAI and A.h-Fs-1 for a degradation time of 5 days. (D) Minimal medium containing AAI and A.h-Fs-1 for a degradation time of 14 days. (E) Minimal medium. B E FIGURE 4 UPLC spectrum analysis of A.h-Fs-1 degradation of AAI. (A) Minimal medium containing AAI (0.515 mg/ml). Frontiers in Microbiology Discussion AAI, naturally present in A. heterotropoides that is broadly used for herbal medicines, can cause nephrotoxicity after being 07 Frontiers in Microbiology frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 A D F G E B C FIGURE 5 Identification of A.h-Fs-1 (Neocosmospora solani). (A) A.h-Fs-1 cultured on plate medium (top: A.h-Fs-1 on the AAI-coated inorganic salt medium plate; bottom: A.h-Fs-1 on PDA medium plate). (B) The microconidia and macroconidia of A.h-Fs-1 cultured on the AAI-coated inorganic salt medium plate. (C) The microconidia of A.h-Fs-1 cultured on the AAI-coated inorganic salt medium plate. (D) The microconidia and macroconidia of A.h-Fs-1 cultured on PDA medium plate. (E) Chlamydospores and hyphae of A.h-Fs-1. (F) ITS phylogenetic tree of A.h-Fs-1; (G) The tef-1α phylogenetic tree of A.h-Fs-1. A B C A C B E D E D D E E F F G G FIGURE 5 Identification of A.h-Fs-1 (Neocosmospora solani). (A) A.h-Fs-1 cultured on plate medium (top: A.h-Fs-1 on the AAI-coated inorganic salt medium plate; bottom: A.h-Fs-1 on PDA medium plate). (B) The microconidia and macroconidia of A.h-Fs-1 cultured on the AAI-coated inorganic salt medium plate. (C) The microconidia of A.h-Fs-1 cultured on the AAI-coated inorganic salt medium plate. (D) The microconidia and macroconidia of A.h-Fs-1 cultured on PDA medium plate. (E) Chlamydospores and hyphae of A.h-Fs-1. (F) ITS phylogenetic tree of A.h-Fs-1; (G) The tef-1α phylogenetic tree of A.h-Fs-1. none of the three demethylases required coenzymes. The degradation product was O-demethylate of AAI and its analogs. This is consistent with the results obtained by Guo (2006), who used Cunninghamella blakesleeana AS3.910 to transform AAI. The observed degradation efficiency and time were similar to those reported by Guo (2006), who used C. brevisiae to biotransform AAI and AAII and performed a specific O-demethylation structural modification of AAI. However, the strain in our experiment isolated from the rhizome of A. heterotropoides is an endophytic fungus that is more conducive to back-staining and colonization, whereas C. blakesleeana is exophytic in A. heterotropoides. After it enters the human body, AAI is reduced to N-hydroxyaristolactam I, which is the activation pathway of carcinogenic effects (Pfau et al., 1990; Chang et al., 2017; Anger et al., 2020). In the detoxification pathway, the redox metabolites of AAI are excreted in the urine and feces (Shibutani et al., 2010). frontiersin.org Discussion The oxidative demethylation product of AAI is 8-Hydroxy AAI, which may be among the metabolites obtained in some previous studies (Levová et al., 2011; Stiborová et al., 2015). The LC–MS showed that although AAI contents were reduced by an order of magnitude according to the degradation product peak, the target values of the secondary ions ([M-H2O-CO2 + NH4] +, [M + NH4] +, [M-H2O-CO2 + H] +, and [M + H] +) were not found. The reason may be that the response value of AAI in mass spectrometry is relatively low, and the response value of its degradation product peak would be too low to display. The specific reason needs to be identified in further study. In plants, demethylases mostly regulate the methylation of DNA and histones (Qiu et al., 2019). Overexpression of plant demethylases may lead to undesirable effects. Studies of demethylase have mainly focused on the effect of methylation modification on epigenetics and some modifications of cytoplasmic proteins through methylation (Li, 2014; Mao, 2014; 08 Frontiers in Microbiology frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 nd developmental tree analysis of ODMs. The amino acid sequences of ODMs of 37 species of plants acid sequences of ODMs of 39 species of Fusarium strains are shown in pink (Fs-ODM-1) and light vided in EXCEL 2.xlsx. FIGURE 6 Comparison of sequence motifs of ODMs and developmental tree analysis of ODMs. The amino acid sequences of ODMs of 37 species of plants are shown in light blue block, and the amino acid sequences of ODMs of 39 species of Fusarium strains are shown in pink (Fs-ODM-1) and light yellow (Fs-ODM-4) blocks. Raw data are provided in EXCEL 2.xlsx. FIGURE 6 Comparison of sequence motifs of ODMs and developmental tree analysis of ODMs. The amino acid sequences of ODMs of 37 species of plants are shown in light blue block, and the amino acid sequences of ODMs of 39 species of Fusarium strains are shown in pink (Fs-ODM-1) and light yellow (Fs-ODM-4) blocks. Raw data are provided in EXCEL 2.xlsx. non-secretory protein and usually not secreted outside the cell. However, studies have shown that non-secretory proteins can be secreted outside the cell in an abnormal mode, which usually occurs in fungi, especially endophytic fungi (Cohen et al., 2020; Nguyen and Debnath, 2020; Zhang et al., 2020; Niu et al., 2021). Discussion (A) SDS-PAGE detection of Fs-ODM-1, Fs-ODM-1Y (induced), Fs-ODM-4, and Fs-ODM-4Y (induced) recombinant protein (columns 1–8 correspond to Fs-ODM-1-37, Fs-ODM-1-37Y, Fs-ODM-1-16, Fs-ODM-1-16Y, Fs-ODM-4-16, Fs-ODM-4-16Y, Fs-ODM-4-37, and Fs-ODM-4-37Y, respectively); (B) SDS-PAGE detection of A.h-ODM-5, A.h-ODM-5Y (induced), NC, and NCY (induced) recombinant protein(columns 1–8 correspond to A.h-ODM-5-16, A.h-ODM-5-16Y, A.h-ODM-5-37, A.h-ODM-5-37Y, NC-16, NC-16Y, NC-37, and NC-37Y, respectively); (C) Western blot (WB) detection of A.h-ODM-5-16Y(induced), Fs-ODM-1-16Y(induced), Fs-ODM-4-16Y(induced), and NC-16Y (induced) recombinant protein. Notes: “−16” means culture at 16°C and “−37” means culture at 37°C. CK was used recombinant 16 tag protein produced by Lianmai Biology. It is a recombinant protein with a size of 68.5 kd and is expressed in E. coli. It is often used as a positive control for WB detection of any of the 16 Tags (include His-tag). FIGURE 8 UPLC profile of AAI degraded by ODMs enzyme at 37°C (left) and 25°C (right). The degradation efficiency of three ODMs is shown in Supplementary Table S3. A B C FIGURE 7 SDS-PAGE and WB detection of ODMs recombinant protein. (A) SDS-PAGE detection of Fs-ODM-1, Fs-ODM-1Y (induced), Fs-ODM-4, and Fs-ODM-4Y (induced) recombinant protein (columns 1–8 correspond to Fs-ODM-1-37, Fs-ODM-1-37Y, Fs-ODM-1-16, Fs-ODM-1-16Y, Fs-ODM-4-16, Fs-ODM-4-16Y, Fs-ODM-4-37, and Fs-ODM-4-37Y, respectively); (B) SDS-PAGE detection of A.h-ODM-5, A.h-ODM-5Y (induced), NC, and NCY (induced) recombinant protein(columns 1–8 correspond to A.h-ODM-5-16, A.h-ODM-5-16Y, A.h-ODM-5-37, A.h-ODM-5-37Y, NC-16, NC-16Y, NC-37, and NC-37Y, respectively); (C) Western blot (WB) detection of A.h-ODM-5-16Y(induced), Fs-ODM-1-16Y(induced), Fs-ODM-4-16Y(induced), and NC-16Y (induced) recombinant protein. Notes: “−16” means culture at 16°C and “−37” means culture at 37°C. CK was used recombinant 16 tag protein produced by Lianmai Biology. It is a recombinant protein with a size of 68.5 kd and is expressed in E. coli. It is often used as a positive control for WB detection of any of the 16 Tags (include His-tag). A A C B B B FIGURE 7 SDS-PAGE and WB detection of ODMs recombinant protein. (A) SDS-PAGE detection of Fs-ODM-1, Fs-ODM-1Y (induced), Fs-ODM-4, and Fs-ODM-4Y (induced) recombinant protein (columns 1–8 correspond to Fs-ODM-1-37, Fs-ODM-1-37Y, Fs-ODM-1-16, Fs-ODM-1-16Y, Fs-ODM-4-16, Fs-ODM-4-16Y, Fs-ODM-4-37, and Fs-ODM-4-37Y, respectively); (B) SDS-PAGE detection of A.h-ODM-5, A.h-ODM-5Y (induced), NC, and NCY (induced) recombinant protein(columns 1–8 correspond to A.h-ODM-5-16, A.h-ODM-5-16Y, A.h-ODM-5-37, A.h-ODM-5-37Y, NC-16, NC-16Y, NC-37, and NC-37Y, respectively); (C) Western blot (WB) detection of A.h-ODM-5-16Y(induced), Fs-ODM-1-16Y(induced), Fs-ODM-4-16Y(induced), and NC-16Y (induced) recombinant protein. Notes: “−16” means culture at 16°C and “−37” means culture at 37°C. CK was used recombinant 16 tag protein produced by Lianmai Biology. Discussion Therefore, we speculated that Fs-ODM-1 and Fs-ODM-4 can be transported outside fungal cells or inside plant cells through other secretory pathways to degrade AAI. We used A.h-ODM-5 as a reference for the in vitro activities of Fs-ODM-1 and Fs-ODM-4. A.h-ODM-5 is similar to the internal reference of A. heterotropoides. The results showed that the degradation efficiency of Fs-ODM-1 and Fs-ODM-4 for AAI was close to that of A.h-ODM-5. Therefore, the strain would offer insights into follow-up re-staining non-secretory protein and usually not secreted outside the cell. However, studies have shown that non-secretory proteins can be secreted outside the cell in an abnormal mode, which usually occurs in fungi, especially endophytic fungi (Cohen et al., 2020; Nguyen and Debnath, 2020; Zhang et al., 2020; Niu et al., 2021). Therefore, we speculated that Fs-ODM-1 and Fs-ODM-4 can be transported outside fungal cells or inside plant cells through other secretory pathways to degrade AAI. We used A.h-ODM-5 as a reference for the in vitro activities of Fs-ODM-1 and Fs-ODM-4. A.h-ODM-5 is similar to the internal reference of A. heterotropoides. The results showed that the degradation efficiency of Fs-ODM-1 and Fs-ODM-4 for AAI was close to that of A.h-ODM-5. Therefore, the strain would offer insights into follow-up re-staining Ma et al., 2020). However, the heterologous overexpression of N. solani demethylases in plants may offer a solution to detoxification. Because ODMs from different sources are quite different, we compared with A.h-ODM-5 from A. heterotropoides to determine the reaction efficiency of Fs-ODM-4 and Fs-ODM-1. If overexpressed, A.h-ODM-5 may affect the plant appearance. Considering the controversy of transgene, it is considered that the of endophytic fungi is more practical. Demethylases from N. solani are closely related to plant demethylases and they can degrade AAI via metabolization, but they cannot enter the plant cell nucleus to participate in epigenetic regulation. A.h-ODM-5 is an ODM that maintains normal life activities in A. heterotropoides. A.h-ODM-5, Fs-ODM-1, and Fs-ODM-4 are no signal peptide. This is a Frontiers in Microbiology 09 frontiersin.org Wang et al. 10.3389/fmicb.2022.917117 and in-depth study. Although these genes are quite different in homology, they play the same role in the structural modification of methyl-containing secondary metabolites, such as AAI. Our results also demonstrate that methylation plays a role in protein– protein interactions and affects protein stability and enzyme activity. These results indicate that demethylases are A B C FIGURE 7 SDS-PAGE and WB detection of ODMs recombinant protein. References ten microorganisms. China J. 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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. Conclusion This research was funded by the National Natural Science Foundation of China (nos. 81773838 and 82104327) and the Fundamental Research Funds for the Central Universities (Beijing University of Chinese Medicine, no. 2020-JYB-ZDGG-037). In this study, we identified an endophytic fungus that is able to decompose the toxic compound AAI of Asarum spp., and screened for candidate genes involved in AAI degradation. The mechanism of AAI degradation by A.h-Fs-1 was uncovered and elaborated. The analysis of N. solani genome and A. heterotropoides transcriptome database indicated demethylases as candidate proteins involved in AAI degradation. We cloned ODMs and used them for in vitro assays, which validated the capability of ODMs to degrade AAI. Our results show that non-histone ODMs play an important role in detoxifying herbal medicine. Further studies on the specific activity and process of AAI degradation by A.h-Fs-1 would facilitate the development of detoxification approach, and provide promising insights into regulation of the content of toxic ingredients in the source materials for herbal medicine. 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. Supplementary material XW and CL conceived and designed the study. XW and QS performed all analyses. XW analyzed the data and prepared the manuscript. XW, CL, GR, and DJ contributed to the writing of the The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.917117/ full#supplementary-material Data availability statement The original contributions presented in the study are included in the article/Supplementary material; further inquiries can be directed to the corresponding authors. Discussion It is a recombinant protein with a size of 68.5 kd and is expressed in E. coli. It is often used as a positive control for WB detection of any of the 16 Tags (include His-tag). FIGURE 7 SDS-PAGE and WB detection of ODMs recombinant protein. (A) SDS-PAGE detection of Fs-ODM-1, Fs-ODM-1Y (induced), Fs-ODM-4, and Fs-ODM-4Y (induced) recombinant protein (columns 1–8 correspond to Fs-ODM-1-37, Fs-ODM-1-37Y, Fs-ODM-1-16, Fs-ODM-1-16Y, Fs-ODM-4-16, Fs-ODM-4-16Y, Fs-ODM-4-37, and Fs-ODM-4-37Y, respectively); (B) SDS-PAGE detection of A.h-ODM-5, A.h-ODM-5Y (induced), NC, and NCY (induced) recombinant protein(columns 1–8 correspond to A.h-ODM-5-16, A.h-ODM-5-16Y, A.h-ODM-5-37, A.h-ODM-5-37Y, NC-16, NC-16Y, NC-37, and NC-37Y, respectively); (C) Western blot (WB) detection of A.h-ODM-5-16Y(induced), Fs-ODM-1-16Y(induced), Fs-ODM-4-16Y(induced), and NC-16Y (induced) recombinant protein. Notes: “−16” means culture at 16°C and “−37” means culture at 37°C. CK was used recombinant 16 tag protein produced by Lianmai Biology. It is a recombinant protein with a size of 68.5 kd and is expressed in E. coli. It is often used as a positive control for WB detection of any of the 16 Tags (include His-tag). FIGURE 8 UPLC profile of AAI degraded by ODMs enzyme at 37°C (left) and 25°C (right). The degradation efficiency of three ODMs is shown in Supplementary Table S3. FIGURE 8 UPLC profile of AAI degraded by ODMs enzyme at 37°C (left) and 25°C (right). The degradation efficiency of three ODMs is shown in Supplementary Table S3. FIGURE 8 UPLC profile of AAI degraded by ODMs enzyme at 37°C (left) and 25°C (right). The degradation efficiency of three ODMs is shown in Supplementary Table S3. and in-depth study. Although these genes are quite different in homology, they play the same role in the structural modification of methyl-containing secondary metabolites, such as AAI. Our results also demonstrate that methylation plays a role in protein– protein interactions and affects protein stability and enzyme activity. 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